BIO LABS / natural science guru

Biology LabPaq / Published by: Hands-On Labs, Inc.

[email protected] / www.HOLscience.com / Toll Free 866.206.0773

A laboratory Manual of Small-Scale Experiments for the independent Study of

general Biology

LabPaq® is a registered trademark of Hands-On Labs, Inc. (HOL). The LabPaq referenced in this manual is produced by Hands-On Labs, Inc. which holds and reserves all copyrights on the intellectual properties associated with the LabPaq’s unique design, assembly, and learning experiences. The laboratory manual included with a LabPaq is intended for the sole use by that LabPaq’s original purchaser and may not be reused without a LabPaq or by others without the specific written consent of HOL. No portion of any LabPaq manual’s materials may be reproduced, transmitted or distributed to others in any manner, nor may be downloaded to any public or privately shared systems or servers without the express written consent of HOL. No changes may be made in any LabPaq materials without the express written consent of HOL. HOL has invested years of research and development into these materials, reserves all rights related to them, and retains the right to impose substantial penalties for any misuse.

Published by: Hands-On Labs, Inc. 3880 S. Windermere St. Englewood, CO 80110

Phone: Denver Area: 303-679-6252 Toll-free, Long-distance: 866-206-0773

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Printed in the United States of America.

The experiments in this manual have been and may be conducted in a regular formal laboratory or classroom setting with the users providing their own equipment and supplies. However, this manual was especially written for the benefit of the independent study of students who do not have convenient access to such facilities. It allows them to perform college and advanced high school level experiments at home or elsewhere by using a LabPaq, a collection of experimental equipment and supplies specifically packaged to accompany this manual.

Use of this manual and authorization to perform any of its experiments is expressly conditioned upon the user reading, understanding and agreeing to fully abide by all the safety precautions contained herein.

Although the author and publisher have exhaustively researched many sources to ensure the accuracy and completeness of the information contained in this manual, we assume no responsibility for errors, inaccuracies, omissions or any other inconsistency herein. Any slight of people, organizations, materials, or products is unintentional.

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Table of Contents 5 To the instructor

6 To the Student

7 How to Perform an Experiment

9 Safety Concerns

11 Science lab Safety Reinforcement Agreement

Experiments 14 Research and The Scientific Method

37 Microscopy and the Metric System

75 Basic Chemistry for Investigating Living Systems

105 Cell Structure and Function: Cell Types and Transport

131 Cell Structure and Function: Tonicity and pH

153 Enzymes

183 Photosynthesis and Respiration

215 Mitosis and Meiosis

238 Genetic Inheritance

266 Human Genetics

285 The Properties of Water

APPENDiX 321 laboratory Equipment and Techniques

323 Potential Laboratory Hazards

326 Material Safety Data Sheets

328 How to Write Lab Notes and Lab Reports

334 laboratory Drawings

336 Final Cleanup Instructions

338 Using Statistics

342 The T-Test

348 The Chi-Square Test

351 Preparing a Water Bath

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To the instructor As an increasing number of students embrace online and independent-study courses, laboratory experiences must remain an integral part of science education. This lab manual’s author and publisher are science educators who welcome electronic technology as an effective tool to expand and enhance instruction. However, technology can neither duplicate nor replace learning experiences afforded to students through traditional hands-on laboratory and field activities. This does not mean that some experiments cannot or should not be replaced or reinforced by computer simulations; but any course of science study must also provide sufficient hands-on laboratory and field experiences to:

● Engage students in open-ended, investigative processes by using scientific problem solving.

● Provide application of concepts students have seen in their study materials, which reinforce and clarify scientific principles and concepts.

● Involve multiple senses in three-dimensional rather than two-dimensional learning experiences that are important for greater retention of concepts and for accommodation of different learning styles.

● Stimulate students to understand the nature of science including its unpredictability and complexity.

● Provide opportunities to engage in collaborative work and to model scientific attitudes and behavior.

● Develop mastery of techniques and skills needed for potential science, engineering, and technology careers.

The knowledge gained from science courses with strong laboratory components enables students to understand, in practical and concrete ways, their own physical makeup, the functioning of the natural world around them, and contemporary scientific and environmental issues. It is only by maintaining hands-on laboratory experiences in our curricula that the brightest and most promising students will be stimulated to learn scientific concepts and avoid being turned-off by lecture- and textbook-only approaches. Physical experimentation may offer some students their only opportunity to experience a science laboratory environment. All students – as potential voters, parents, teachers, leaders, and informed citizens – will benefit from a well-rounded education that includes science laboratory experiences when it is time for them to make sound decisions affecting the future of their country and the world.

This lab manual can be used by all students, regardless of the laboratory facilities available to them. The experiments are based on the principles of micro-scale science which have been successfully used in campus laboratories for decades. LabPaq’s micro-scale experiments can also be performed at home, in a dorm room, or at a small learning center that lacks a formal laboratory.

Introduction

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To the Student Science is a way of learning about our natural world and how it works by testing ideas and making observations. Learning about the characteristics of the natural world and how those characteristics change and interact with each other makes it easier to understand ourselves and our physical environment. Also, it helps us make the multitude of personal and global decisions that affect our lives and our planet. Science credits are impressive on an academic transcript and your science knowledge may create some unique job opportunities.

What are Micro-Scale Experiments?

You may be among the growing number of students to take a full-credit, laboratory science course through independent study, due to the development and perfection of micro-scale and small- scale experimentation techniques over the past half century. While experimentation on any scale is foundational to fully understanding science concepts, science courses in the past have required experimentation to be performed in the campus laboratory due to the potential hazards inherent in traditional experimentation.

Potential hazards, increasing chemical, specimen, and science equipment costs, and environmental concerns made high schools, colleges, and universities reexamine the traditional laboratory methods used to teach science. Scientists began to scale down the quantities of materials and the size of equipment used in experiments and found reaction results remained unchanged.

Over time, more and more traditional science experiments were redesigned to be performed on micro and small scales. Educational institutions eventually recognized that the scientific reaction, not the size of the reaction, facilitates learning. Successive comparative assessments have proven that students’ learning is not impaired by studying small-sized reactions. Many assessments even suggest that science learning is enhanced by small-scale experimentation.

In the mid-1990s, Dr. Peter Jeschofnig of Colorado Mountain College, pioneered the development of LabPaqs: academically aligned, small-scale experiments that can be performed at home. Hands-On Labs, Inc. has subsequently proven that students can perform LabPaq’s rigorous science experiments at home and still achieve an equivalent, if not higher, level of learning than their campus-based peers.

Introduction

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How to Perform an Experiment

Although each experiment is different, the process of preparing, performing, and recording an experiment is essentially the same.

Choose the Right Place for Your Home Laboratory: The best place to perform at-home experiments will be determined by the nature of the individual experiments. However, this place is usually an uncluttered room where a door can be closed to keep out children and pets; a window or door can be opened for fresh air, ventilation, and fume exhaust; there is a source of running water for fire suppression and cleanup; and there is a counter or tabletop work surface. A kitchen usually meets all these requirements. Sometimes the bathroom works too, but it can be cramped and subject to interruptions.

Review each experiment before starting any work to help you select the most appropriate work area. Because some of the equipment and supplies in your LabPaq may pose dangers to small children and animals, always keep safety in mind when selecting a work area, and always choose an area where you cannot be disturbed by children or pets.

Use a Lab Partner: While the experiments in the LabPaq can be performed independently, it is often fun and useful to have a lab partner to discuss ideas with, help take measurements, and reinforce your learning process. Whether your partner is a parent, spouse, sibling, or friend, you will have to explain what you are doing, and in the process of teaching another, you will better teach yourself. Always review your experiments several days ahead of time so you have time to line up a partner if needed.

Read the Entire Experiment before You Start: Knowing what you are going to do before you do it will help you organize your work and be more effective and efficient.

Review Basic Safety: Before beginning work on any experiment, reread the lab manual’s safety sections, try to foresee any potential hazards, and take appropriate steps to prevent safety problems.

Organize Your Work Space, Equipment, and Materials: It is hard to organize your thoughts in a disorganized environment. Assemble all required equipment and supplies before you begin working.

Outline Your Lab Notes: Outline the information needed for your Lab Notes and set up any required data tables before the experiment, to make it easier to enter observations and results as they occur. LabPaq CDs normally include a Report Assistant containing .rtf files of each experiment’s questions and data tables. These files can be copied and pasted into your Lab Notes to facilitate your compilation of data and text information.

Perform the Experiment According to Instructions: Follow all directions precisely in sequential order. This is not the time to be creative. Do not attempt to improvise your own procedures!

Introduction

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Think About What You Are Doing: Stop and give yourself time to reflect on what has happened in your experiment. What changes occurred? Why? What do they mean? How do they relate to the real world of science? This step can be the most fun and often creates “light bulb” experiences of understanding.

Cleanup: Always clean your laboratory space and laboratory equipment immediately after use. Wipe down all work surfaces that may have been exposed to chemicals or dissection specimens. Blot any unused chemicals with a paper towel or flush them down the sink with generous amounts of water. Wrap dissection specimens in newspaper and plastic and place them in a sealed garbage can. Discard used pipets and other waste in your normal trash. Return cleaned equipment and supplies to their LabPaq box and store the box out of reach of children and pets.

Complete Your Work: Complete your Lab Notes, answer the required questions, and prepare your Lab Report. If you have properly followed all the above steps, the conclusion will be easy.

NOTE: The Appendix section of this manual contains valuable information regarding equipment and techniques specific to the discipline you are studying. Please take the time to review this section before beginning experimentation.

Introduction

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Safety Concerns CAUTION for Women:

If you are pregnant or could be pregnant, you should seek advice from your personal physician before doing any type of science experimentation.

You, as a responsible science student and researcher, are solely responsible for safely storing and using your LabPaq materials and for conducting your experiments in a safe and responsible manner.

Items in your LabPaq can be especially dangerous to children and pets, so the LabPaq should always be kept safely stored out of their reach. The LabPaq may contain acids or other chemicals that can cause burns if mishandled plus serious illness and/or death if consumed.

Many LabPaq items are made of glass and/or have sharp edges that pose potential risks for cuts and scratches. While LabPaq thermometers do not contain mercury, they might still break and cause injury. LabPaqs contain small items and materials that could cause choking, injury, or death if misused.

Experimentation may require you to climb, push, pull, spin, and whirl. While these activities are not necessarily dangerous, they can pose hazards which means you should always undertake these activities cautiously and with consideration for your surroundings. If you need to climb to take measurements, make sure any stool, chair, or ladder you use is sturdy and take ample precautions to prevent falls. It is wise to have a partner help keep you stable when you must climb. Be especially aware of experimental equipment that you must put in motion, and act cautiously to ensure that items cannot go astray and cause injury to people or property.

If you or anyone accidentally consumes or otherwise comes into contact with a substance that could be toxic or cannot be easily washed away, immediately call:

The National Poison Control Center: 1-800-222-1222

Your eyesight is precious and should be protected against chemical spills or splashes as well as flying objects and debris. Always wear safety goggles when working with chemicals of any kind and when working with non-chemical objects that could possibly fly into your eyes.

Since chemicals, dirt, and germs are often involved in laboratory experiments, you should never eat or smoke in your laboratory area. Protect your body by keeping your hair tied back from your face and by wearing old clothing that fully covers your arms, legs, and feet.

You also need to protect your home furnishings from damage during your experimentation. Cover your work surface with plastic or paper towels when appropriate to prevent ruining furniture and to aid in cleanup.

Introduction

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The best safety tools you have are your own mind and intellectual ability to think and plan. After previewing each experiment, carefully think about what safety precautions you need to take to experiment safely, and then take them!

Since it is impossible to control students’ use of this lab manual and related LabPaqs or students’ work environments, the author(s) of this lab manual, the instructors and institutions that adopt it, and Hands-On Labs, Inc. – the publisher of the lab manual and the producer of LabPaqs – authorize the use of these educational products only on the express condition that the purchasers and users accept full and complete responsibility for all and any liability related to their use of same. Additional terms authorizing the use of a LabPaq are contained in its Purchase Agreement available at www.HOLscience.com.

Introduction

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Science lab Safety Reinforcement Agreement Any type of science experimentation involves potential hazards, and unforeseen risks may exist. The need to prevent injuries and accidents cannot be overemphasized!

Use of this lab manual and any LabPaqs are expressly conditioned upon your agreement to follow all safety precautions and accept full responsibility for your actions.

Study the safety section of this lab manual until you can honestly state the following:

 Before beginning an experiment, I will first read all directions and then assemble and organize all required equipment and supplies.

 I will select a work area that is inaccessible to children and pets while experiments are in progress. I will not leave experiments unattended and I will not leave my work area while a chemical equipment is set up unless the room is locked.

 To avoid the potential for accidents, I will clear my home laboratory workspace of all non- laboratory items before setting up equipment and supplies for my experiments.

 I will never attempt an experiment until I fully understand it. If in doubt about any part of an experiment, I will first speak with my instructor before proceeding.

 I will wear safety goggles when working with chemicals or items that can get into my eyes.

 I know that except for water, most solvents, such as toluene, alcohols, acetone, ethers, and ethyl acetate are highly flammable and should never be used near an open flame.

 I know that the heat created when water is added to concentrated acids is sufficient to cause spattering. When preparing dilute acid solutions, I will always add the acid to the water – rather than the water to the acid – while slowly stirring the mixture.

 I know it is wise to wear rubber gloves and goggles when handling acids and other dangerous chemicals; I should neutralize acid spills with sodium bicarbonate; and I should wash acid spilled on skin or clothes immediately with plenty of cold water.

 I know that many chemicals produce toxic fumes and that cautious procedures should be used when smelling any chemical. When I wish to smell a chemical, I will never hold it directly under my nose, but will use my hand to waft vapors toward my nose.

 I will always handle glassware with respect and promptly replace any defective glassware. Even a small crack can cause glass to break, especially when heated. To avoid cuts and injuries, I will immediately dispose of any broken glassware.

Introduction

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 I will avoid burns by testing glass and metal objects for heat before handling. I know that the preferred first aid for burns is to immediately hold the burned area under cold water for several minutes.

 I know that serious accidents can occur when wrong chemicals are used in an experiment. I will always read labels before removing chemicals from their containers.

 I will avoid the possibility of contamination and accidents by never returning an unused chemical to its original container. To avoid waste I will try to pour only the approximate amount of chemicals required.

 I know to immediately flush any chemical spill on the skin with cold water and consult a doctor if required.

 To protect myself from potential hazards, I will wear long pants, a long-sleeved shirt, and enclosed shoes when performing experiments. I will tie up any loose hair, clothing, or other materials as well.

 I will never eat, drink, or smoke while performing experiments.

 After completing all experiments, I will clean my work area, wash my hands, and store the laboratory equipment in a safe place inaccessible to children and pets.

 I will always conscientiously work in a reasonable and prudent manner to optimize my safety and the safety of others whenever and wherever I am involved with any type of science equipment or experimentation.

I am a responsible adult who has read, understands, and agrees to fully abide by all safety precautions prescribed in this lab manual for laboratory work and for the use of a LabPaq. Accordingly, I recognize the inherent hazards associated with science experimentation; I will always experiment in a safe and prudent manner; and I unconditionally accept full and complete responsibility for any and all liability related to my purchase and/or use of a science LabPaq or any other science products or materials provided by Hands-On Labs, Inc. (HOL).

____________________________________________________ ____________ Student’s Name (print) and Signature Date

Introduction

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LabPaq by Hands-On Labs

ExpErimEnts

Research and The Scientific Method Margaret E. Vorndam, M.S. Version 42-0116-00-01

Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.

Experiment Summary:

Students will use the scientific method, controls, double-blind experimentation, and statistics to determine the validity of data. Students will investigate whether a subject’s dominant hand can be used to predict which thumb will be placed on top when hands are clasped. They will learn how to write a formal lab report.

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ExpErimEnt

Objectives ● To outline an approach to explore a scientific problem

● To explain the rationale of the scientific method

● To use simple statistics to compare data results

● To write a laboratory report

Time Allocation: Four to eight hours total.

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Experiment Research and The Scientific Method

materials

MATERIALS FROM: QTy ITEM DESCRIPTION: Student Provides 1 Paper 1 Pen or pencil 1 Computer with word processing and spreadsheet programs 1 Calculator 50 Volunteers

Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq.

Safety Issues: None

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Experiment Research and The Scientific Method

Discussion and review Science begins with observation and inquiry based on that observation. While there are questions that we will possibly never be able to answer satisfactorily such as “How did the universe begin?” most questions do have answers that can be researched. Theoretically, all questions that we pose could be considered to be scientific because we are biological creatures, and the world/ universe around us provides the items that we require to live – shelter, food, water, and air. Although questions about how the stock market functions may seem to be far removed from scientific inquiry, those questions are generated by the barter system that provides shelter, food, water, and air for us. Other animals may use systems that supply their needs in a parallel, though through a much less complex way.

The acquisition of new scientific knowledge depends on free and ready access to prior scientific knowledge of a chosen research topic. A scientist is interested in exploring a scientific problem or an area where knowledge is incomplete – the unknown. She first explores the literature to discover what is already known about the subject (background information), and then poses a hypothesis regarding the problem. She then plans and conducts a research project that is capable of supporting or refuting her hypothesis. If she is able to support the hypothesis, she can follow up with more research to explore further. Finally, she will seek to publicize her research findings at a scientific conference and/or submit a publication to a scientific journal. Not only will she be publicly credited with the new information, but she will make her findings available for other scientists to test, critique, and use in further research on the topic. Thus, the cycle of information continues with past research informing new research efforts. The overall result is the total body of knowledge that comprises all of science.

Over time, scientists have developed a global approach to research that has informed investigative methodology. This method had its roots in the inquiry-driven teachings of Socrates and developed more formally as a logical approach to critical thinking-driven research during the Age of Enlightenment. Although today scientists and theoreticians argue about variations applicable to specific problems, the scientific method has been accepted as the basis of scientific inquiry.

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Experiment Research and The Scientific Method

Exercise 1: The Scientific Method Search Key Words: Scientific method, scientific research.

For this laboratory, a simple research project will be conducted, followed by the construction of a formal laboratory report. We will first illustrate the step-by-step process involved in the formulation and conduct of a simple exercise. Then, we will look at the process involved in the construction of a formal laboratory report which is a way to present research in an organized manner and is similar to the format that one would use to author a research article that could be submitted to a scientific journal.

First, let us look at a simple example of a biological research study about the eating habits of a common toad.

A. Make an Observation and Pose a Question: The scientific method process begins with an idea: Propose a problem and formulate a statement about the problem. For instance, this problem might be of interest: “It is generally believed that toads of genus and species Bufo bufo eat insects and minnows. This researcher is interested in investigating whether B. bufo prefers insects over minnows, if presented with a choice.”

B. Research Existing Information: All scientific research builds on past research. So, the question would be researched in the scientific literature to find out what is already known about it. Literature research can be done online, but library services such as Biological Abstracts1 or EBSCO2 should also be used to be as comprehensive as possible. Use search terms such as “B. bufo diet” or a similar descriptor. The research may or may not indicate that there is existing information on the question. Whatever the case, summarize the findings of this preliminary research in the background section of the final report and provide references to the existing literature in the reference section at the end of the report.

C. Formulate a Testable Hypothesis: The literature search may indicate that there is little existing information on the problem or that someone else has already answered all or part of the problem. If so, revise the initial question based on pre-existing knowledge.

Now, formulate a testable hypothesis (H1). Do not attempt to “prove” a hypothesis. Rather, attempt to either support or reject the hypothesis. Either outcome is viable, though there may not be much interest in a journal article that presents a rejected hypothesis! There are actually a minimum of two hypotheses to test:

1 Biological Abstracts, BIOSIS, http://www.biosis.org, May 2002. Two Commerce Square, 2001 Market Street, Suite 700, Philadelphia, PA 19103-7095 USA phone: 1-800-523-4806 (USA and Canada). E-mail: [email protected] 2 EBSCO Information Services, http://www-us.ebsco.com/home/default.asp, May 2002. Suite 120, 2801 Youngfield St., Golden, CO 80401-2264, Phone: (303) 237-1753/(800) 727-1077. E-mail: [email protected]

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Experiment Research and The Scientific Method

H1= “When given a choice, B. bufo has a food preference for either insects or minnows (amount of insects consumed ¹ amount of minnows consumed).”

Ho is the “null” hypothesis, or the hypothesis that is accepted if the H1 hypothesis is rejected. Generally, this hypothesis is understood as the equal assumption, so it is not included it in the report.

Ho = “When given a choice, B. bufo has NO food preference for either insects or minnows (amount of insects consumed ≈ amount of minnows consumed).”

H1 is the alternate hypothesis that will be tested. For testing purposes, the condition to be tested is never equal to another condition, but it can be ‘greater or less than’ or ‘not equal to’ or ‘greater than’ or ‘less than.’ At this point, a scientist might wish to identify grant sources and obtain financial support from an interested person or group that is willing to fund the research project!

Hypothesis construction includes planning how to test the hypothesis. So, the scientist must develop a sound experimental framework for the research plan. It is a good idea to have the plan reviewed by knowledgeable researchers who may be able to suggest revisions that will increase the value of the research. Also, where the planned research may deal with comparative treatments, such as testing a new drug, a researcher may also want to include a “control”, such as a placebo sugar pill, that provides baseline information to which outcomes can be compared for treatment versus no treatment scenarios.

In the B. bufo example, a control treatment would not necessarily be needed, since responses to two different treatments are measured. Outcomes can readily be differentiated through comparative statistics. Of course, the Bufo study would require that each toad (we will explain why one is not enough later) be as similar as possible to the others in the study and that the aspects of the study be handled the same. Why?

D. Planning the Experimental Design: This step is the single most important part of research. Good experimental design is critical if data are to be valid. Take the time to do experimental design planning carefully.

Several different experimental designs exist. Designs also differ by discipline. Thus, the approach used in a sociological investigation may be differently designed than an approach used in a scientific investigation, but the basic structure, forming a testable hypothesis, managing for variables, etc., remains the same.

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Experiment Research and The Scientific Method

There is a place for accepting hypotheses based on the results of repetition in scientific research. This approach is referred to as the inductive method. Numerous observations should be made over a period of time. After the same cause-and-effect process is observed repeatedly, a hypothesis can be accepted as true inductively. No formal testable hypothesis was set up, but there is enough experiential evidence to accept that an outcome will predictably result from a specific action. We all learn this way; since we were born we’ve learned from experience gained from the positive and negative effects that result from our own actions.

In the hypothetico-deductive method that we are exploring in this experiment, the student in the role of researcher will formally set up a testable hypothesis as we have discussed in the section above. This testable hypothesis must be potentially disprovable or supportable. Think of it as formally setting up an “if…then…” statement. Hypothesis construction includes planning how to test the hypothesis. So, develop a sound experimental design.

There are several types of experimental design from which one could choose. Each can be applied to specific research questions. So, before beginning to plan the design, assess these design options:

● Should a control group be used in addition to the actual treatment group? For instance, if the effectiveness of a new drug for migraine headaches is being assessed, how will effectiveness be judged? The researcher could choose to set up two groups of people selected from a population of people who suffer from migraine headaches.

The treatment group will be given the new drug, while the control group will be treated with a placebo (such as a sugar pill that is known to be safe and will have no effect on headaches) that looks like the new drug. Collect data from both groups and then determine whether there is a reported difference in headache frequency between the two groups. The control group enables the researcher to make a statement about how not using the drug versus how using the drug will affect headache occurrence. One could also assess the possibility of drug side effects by comparing the experiences of the two groups.

If the experiment is designed so that the researcher who passes out the medication to the two groups does not know which medication is the drug or the placebo, the experiment is called a double-blind experiment. This experimental design can eliminate researcher bias because it won’t be known whether the new drug was more effective than the placebo until the data has been collected and compared. The researcher will not be able to have any preconceived idea about what the outcome of the experiment should be.

● Are the test subjects selected all representative of the universal population of the group? In other words, if a researcher repeated this study over and over with different groups of test subjects, would the outcomes be reliably predictable of the universal population?

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Experiment Research and The Scientific Method

● How many test subjects should be included in the experimental design? For example, if data about hind foot longest toe lengths in a population of toads are collected, how many toads have to be sampled in order to be able to support a hypothesis related to toad toe length in general and representative of the universal population of toads? Would one have more confidence in the data if the researcher sampled and obtained results from only five toads out of a population of 500 toads or if the researcher sampled and obtained results from 50 toads? Why? Because the larger sample size will give more confidence in the results by reducing the possibility of sampling bias. However, will sampling 500 subjects increase the confidence in the data more than sampling just 50 toads? At some point, more exact data are not obtained; it just becomes more expensive or time-consuming to collect data. So, determine how many subjects are enough to provide good data, but not so many that effort is wasted in doing data collection. Although we will not explore the subject of sample size further, the student can obtain more information about adequate sample size through the study of statistical probability.

● Plan the design to statistically assess the different results that might be obtained from the two test groups. The application of statistical probability to data will allow one to decide whether data results are sufficiently similar or different enough to support or reject the hypothesis. There are many types of statistical tests available that scientific researchers use to compare their data results, but using tests of probability, such as chi-square, the t-test, the test of correlation (r), or analysis of variance will be adequate in hypothesis testing of the type that we are exploring here.

So, let us turn again to our minnow or insect-eating toad example. In the B. bufo example, a control population is not necessarily needed since responses are being measured between two different treatments (minnows vs. insects), and the researcher can differentiate between the two outcomes readily by using a statistical approach. One can offset bias by:

● ensuring that each toad is given similar sized insects and minnows, or that adjustment be made to account for the caloric intake of insects vs. minnows

● that the choices of minnows or insects are presented simultaneously or possibly at equally spaced intervals

● that the toads are of similar size, and so on

All of these variables weigh in the experimental design and must be considered to ensure consistent data results. The experimental design must be explained in the formal report so that other researchers can repeat the study if they choose to do so.

Before beginning an experiment take the time to write out the experimental Procedures in detail and develop the forms that will be needed to collect data. This will provide a written guide from which to work.

E. Observe\Experiment: Now, conduct the research study. Keep detailed notes of the research and the observations made. There may be other factors that explain some of the observed outcomes, and interested researchers may wish to duplicate the study to confirm results. Thus, accurately recorded observations are important!

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Experiment Research and The Scientific Method

For example, the research plan may require that the toads be presented with equal amounts each of insects and fish. Then, the scientist observes which food is chosen and records the information.

F. Collect/Analyze Data: A laboratory notebook is an essential tool in scientific research. Notes written on paper towels, etc., are not acceptable. Every observation, no matter how mundane, should be recorded in a dedicated laboratory notebook during the conduct of an experiment. Any deviation from the experimental design plan, no matter how minor, should be recorded along with the rationale for the deviation. Why? A researcher may discover that other factors explain some of the data outcomes, and interested researchers may wish to duplicate the study to confirm the results. Thus, accurately recorded observations are very important as a way to document the study conduct and explain anomalous data.

Data records can also be kept on a computer spreadsheet or database. Spreadsheet software often provides access to basic statistical testing that can be useful. Statistical tools are powerful in providing insight into data groupings and are definitive for empirically justifying outcomes. We will turn to that discussion in a moment.

It is also important to keep a copy of experimental notes at a different location. If a fire or other mishap occurs that destroys the study notebook, a back up of the study conduct and data results will remain.

g. Draw a Conclusion: Good data analyses will either confirm or reject the hypothesis, H1, or they may be inconclusive. If an outcome cannot be established, then refinement of the research Procedure is possibly needed. In this special case, if H1 is rejected, then the scientist can state that Ho is accepted.

Statistics Rule! Although a complete statistics presentation is not included here, it is important to understand the application of statistics to scientific research. Statistical handling of data results can provide a high level of confidence in research conclusions. Thus, consider statistical use in research to make it more powerful.

Chi-squares, t-tests, standard deviations, confidence levels, and analysis of variance tests are statistical tools that may be appropriate to add robustness to data results. We will discuss means as the simplest versions of statistical handling.

Means: If we have two treatments of data results such as number of insects eaten by a toad versus number of fish eaten by the same toad, we can calculate the mean number of each food type eaten by the population of toads. First compute the mean for insects:

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Experiment Research and The Scientific Method

Sample Experimental Data

Toad # Treatment 1 # insects eaten

Treatment 2 # minnows eaten

Mean# insects eaten per toad = # insects eaten by toad 1 + # insects eaten by toad 2

+ ….etc.

÷ total # toads

1 3 6 2 5 5 3 2 8 4 4 6 5 3 7 Mean 17 ÷ 5 = 3.4 32 ÷ 5 = 6.4

Next, we do the same calculation for the number of minnows eaten by each toad to determine that mean value. We then compare the two means for similarity to decide whether the toads have eaten the same amounts of insects and minnows, or whether the toads prefer one food over the other.

The Dominant Thumb Experiment

We all have physical characteristics, such as eye color, hair color, height, etc., that are determined by the genes we inherited from our parents. We also inherit genes for handedness, whether

we tend toward right-handedness or left-handedness. We might also expect that genetic inheritance dictates that when we clasp our hands, the thumb of our dominant hand may overlap the non-dominant hand’s thumb. This experiment will determine whether for thumbs of clasped hands, there is a relationship between right-handedness or left-handedness and which thumb is clasped on top of the other thumb. Our question is whether one inherited trait always influences another closely related, trait.

In this exercise, the student will be conducting an experiment by using the scientific method. Some of the information is provided. The student as researcher will have to conduct research to determine whether a selected hypothesis is accepted or rejected. Steps to complete the study are numbered below.

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Experiment Research and The Scientific Method

procedure 1. Background: When investigating a problem for experimentation, a good researcher is

interested in what might already be known about the problem. Do a brief library and/or online search to determine what is already known about this problem. Note that this search need not be as exhaustive as if a formal research project were being conducted. Typical search terms to use when searching the Internet might be handedness, thumb dominance, linked inherited traits, and like terms.

2. Statement of Problem: This experiment will investigate whether the subject’s dominant hand, left or right, can be used to predict which thumb will be placed on top when hands are clasped.

3. Setting up a Hypothesis: A hypothesis is a statement that the researcher constructs about what she believes will happen. It is not right or wrong. It will be tested to determine whether the hypothesis is supported by the collected data or rejected. Which of the following possible hypotheses do you wish to test?

a. If a person is right-handed, the right thumb will be on top when the hands are clasped. If the person is left-handed, the left thumb will be on top when the hands are clasped. In this hypothesis, we believe that the gene for handedness influences the gene for dominant thumb. What will be the mathematical equivalent of this statement?

b. There is no correlation between which thumb is on top and the hand dominance when the hands are clasped. In this hypothesis, we accept that these two characteristics are genetically unrelated. What will be the mathematical equivalent of this statement?

4. Research Design and Procedure for Testing the Hypothesis: Choose 50 volunteers as subjects for this experiment. When choosing the volunteers consider how a researcher might bias the experimental outcome and how to avoid doing so. If the student is unable to choose 50 volunteers, defend the choice of the number of volunteers that are proposed for sampling. How can one prove that the number of volunteers used is an adequate sample size to test the hypothesis?

a. For this experiment, the student will be asking the subjects, without informing them of the purpose of the study, to clasp their hands together. Then, observe which thumb is on top. Ask the subjects whether they are right-handed or left-handed. Why is it important not to tell the subjects ahead of time why they are being asked to clasp their hands?

b. Record the data for each subject in Data Table 1. Maintain this table with laboratory noted, but do not include it in your report. Have it available if anyone wishes to more fully review your results.

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Experiment Research and The Scientific Method

Data Table 1: Data Results

Subject # Left Thumb Dominant Left-Handed Right Thumb Dominant Right-Handed

1 2 3 4 5 6 Etc…..50

c. Compile the data results from Data Table 1 to Data Table 2 summary. In the laboratory report to follow (Exercise 2, below), provide a paragraph with a text description of the data summary in Data Table 2.

Data Table 2: Summary of Data Subjects Number % of Total Subjects* Right-handed with right thumb dominant Right-handed subjects with left thumb dominant Left-handed subjects with right thumb dominant Left-handed subjects with left thumb dominant Total Number of experimental subjects

Number of subjects per category divided by total subjects. Example: if 5 of 50 total subjects were found in a category, the calculation would be 5 ÷Total number of subjects or 5/ 50 = .10 x 100 = 10%

100%

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Experiment Research and The Scientific Method

Exercise 2: Writing a Laboratory Report Search Key Words: Scientific experiment report, scientific writing, scientific writing style, Council of Biology Editors, reference citation.

All scientists write technical reports, and those scientists who write well are highly prized by their employers. Writing skills are an important consideration in job performance in scientific fields. Thus, it is important that these skills be developed. Although there may be some variation between instructors about how a laboratory report should be structured, generally all include the parts that are outlined below. Refer to reputable online sources for further information on technical writing and laboratory report writing.

Keep a laboratory notebook to record the details of an experiment. The notebook is an important asset when the laboratory report is written. The notebook is written as the experiment proceeds and should contain all pertinent information including what equipment is used for each part, each Procedure followed, the concentrations and amounts of any chemicals added, drawings of the setup of the experiment, and, most importantly, observed outcomes. At the conclusion of the experiment, the notebook provides all of the details that will be needed to write the laboratory report and to repeat the results observed, if necessary.

Style Rules for Scientific Reports: The Council of Science Editors3 has developed the style rules to use in scientific writing, and in particular, biological writing. Writing in the sciences uses different style rules than writing in the Humanities. When writing up research in the sciences, use the style rules that apply to scientific writing. In general:

1. Write in the third person, past tense mode, using a passive, impersonal style. For example, instead of writing “I measured the temperature of the solution . . .” write, “The temperature of the solution was measured . . .” The emphasis should be on the experiment, not on the experimenter.

2. Write in a textual format, not in a listing format. Where tables or figures are used for elaboration, explain the significance of their use. Also explain the tables’ or figures’ data within the text. A reviewer may not interpret the data as the author/researcher intended them to be interpreted, so write it out.

3. Be very concise and detailed. Reread and tighten the report prose until it is to the point and clear to the reader.

4. Write in complete sentences.

5. Use a word processor. Check for and correct spelling and grammatical errors. Word processors have spell checkers and grammar checkers.

6. Use headings to divide the logical sections of the report.

3 Council of Science Editors. Scientific Style and Format: The CSE manual for authors, editors, and publishers. 7th ed. 121Sunset Hills Road, Reston, VA 20190; 2006. 680 pp.

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Experiment Research and The Scientific Method

7. Avoid using quotes unless they are critical to the experiment and the report.

8. Read the paper out loud. If it doesn’t make sense verbally, it doesn’t make sense in text.

9. Never change what was recorded in the laboratory notebook. Instead, correct an entry by marking a single slanted strike through it. Add the date of revision and your initials. Why is this point important?

Parts of a Scientific Laboratory Report: A formal laboratory report should include the following sections in the order listed. Use the headers above each section so that a reviewer can easily follow the transition from one section to the next.

1. Title Information: Including name of researcher, Sponsoring Organization, Date.

2. Abstract: A brief description of the experiment that includes the purpose, the key result, the most significant point, and the major conclusion of the experiment. This section appears first, but it is written last, after the rest of the report is finished.

3. Introduction:

a. Background Information – What is known about the experiment prior to beginning it? The student should consult library or online sources for research on the topic of interest and outline it here. Cite references consulted and used for information at the end of the report in the References section.

b. Purpose – Why is the experiment being done? What new knowledge will be sought?

c. Hypothesis – What will be tested by doing the experiment? What are the underlying assumptions or points of previous research that have lead the researcher to this hypothesis?

4. Procedures:

a. Equipment Used – In sentence form, what equipment is required to do the experiment? Elaborate in enough detail so that another researcher can successfully repeat the study, but omit non-essential detail. For instance, the size of a beaker is not vital information unless it is necessary for an outcome.

b. Collection of Data – In sentence form, describe how the experiment was conducted, so that another researcher could repeat it exactly.

5. Results:

a. graph or Table – Present the summary results of the data in graphic or tabular format. Note that the detailed results should be maintained in the notebook, but do not include them in the formal report.

b. Description/Interpretation of Data – Write a paragraph that verbally presents the graph or table summary and elaborate on it. Present any calculations clearly, and show each step in detail.

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Experiment Research and The Scientific Method

6. Discussion: Support of Hypothesis – Make a statement as to whether the experiment supported or refuted the initial hypothesis.

7. Explanation/Conclusion:

a. How did the experiment support the hypothesis?

b. If it did not support the hypothesis, then how did the experiment refute the hypothesis? How might the researcher explain this outcome?

c. What was proven or disproved by the experiment? What is its importance? The outcome is just as valid if the hypothesis is not supported as if it were supported.

8. References:

a. What resources, websites, texts, lab manuals, etc., were used to inform the experiment?

b. If background research was done to find out what was known about the experiment before beginning, what are the sources that were cited in the background section? Use correct citation syntax when listing references.

procedure 1. Write a formal laboratory report using the data that were collected in Exercise: The Scientific

Method. Incorporate those results and Discussion assignment answers and tables, as appropriate.

2. Use correct style rules and use the same layout to complete your report as outlined in Parts of a Scientific Laboratory Report above.

3. Check the report for typographical errors and incomplete sentences. All word processing software has spelling and grammar checking software, so use it to correct the report.

4. Correctly cite all references. Use the citation style used by scientific writers.

5. Check the report one last time.

a. Did you check entire report for spelling and grammar errors?

b. Did you avoid using “I,” “me,” and “my.” Instead, used third person statements such as “This researcher….”?

c. Are all sections of the laboratory report filled in with the correct information?

d. Are all parts of the laboratory report complete?

i. Abstract at top of report is a mini-presentation of the report, including results, in only a few sentences?

ii. Background information included about previous studies done on this topic?

iii. Hypothesis listed?

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Experiment Research and The Scientific Method

iv. Research Procedures listed clearly enough that someone else could repeat the study?

v. Summary table and paragraph of description about table is included in the data sec- tion?

vi. No lists are included. Only complete sentences are used throughout the report?

vii. Results section contains statement about whether the data supported or did not support your hypothesis? Are statistics included and explained that will support the hypothesis statement?

viii. Are reference citations entered in correct format?

Research and The Scientific Method Margaret E. Vorndam, M.S. Version 42-0116-00-01

Lab report Assistant This document is not meant to be a substitute for a formal laboratory report. The Lab Report Assistant is simply a summary of the experiment’s questions, diagrams if needed, and data tables that should be addressed in a formal lab report. The intent is to facilitate students’ writing of lab reports by providing this information in an editable file which can be sent to an instructor.

Observations

Data Table 1: Data Results

Subject # Left Thumb Dominant Left-Handed Right Thumb Dominant Right-Handed

1 2 3 4 5 6 Etc…..50

Data Table 2: Summary of Data Subjects Number % of Total Subjects* Right-handed with right thumb dominant Right-handed subjects with left thumb dominant Left-handed subjects with right thumb dominant Left-handed subjects with left thumb dominant Total number of experimental subjects

Number of subjects per category divided by total subjects. Example: if 5 of 50 total subjects were found in a category, the calculation would be 5 ÷ Total number of subjects or 5 ÷ 50 = .10 x 100 = 10%

100%

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Experiment Research and The Scientific Method

Data Table 3: Thumb Dominance Results

Handedness and Thumb on top

Combined Percentages from Table 2

Right-handed, Right Thumb

Left-handed, Left Thumb

Right-handed, Left Thumb

Left-handed, Right Thumb

Exercise 1: The Dominant Thumb Experiment results A. During the background examination of the literature, what was discovered about what is already known concerning this problem?

B. If the Internet was used as a background information source, how can the researcher be sure that the information presented on the sites is correct? How could one determine whether one could safely accept the site as a good source of information?

C. What is the mathematical equivalent of the chosen hypothesis?

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Experiment Research and The Scientific Method

D. If 50 subjects were not interviewed, how can the number of volunteers be justified as an adequate sample size to test the hypothesis? If 50 subjects were interviewed, how does the researcher know that this sample size is large enough to effectively test the hypothesis?

E. Why is it important NOT to tell subjects what is being determined before they clasp their hands?

F. Combine the percentages for same hand/thumb (right handed/right thumb dominant + left hand/left thumb dominant) and opposite hand/thumb (right handed/left thumb dominant + left hand/right thumb dominant) in another table like Data Table 3.

G. Write a paragraph that verbally presents the data summarized in Data Tables 2 and 3.

Questions A. What was the hypothesis? Do the experimental data results support or refute the selected hypothesis?

B. Why should the data for same handedness-thumb and different handedness-thumb be combined for purposes of analysis?

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Experiment Research and The Scientific Method

C. Recheck all steps, evaluate the total experiment, and draw implications from the conclusions. Is the experiment valid enough to reach a conclusion?

D. How can the conclusion be supported by using statistics?

E. Are the results meaningful?

F. Were enough subjects used to be conclusive?

G. What experimental conditions are responsible for the results that were obtained?

H. What follow-up experimentation might be of use, based on this experiment?

I. If the obtained data results do not agree with those that are provided in the example above, which set would you believe? Why?

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Experiment Research and The Scientific Method

Exercise 2: Writing a Laboratory Report results Your laboratory report will be the result for this exercise.

Questions A. Propose a hypothesis for a hypothetical experiment and outline the experiment that would be conducted to test it. Use the steps of the scientific method to organize the outline.

B. Read a scientific research paper in a scientific journal. Identify the parts of the paper that address aspects of the scientific method. Find the parts that would be considered the abstract, background information, the hypothesis, the Procedure, the data, and the conclusion. Did the experiment include a control? Why or why not? Is a control always necessary?

C. Verify what you have learned by answering the following questions:

1. What are the essential elements of the scientific method?

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Experiment Research and The Scientific Method

2. What is a hypothesis?

Can more than one H1 be tested? Can there be an H2?

3. Can a hypothesis be proven? Why or why not?

4. Is it sufficient to do a single experiment to test a hypothesis? Why or why not?

5. What statistical techniques can be used to add robustness to the data results?

6. Why is it easier to test only one substance at a time or only one action at a time when doing an experiment?

7. Is it possible to test more than one outcome at a time? What revisions in the testing approach would need to be made to do this?

8. What is the purpose of a control in an experiment? Does every experiment require a control?

D. Do the following statements represent a hypothesis, conclusion, or theory?

1. The data show that various vaccines protect people from disease.

2. All living things are made of cells.

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Experiment Research and The Scientific Method

3. e = mc2

4. The instructor loves chocolate chip cookies! (Okay, just checking that you’re still awake, but you could test this! It would definitely require multiple tests!)

E. What applications might the Scientific Method have to daily life? Give an example.

Laboratory summary What have you learned from doing this laboratory?

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Microscopy and the Metric System Margaret E. Vorndam, M.S. Version 42-0090-00-01

Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.

Experiment Summary:

Students will use the metric system to measure length, volume, mass, and temperature. Students will learn how to convert between Metric and US customary units and identify the types of equipment and glassware used in measurement. In addition, students will learn about the different types of microscopes and their functions, the parts of the microscope, and how to calculate total magnification, depth of field, and field of view. They will practice using a microscope by preparing a wet-mount slide, a slide of cheek cells, and a slide of onion cells.

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ExpErimEnt

Objectives ● To perform US customary to metric and Fahrenheit to Celsius temperature scale conversions

● To suggest the correct microscope to use in a particular situation

● To operate a microscope

● To prepare a simple specimen wet mount and view it under a compound light microscope

● To outline the use of common experiment instruments

Time Allocation: Four to eight hours total.

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Experiment Microscopy and the Metric System

materials MATERiAlS

FROM: QTy ITEM DESCRIPTION:

Student Provides 1 Cutting board 1 Glass to hold water 1 Object to measure 1 Onion 1 Disposable magazine or newspaper 1 Isopropyl alcohol 1 Paper towels

1 Piece of white paper 1 Sugar 1 Toothpicks 1 Liquid detergent for cleaning 1 Tap water of varied temperatures 1 Thread, 1” long pieces (three colors) 1 Microscope 1 Yardstick with metric scale (optional) 1 Apron From labPaq 2 Beaker, 50 mL, plastic 1 Cylinder, 50 mL - Graduated, Plastic

1 Dissection-kit with 7-tools - including the following: Bent Probe, Dropping Pipette, Probe, Ruler in pocket, Scalpel with 2 Blades - Note blades are in the pocket, Scissors, Tweezers

1 IKI Indicator, 2.1% - 13 mL in Glass Vial in Bubble Bag 10 Pipette, Long Thin Stem 1 Digital scale

1 Slide, blank (10 pcs) 1 Slide - Cover Glass - Cover Slip Cube

1 Prepared Slide, any 1 Gloves, Disposable (1 pair) 1 Thermometer-in-cardboard-tube 1 Lens-paper-pack-50-sheets

Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq.

Safety Issues: Specifically review Cutting, Chemical, and Glassware safety issues.

Lab Hint: When using IKI iodine indicator solution – wear apron, safety glasses, and rubber gloves plus cover work surfaces with white plastic garbage bags to avoid stains.

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Experiment Microscopy and the Metric System

Exercise 1: Measuring Length, Weight, Volume, and Temperature Search Key Words: metric, metric measurement, experiment equipment, metric length, metric weight, metric volume, temperature, Celsius, metric conversion.

Metric Terminology and Unit Conversions: Scientific measurement is based on the International System of Units commonly referred to as the metric system. A French vicar, Gabriel Mouton, conceived of the system around 1670. In 1790, Louis XVI authorized the use of the system for scientific pursuits, and France was the first country to adopt the metric system as its formal method of measurement in 1795. Today, the metric system is used worldwide, and many countries have adopted it as their measurement method.

Students in the United States generally have not had the opportunity to use the metric system in their daily lives. Although the United States adopted the system in 1972, it has never actually used it as the unit of measurement. However, it is important for students of the sciences to understand the metric system and its relative relationship to the US customary system that is commonly used in the United States. Students may be familiar with computer storage terminology which uses metric, as in byte, megabyte, gigabyte, etc.

The versatility of the metric system is attributable to its decimal system base. For example, the centimeter is used for measuring length; it is abbreviated as cm, and 2.54 cm = 1 inch. Ten centimeters are equal to one decimeter (dm). Ten dm (or 100 cm) are equal to one meter (m). There are similar relations for weight and volume measurements. We will examine each of these in turn. But first, let us look at the relationships between the metric and US customary systems and relationships within the metric system. Not all types of measurements use every increment of the metric system. For instance, in the length category, cm and m are commonly used, but dm is not. Convention dictates that liter (L) is always capitalized. Periods are not used after units, except at the end of a sentence, of course.

The metric system is based on the following prefix terms that are associated with changes in the powers of 10.

Metric Prefixes

Pico Nano Micro Milli Centi Deci Deka Hecto Kilo Mega giga

Symbol p n m(mu) m c d da h k M G

Power of 10 10-12 10-9 10-6 10-3 10-2 10-1 10 102 103 106 109

Mathematical Equivalent 0.000001 0.001 0.01 0.1 1 10 100 1,000 1,000,000

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Common Units of Metric Measurement

length nm mm mm cm Meter, m km Weight pg ng mg mg Gram, g kg Volume mL mL Liter, L

Note: The grey areas above are not commonly used as a unit for that particular measurement.

The student must also be familiar with conversions between different powers of 10. When doing conversions, determine the conversion factor first and then do the calculation. Conversions can be determined using proportional equations. For instance, how do we determine the number of mL in 5 liters of fluid?

First, determine the conversion factor. The direction of the conversion determines which unit is in the numerator and which is in the denominator. The numerator will be the unit that we are converting to, and the denominator will be the 1 unit equivalent for the unit we are converting from. It is easy to remember that the units that we are converting from must cancel out, which is accomplished by dividing by that unit. Thus, the unit we are converting from must appear in the denominator.

We know that there are 1000 mL in 1 L, so in this example, the needed conversion factor is 1000 mL/1 L.

The proportional equation will be: 1,000 mL = X mL__

1 L 5 L

Thus: X mL = 1,000 mL/1 L x 5.0 L and 5.0 L x (1000 mL/L) = 5000 mL, with the liter units canceling out each other.

This equation can also be written as: 5.0 L x 103 mL/L = 5 x 103 mL.

How many mL (microliters) are there in 2.4 liters? There are 1,000,000 mL, also written as 1x106 mL, in 1 L. The conversion factor will be 1x106 mL/1 L, so:

_1x106 mL_ = __X mL__

1 L 2.4 L

2.4 L x (1x106 mL/L) = 2,400,000 mL = 2.4 x 106 mL

If the student is familiar with decimal and power of 10 conversions, this is straightforward. Shift the decimal point the appropriate number of places right or left, depending on the direction of the conversion.

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Try the following conversions for practice.

240,000 ng = ______mg = ______g 50 cm = ______mm = _______m

The table below provides comparative metric units to US customary units.

Metric and US Customary Unit Equivalents1

US Customary Unit = Metric Unit Metric Unit = US Customary Unit

1 inch 2.54 cm 1 cm 0.3937 inch 1 yard 0.91 m 1 m 39 inches = 1.09 yd 1 mile 1.61 km 1 km 0.62 mile

1 ounce 28.35 g 1 g 0.04 oz 1 pound (16 oz) 0.45 kg 1 kg 2.20 pounds 1 cubic inch 16.39 cm3 1 cm3 (= 1 mL) 0.06 in3

1 fluid ounce 29.57 mL 1 mL 0.03 fl oz 1 pint (liquid) 0.47 L 1 L 2.13 pt (liquid)

1 gallon (liquid) 3.79 L 1 L 0.26 gal. = 1.1 qt (liquid)

To convert units from US customary to metric, and vice versa, use the comparative table values, setting up the conversion factor as above for metric conversions. For instance, to determine the number of meters in 2 yards:

0.9144 m = ___X m__ and 2 yards x (0.9144 m/yd) = 1.8288 m.

1 yard 2 yards

And to determine the number of mL in 3.5 oz:

29.574 mL = _X mL__ and 3.5 fluid oz x (29.574 mL/oz) = 103.5 mL 1 fluid oz 3.5 fluid oz

1 Note that there are unit conversion differences between dry and liquid measurements in the US system of weights and measures. Also, the United Kingdom (Imperial) and the US measurements differ in some cases. We use liquid volumes and US scales for the units in this laboratory.

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procedure 1. Length: A metric ruler is useful for measuring items of length. The ruler below measures in

mm, indicated by the small mm near 0.

a. How many mm are there in 1 cm? _____ In a meter (m)? _______

(Ruler is not to scale. See ruler in dissection kit.)

b. Locate a measurable object to use for this exercise. If the object is long, obtain a yardstick that includes a cm scale; they can be found in local hardware stores.

c. Record the length of the object below and do the conversions:

Name of object _________________

_______ cm = ________ mm = _________ m

2. Volume: The most commonly used metric measures of volume are the liter (L) and the milliliter (mL). Note the capitalized “L,” a scientific style for reference to liter.

Volume can also be expressed as length x width x height, resulting in a cubic cm (cm3), cubic m (m3), etc., designation. One mL of liquid is equivalent to 1 cubic centimeter (cm3) and is commonly written as cc for cubic centimeter. The cc designation is extensively used in the medical field.

Liquid volumetric measuring devices include calibrated graduated cylinders, volumetric flasks, burets, or graduated pipettes. There are many specialized variations of these basic tools in existence in today’s laboratory, including automated versions.

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Experiment Microscopy and the Metric System

Graduated Cylinder

Graduated Pipette Buret Volumetric Flask Not to Scale

Techniques for liquid Measurement

It is important to be as accurate as possible when measuring liquid using calibrated volumetric equipment. Since liquids tend to be slightly drawn up around the edges of the volumetric cylinder or flask due to capillary action, a bowl-shaped surface is created. This curved surface at the top of the liquid is called the meniscus. Always ensure that the bottom of the liquid’s meniscus is at the desired mark on the flask or cylinder. The meniscus should be even with the volumetric mark when looking at it from a parallel, eye-level position.

Practice adding tap water to the graduated cylinder. Select a mL level, and hold the cylinder mark for that mL at the level of the eye. Add water until the bottom of the meniscus is at the desired mL mark. A dropper or long-stemmed pipette can help to control the amount of liquid added, but with practice, students should become proficient at accurately pouring from a beaker.

Always pour an approximate volume of liquid into a clean beaker and then from the beaker into the volumetric flask or graduated cylinder. This will minimize contamination of the parent liquid source. Dispose properly of any left over liquid. Do NOT pour it back into the original container. Why?

When using a pipette or dropper to measure liquid, pour an aliquot into a clean beaker and then draw up the liquid from the beaker into the pipette. NEVER try to draw up chemicals by mouth. Why?

3. Weight: The most commonly used metric measures of weight are the gram (g), the kilogram (kg), and the milligram (mg). Metric weight is measured using a scale. Scales can be either digital or balance types and exist in all shapes and sizes. Calibrated weight sets of known

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weights can be purchased to ensure that experiment scales weigh correctly and do not drift from true measurements.

Specialized equipment such as spatulas and weighing papers are used to keep the weighing platform clean and to efficiently transfer the weighed solid material to a final container. As with liquids, it is always a good idea, but not as often done, to place an approximate aliquot of the solid material into a beaker and to use a clean knife to transfer the solid to the tared2 weighing paper on the scale. Liquids are weighed also. In this case, the container is tared and then the liquid is added to the container until the final weight is achieved.

Calibrated Weight Set Balance Scale Digital Scale

Use a small piece of paper and the scale from the LabPaq to measure out exactly three grams of sugar. Make sure to tare the scale with the paper before adding the sugar. Why must the scale be tared before adding the sugar?

2 Taring is the weighing of a measuring paper or container before it is filled with the material to be weighed so the final weight can be adjusted for the weight of the paper or container. On some scales, especially digital ones, the readout can be adjusted to zero before adding the material to be weighed. Thus, the scale is first calibrated with an empty platform at exactly 0.000 g. Next, the empty weighing container is placed on the scale and the readout is reset (tared) to zero. Then the scale will show only the exact weight of the item being weighed. With other scales and the hanging scale in the LabPaq, the weight of the empty weighing container is first measured and recorded. Then the material to be weighed is placed in the container and the total gross weight is measured and recorded. The net weight of just the material equals the gross weight less the weight of the container. (gross wt. – container wt. = sample wt.)

1. Cut a small square of white paper and place it on the scale.

2. Tare the scale by pressing the Φ/T button so that the scale reads 0.0 g.

3. Add sugar until the scale reads 3.0 g.

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Experiment Microscopy and the Metric System

4. Temperature: Temperature is measured in degrees Celsius which is abbreviated as °C, and also known as centigrade which means 100 graduations. The Celsius system is based on the properties of water.

Pure water freezes at 0°C and boils at 100°C under conditions of standard temperature and pressure (STP). STP equals 0°C, equivalent to 273 Kelvin3 or 32°F, and 760 mm Hg,4 or 1 atmosphere of pressure which is understood to be equivalent to sea level or 0 feet of altitude. STP must be specified for temperature when appropriate, because boiling point depression occurs with increasing altitude due to the drop in atmospheric partial pressure.

When converting from °F to °C, the adjustment must include a ratio shift of the Fahrenheit scale into the Celsius scale (32°F = 0°C and 212°F = 100°C). There are 9°F for every 5°C. We must also take into account that the Fahrenheit scale is 32°F at 0°C. The conversion from °F to °C is thus:

°C = (5°C/9°F) x (X°F – 32°F).

For example, to convert 47°F to the equivalent °C:

°C = (5°C/9°F) x (47°F – 32°F)

= (5°C/9°F) x 15°F

= (5°C x 15°F) ÷ 9°F

= 75°C ÷ 9 = 8.3°C

Practice converting the following with this conversion formula:

45°F = ______°C 62°F = ______°C 98.6°F = ______°C

Use a Celsius thermometer to measure the °C temperature of several different aliquots of cold and warm tap water. Make sure to allow the thermometer to remain until the temperature is stable and no longer changes. Record the temperatures:

________°C ________°C ________°C

3 Kelvin temperature scale is also used by scientists. 0 K is equal to -273.15°C, which physicists believe to be the lowest temperature possible in the universe. 4 Hg, is the chemical symbol for the element, mercury. Climatologists measure air pressure in mm of mercury. One atmosphere of air pressure at 0 feet elevation (sea level) is equivalent to a column of Hg 29.921 inches tall = 760 mm Hg.

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Questions A. What laboratory equipment would be used to measure the following items?

5 g flour 36 mL water The length of a frog’s leg 36 g water 38ºC Volume of a turtle* 125ºF Volume of blood Weight of a plant Weight of blood Temperature of a fish’s body Temperature of blood

This answer may require some creativity. How could it be done?

B. Provide the calculation steps, including the conversion factor that would be needed to convert the following measurements, and the final answers. Use U.S. and liquid units where appropriate.

248 g = _______ mg 145,000 μL = _______mL 536 mL = _______ cc 0.372 kg = _______ g 0.75 L = _______ μL 20.39 cm = _______ m

C. Provide the calculation steps, including the conversion factor that would be needed to convert the following measurements, and the final answers. Use U.S. and liquid units where appropriate.

3 cups = __________ L 7,893 mg = __________ lb 2.25 oz = __________ cc 36ºC = __________ ºF 145,000 uL = __________ tsp 96ºF = __________ ºC

D. What advantages does the metric system have over the English method of measurement? What are the disadvantages?

E. Outline the steps necessary to accurately weigh 3.5 g of starch.

F. Outline the steps necessary to accurately pipette 5 mL of distilled water.

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Exercise 2: Microscopy Search Key Terms: Microscope, stereomicroscope, dissecting microscope, scanning electron microscope, transmission electron microscope, compound light microscope, cheek cells.

Introduction: Antonie van Leeuwenhoek will forever be famous for perfecting the first working microscope. Until he began developing microscopes in 1670, previous microscopes in use since the 1590s were only capable of magnification up to 20x (20 times or 20 power). Leeuwenhoek was able to produce a simple microscope that magnified to over 200x (200 times or 200 power). He reported the existence of bacteria in 1683 when he viewed white material scraped from his teeth with his microscope. Viewing the micro-universe has proceeded from those first attempts.

The dissecting microscope, or stereoscope, efficiently magnifies objects in the range from 10x to 40x. It provides a view of organisms for purposes of closer, more detailed examination or dissection. Typical subjects might be chicken eggs, organ parts, or flower parts. Dissecting microscopes are used to magnify specimens of sizes 10 µm to 0.1 m; the equivalent in inches is 0.0004 to 3.9 inches.

If a dissecting microscope has two ocular lenses on separate body tubes, it is referred to as a binocular dissecting microscope, or a stereoscope. The subject is lighted from the side, not from the bottom.

The compound light microscope effectively magnifies in the range of 40x to 2000x. If an object under view is 10 nm in length without any magnification, what will be its viewing size at 40x? ______ at 2000x? ______. What is the equivalent size at these magnifications in inches? ______. Show your calculations.

Typical specimens used with compound light microscopes range in size from 200 nm to 5 mm; this can include large cell organelles, bacteria, and developing frog eggs. Light microscopes employ a light beam to view the specimen. In compound light microscopes, the light comes from below the specimen; thus, specimens must be small or thinly sliced to obtain a good image. We will look closer at this microscope below.

The scanning electron microscope (SEM) employs electron bombardment to image very small specimens. The electrons passing through a three-dimensional specimen are “read” using a detection device, and a computer reconstructs the specimen image from the information gathered by the scanning process. Scanning power can reach 200,000x, and provide great detail. The transmission electron microscope (TEM) allows internal investigations of prepared specimens and has a higher magnification range than the SEM. Typical subjects may be genetic material, large molecules, viruses, and cellular organelle detail. Electron microscopes are used to image specimens that range from 1 nm to 100 µm in size. What is the equivalent in inches? ______. Show your calculations.

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Compound light microscopes are so named because two sets of lenses are used during the viewing process. The ocular lens is nearest the eye, and the objective lens is nearest the specimen. Light, either from a focused mirror or electric source, is sent from below the specimen through the lenses to the eye. Light transmits through thin sections of the specimen, but is differentially transmitted through thicker portions of the specimen. Stains, dyes, and contrast adjustment can be used to optimize specimen viewing.

A microscope is well designed to serve its function, and should be treated with the care necessary to maintain it in working order. When moving a microscope, always support it with one hand under the base, and use the other hand to hold the microscope arm. Use lens paper to clean the lenses and light sources. The stage, arm, and base can be wiped with a soft, damp cloth. When not in use, the microscope should be stored in its case or covered to protect it from dust. Store it in a dry area away from temperature extremes and vibration.

procedure 1. Parts of the Compound Light Microscope: Refer to a microscope as this section is read. Label

the microscope diagram that follows as the examination of the microscope proceeds.

a. Eyepiece (Ocular Lens): The magnification power is stamped on the outside of the lens. What is the power of the ocular lens? ________. Microscopes may have interchangeable ocular lenses of different magnification.

b. Body Tube: Holds the ocular and objective lenses at the correct focal distance.

c. Arm: Used to transport microscope and hold the body tube.

d. Nosepiece: The revolving device that holds the objective lenses. May also be referred to as the turret.

e. Objective Lenses: Consists of one or more lenses:

i. The scanning power objective lens is the shortest of the lenses. What is its power? ______

ii. The low-power objective is slightly longer than the scanning objective. What is its power? ______

iii. The high-power objective is longer than the low-power objective. What is its power? ______

iv. The oil immersion objective is the longest lens. Its power is approximately 95x to 100x. Not all microscopes have an oil immersion objective.

f. Focus Adjustment Knobs: One or two knobs are used to focus the lens system.

i. The coarse-focus knob is used only with the scanning power objective to bring specimen into view. All microscopes have a coarse focus.

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ii. The fine-focus knob is used to adjust lenses to optimal viewing range. Used with all other objectives. (Optional)

g. Condenser: A lens system below the stage used to focus the light entering from beneath the stage. (Optional)

h. Diaphragm: Controls the amount of light entering from beneath the stage. (Optional)

i. Light source: A lamp or mirror used to direct light up toward the stage.

j. Stage & Stage Clips: The area below the objective lens that securely holds a slide in place for viewing.

k. Base: Supports the working parts of the microscope. Used for transporting microscope.

Label this microscope diagram with the appropriate part names and their functions:

a____________________________________

b____________________________________

c____________________________________

d___________________________________

e____________________________________

f____________________________________

g____________________________________

h____________________________________

i_____________________________________

Parts not included in microscope are: _______

_____________________________________

2. Focusing the Microscope:

a. Place the microscope on a stable surface. If the microscope has a mirror to focus the light source, use it near bright lighting for best viewing. But DO NOT USE direct sunlight as the light source or severe eye damage may result.

b. Rotate the lowest power (scanning) objective lens into line with the ocular lens.

c. Turn the coarse adjustment knob to raise the body tube to its highest position so that the objective lens is well above the stage.

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d. Mirror light source: While looking through the ocular lens, adjust the mirror for maximum light entry through the stage area. A bright light should be observed through the ocular lens when the mirror is in the correct position.

e. Electric light source: Plug the cord into an electrical outlet.

f. Attempt to keep both eyes open, even with a single ocular lens, to minimize the eyestrain caused by squinting.

g. Place a prepared slide on the stage. Try to locate the slide subject in the center of the viewing area. Secure the slide in place with the stage clips.

h. While watching from the side of the microscope and using the coarse focus knob, lower the scanning objective lens to its lowest point above the slide. Take care not to put pressure on the slide.

i. While looking through the ocular lens, slowly raise the scanning lens using the coarse focus knob until the subject material on the slide becomes clearly visible.

j. If the microscope has a fine adjustment knob, use it to fine-tune the view of the subject material. Since the field of vision decreases at higher viewing magnifications, ensure that the subject material is centered below the lens. Move the slide around on the stage until the material is within the field of view and centered on the stage.

k. If the microscope has either or both a diaphragm and/or condenser, adjust the settings of these items to optimize the view of the subject material on the slide. Low viewing light may be preferable, in some instances, to increase the contrast of the slide subject.

l. Compound light microscopes are parfocal, meaning that if the subject is in focus at low power, it should remain nearly in focus when the objective is switched to higher power.

m. Using the settings established for the scanning view above, move the low power (second shortest lens tube) objective into place. The slide subject should still be viewable. If the subject is not in focus, use the adjustment knob to slightly refocus the view, taking care not to crush the cover slip on the slide. Then, view the subject with the higher power objective lens (longest lens tube) using the same method. If the microscope has a coarse and fine adjustment knob, never use the coarse adjustment knob with higher power lenses.

n. If the microscope includes an oil immersion lens, place a drop of immersion oil on the slide cover slip before rotating the lens into place. The function of the oil is to minimize light diffraction through the slide and subject so that greater detail can be seen. After using the oil immersion lens, clean excess oil off of the lens and the slide with a lens cloth. Never tilt a microscope when using oil or if viewing a wet slide. Why?

o. When the observation is complete, the scanning lens should be rotated into place, and the body tube should be raised with the coarse focus knob to minimize the possibility of damaging the slide. The slide can then be removed safely.

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3. Operating the Microscope:

a. Obtain a clean slide and cover slip from the slide box. Place the slide and cover slip separately on a paper towel or other soft surface to reduce the possibility of scratching them.

b. With scissors, cut a letter “e” from an old magazine or newspaper.

c. Place the letter in the center of the slide.

d. Follow the instructions in Section 6 below to make a wet mount of the letter.

e. Following the directions outlined above under Handling and Focusing the Microscope, place the prepared slide on the microscope stage. Leave the scanning lens in place and focus so that the letter is clearly viewable. Make drawings of the letter in the boxes below as instructed.

Side of the slide furthest away from student

Look from the side of the microscope, view and then draw the letter, as it appears on the slide on the stage.

Draw the letter as it appears when viewing it through the microscope.

Side of the slide closest to student f. What is observed? Microscopes invert the image on the slide. This means that the subject

will appear to be 180° rotated and reversed from the actual image viewed on the slide.

g. While viewing the letter through the lenses, move the slide slightly. What do you observe about the movement of the letter and slide when viewed through the lenses?

h. Use the directions above to view the letter at the higher objective powers. On the drawing made above, circle the portion of the letter that is viewable as successively higher power observations are made. What is your conclusion about what happens when higher power objectives are used?

4. Total Magnification Calculation: Typically, the ocular lens of a microscope will be 10x, but it may be higher or lower. The power is recorded on the side of the lens.

a. Set up a data table similar to Data Table 1.

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Data Table 1: Calculating Magnification Ocular Lens Magnification x

Objective Lenses Magnification = Total Magnification

b. What is the ocular lens power of the microscope that you are using? It may be 10x or 15x. Record it in Table 1.

c. The objective lenses also have the magnification power recorded on their sides. What powers do the objective lenses on the microscope have? Record them in Table 1.Now, calculate the total magnification of the viewing area by multiplying the power of the ocular lens with that of the objective lens in use. For instance, if a microscope has a 10x magnification ocular lens and a 4x objective lens in place for viewing, the total magnification will be 40x (10x multiplied by 4x). What other view magnifications are possible with the microscope? Calculate the total magnification for each set of lenses in Table 1.

5. Diameter of Field:

a. Set up a data table similar to Data Table 2: Diameter of a Viewing Field.

Data Table 2: Diameter of a Viewing Field Magnification (ocular x objective lens’ powers)

mm diameter of field of view

μm diameter * of field of view

Scanning lens low Power lens High Power lens

With the low-power objective in viewing position, place a short transparent metric ruler on the stage.

b. While viewing the ruler through the lenses, measure the low-power diameter of field of view in mm. Convert this measurement to μm and record in Table 2.

c. Switch to the other higher power objectives, noting the diameter, in mm, for each in Table 2. Convert measurements to μm. How might this information be useful when viewing microscopic subjects?

6. Preparing a Wet-Mount Slide: A wet-mount technique is often used when mounting a specimen on a slide for viewing. This technique holds the specimen in place with a glass or plastic cover slip and minimizes light scattering through the slide. Read through this section and then practice wet mounting with water until an air bubble-free slide is achieved.

a. Place the specimen in the center of the slide with tweezers or, in the case of aquatic specimens, a dropper, and add a drop of water using a dropper.

b. Place an edge of the cover slip along the outer edge of the water drop on the surface of the slide.

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c. Slowly allow the cover slip to drop into place. This should remove the air and minimize the number and size of air bubbles trapped under the cover slip. Use a dissecting needle to control the fall of the cover slip.

d. If a stain is required to enhance the image on the slide, use the same technique, but use the stain instead of a water drop as the mounting liquid.

e. Wipe off the bottom of the slide before placing it on the stage.

7. Depth of Field: Prepare a wet-mount slide of three differently colored crossed threads using the wet mount technique described above. Place the slide on the microscope stage with the thread crossing area in the center of the viewing area. Focus carefully, moving the scanning objective lens up and down, taking care not to break the cover slip.

Depth Thread Color Top

Middle Bottom

Record the order of the threads in this table. Note that, when one thread is in focus, the others appear blurred. Why? When you focus on another thread, what happens to the thread that you were viewing?

Switch to high power and focus on one thread, then focus on another thread. What do you notice about the depth of field? Can you see as much of the thread in focus at the high power as you could at the low-power magnification?

8. View an animal cell:

a. Sanitize a clean toothpick with alcohol and use it to lightly scrape a small amount of epithelial cells from the inside of cheek.

b. Place the scraped material in the center of a clean slide.

c. Add one drop of IKI iodine indicator solution stain to the material on the slide. Use a toothpick to slightly swirl the drop, without spreading it, to separate the cells.

d. Cover with a cover slip.

e. Observe the prepared slide under the microscope, beginning with the scanning lens and then proceeding to higher magnification levels. Locate the nucleus in several cells. Locate the cytoplasm and the plasma membrane. On a sheet of paper, make a drawing of a few cells, and label the observed parts.

9. View a plant cell:

a. Using the dissection kit’s scalpel, remove a small piece of live skin membrane from the surface of an onion. The membrane is located just underneath the outer dead protective layers of onion skin and will appear to be nearly transparent, slightly moist, and very

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pliable. The membrane represents the epithelial cells common to plants.

b. Place the thin membrane layer flat on a clean slide. It may help to place a drop of the IKI iodine indicator solution stain on the slide first and then place the membrane piece in it. This will enable the membrane to lie out flat instead of clumping.

c. Place a small amount of the stain on the top surface and add a cover slip.

d. Observe the slide under the microscope, beginning with the scanning lens and then proceeding to higher magnification levels. Locate the cell wall and the nucleus in several cells. Make a drawing of a few cells and label the observed parts.

e. Count a column of cells, stacked end-to-end, across the field of vision under high-power magnification.

f. Based on the field of vision measurement you calculated above, compute the average length of one cell in the column of cells with this formula:

μm average length of cell = μm diameter of field of view ÷ total number of cells in the column.

g. What differences were noted between the animal cells and the plant cells? How do the differences dictate the form of the organism?

Now that you have completed this lab, make sure you read the lab for next week. This will help you plan your time better. Take some time and highlight anything you will need to prepare in advance. As you read the lab, write out a hypothesis for each exercise.

Questions A. What is the purpose of staining cells before viewing them under a microscope?

B. What type of microscope would you use to view the following organisms? There may be more than one correct response for each.

Strep Throat culture Mitochondria in an animal cell

Structure of a bird feather

Chloroplasts in a leaf cell

Earthworm digestive system

Enterococcus bacteria

DNA structure in the nucleus

Spores from a mushroom

Cells from plant leaf Herpes simplex

C. Summarize the capabilities of each of the microscopes listed in Table 3 below.

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Table 3: Summary of Microscope Capabilities

Type of Microscope

Type of Detection Beam

Magnification Range, metric

Examples of Viewable Specimens

Dissecting Compound Light Scanning electron Transmission electron

Exercise summary A. What have you learned from doing this exercise?

B. Why is the information presented in this exercise intrinsic to all future studies in biology?

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Microscopy and the Metric System Margaret E. Vorndam, M.S. Version 42-0090-00-01

Lab report Assistant This document is not meant to be a substitute for a formal laboratory report. The Lab Report Assistant is simply a summary of the experiment’s questions, diagrams if needed, and data tables that should be addressed in a formal lab report. The intent is to facilitate students’ writing of lab reports by providing this information in an editable file which can be sent to an instructor.

Observations

Data Table 1: Calculating Magnification Ocular Lens Magnification x

Objective Lenses Magnification = Total Magnification

Data Table 2: Diameter of a Viewing Field Magnification (ocular x objective lens’ powers)

mm diameter of field of view

μm diameter * of field of view

Scanning lens low Power lens High Power lens

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Exercise 1: Measuring Length, Weight, Volume, and Temperature Try the following conversions for practice.

240,000 ng = mg = g 50 cm = mm = m

procedure 1. Length: A metric ruler is useful for measuring items of length. The ruler below measures in

mm, indicated by the small mm near 0.

a. How many mm are there in 1 cm? , in a meter (m)?

(Ruler is not to scale. See ruler in dissection kit.)

b. Locate a measurable object to use for this exercise. If the object is long, obtain a yardstick that includes a cm scale; they can be found in local hardware stores.

c. Record the length of the object below and do the conversions:

Name of object _________________

Volume: Always pour an approximate volume of liquid into a clean beaker and then from the beaker into the volumetric flask or graduated cylinder. This will minimize contamination of the parent liquid source. Dispose properly of any leftover liquid. Do NOT pour it back into the original container. Why?

When using a pipet or dropper to measure liquid, pour an aliquot into a clean beaker and then draw up the liquid from the beaker into the pipet. NEVER try to draw up chemicals by mouth. Why?

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Weight: Use the pen scale from the lab kit to measure out exactly three grams of sugar. Make sure to tare the bag before adding the sugar. Why must the bag be tared before adding the sugar?

How is the weight of the bag accounted for when the sugar is weighed?

Temperature:

Practice converting the following with this conversion formula:

45°F = __°C 62°F = __°C 98.6°F = __°C

45°F = 62°F = 98.6°F =

Use a Celsius thermometer to measure the °C temperature of several different aliquots of cold and warm tap water. Make sure to allow the thermometer to remain until the temperature is stable and no longer changes. Record the temperatures:

________°C ________°C ________°C

Questions A. What laboratory equipment would be used to measure the following items?

5 g flour 36 mL water

The length of a frog’s leg 36 g water

38ºC Volume of a turtle*

125ºF Volume of blood

Weight of a plant Weight of blood

Temperature of a fish’s body Temperature of blood *This answer may require some creativity. How could it be done?

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B. Provide the calculation steps, including the conversion factor that would be needed to convert the following measurements, and the final answers. Use U.S. and liquid units where appropriate.

248 g

= _______ mg 145,000 μL

= _______mL

536 mL = _______ cc 0.372 kg = _______ g

0.75 L

= _______ μL 20.39 cm = _______ m

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C. Provide the calculation steps, including the conversion factor that would be needed to convert the following measurements, and the final answers. Use US and liquid units where appropriate.

3 cups = __________ L 7,893 mg = __________ lb

2.25 oz = __________ cc 36ºC = __________ ºF

145,000 uL = __________ tsp 96ºF = __________ ºC

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D. What advantages does the metric system have over the English method of measurement? What are the disadvantages?

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E. Outline the steps necessary to accurately weigh 3.5 g of starch.

F. Outline the steps necessary to accurately pipet 5 mL of distilled water. Pour an aliquot of distilled water into a clean beaker.

Exercise 2: Microscopy The compound light microscope effectively magnifies in the range of 40x to 2000x. If an object under view is 10 nm in length without any magnification, what will be its viewing size at 40x?

at 2000x?

What is the equivalent size at these magnifications, in inches? Show your calculations.

The scanning electron microscope (SEM) employs electron bombardment to image very small specimens. Electron microscopes are used to image specimens that range from 1 nm to 100 µm in size. What is the equivalent in inches? ______. Show your calculations.

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procedure 1. Parts of the Compound Light Microscope: Refer to a microscope as this section is read. Label

the microscope diagram that follows as the examination of the microscope proceeds.

a. Eyepiece (Ocular Lens): The magnification power is stamped on the outside of the lens. What is the power of the ocular lens? Microscopes may have interchangeable ocular lenses of different magnification.

b. Body Tube: Holds the ocular and objective lenses at the correct focal distance.

c. Arm: Used to transport microscope and hold the body tube.

d. Nosepiece: The revolving device that holds the objective lenses. May also be referred to as the turret.

e. Objective Lenses: Consists of one or more lenses:

i. The scanning power objective lens is the shortest of the lenses. What is its power?

ii. The low-power objective is slightly longer than the scanning objective. What is its power?

iii. The high-power objective is longer than the low-power objective. What is its power?

Label this microscope diagram with the appropriate part names and their functions:

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a

b

c

d

e

f

g

h

i

Parts not included in microscope are:

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2. Focusing the Microscope:

If the microscope includes an oil immersion lens, place a drop of immersion oil on the slide cover slip before rotating the lens into place. The function of the oil is to minimize light diffraction through the slide and subject so that greater detail can be seen. After using the oil immersion lens, clean excess oil off of the lens and the slide with a lens cloth. Never tilt a microscope when using oil or if viewing a wet slide. Why?

3. Operating the Microscope:

a. Obtain a clean slide and cover slip from the slide box. Place the slide and cover slip separately on a paper towel or other soft surface to reduce the possibility of scratching them.

b. With scissors, cut a letter “e” from an old magazine or newspaper.

c. Place the letter in the center of the slide.

d. Follow the instructions in Section 6 below to make a wet mount of the letter.

e. Following the directions outlined above under Handling and Focusing the Microscope, place the prepared slide on the microscope stage. Leave the scanning lens in place and focus so that the letter is clearly viewable. Make drawings of the letter in the boxes below as instructed.

Side of the slide furthest away from student

Look from the side of the microscope, view and then draw the letter here, as it appears on the slide on the stage.

Draw the letter here as it appears when viewing it through the microscope.

Side of the slide closest to student

f. What is observed? Microscopes invert the image on the slide. This means that the subject will appear to be 180° rotated and reversed from the actual image viewed on the slide.

g. While viewing the letter through the lenses, move the slide slightly. What do you observe about the movement of the letter and slide when viewed through the lenses?

h. Use the directions above to view the letter at the higher objective powers. On the drawing made above, circle the portion of the letter that is viewable as successively higher power observations are made. What is your conclusion about what happens when higher power objectives are used?

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4. Total Magnification Calculation: Typically, the ocular lens of a microscope will be 10x, but it may be higher or lower. The power is recorded on the side of the lens.

a. What is the ocular lens power of the microscope that you are using? It may be 10x or 15x. Record it in Table 1.

b. The objective lenses also have the magnification power recorded on their sides. What powers do the objective lenses on the microscope have? Record them in Table 1.

c. Now, calculate the total magnification of the viewing area by multiplying the power of the ocular lens with that of the objective lens in use. For instance, if a microscope has a 10x magnification ocular lens and a 4x objective lens in place for viewing, the total magnification will be 40x (10x multiplied by 4x). What other view magnifications are possible with the microscope? Calculate the total magnification for each set of lenses in Table 1.

Table 1: Calculating Magnification

Ocular Lens Magnification x Objective Lenses Magnification = Total Magnification

5. Diameter of Field:

a. With the low-power objective in viewing position, place a short transparent metric ruler on the stage.

b. While viewing the ruler through the lenses, measure the low-power diameter of field of view in mm. Convert this measurement to μm and record in Table 2.

c. Switch to the other higher power objectives, noting the diameter, in mm, for each in Table 2. Convert measurements to μm. How might this information be useful when viewing microscopic subjects?

Table 2: Diameter of a Viewing Field

Magnification (ocular x objective lens’

powers)

mm diameter of field of view

μm diameter * of field of view

Scanning lens low Power lens High Power lens

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6. Depth of Field: Prepare a wet mount slide of three differently colored crossed threads using the wet-mount technique described above. Place the slide on the microscope stage with the thread crossing area in the center of the viewing area. Focus carefully, moving the scanning objective lens up and down, taking care not to break the cover slip.

Record the order of the threads in this table. Note that, when one thread is in focus, the others appear blurred. Why? When you focus on another thread, what happens to the thread that you were viewing?

Depth Thread Color

Top Middle Bottom

Switch to high power and focus on one thread, then focus on another thread. What do you notice about the depth of field? Can you see as much of the thread in focus at the high power as you could at the low power magnification?

7. View an animal cell:

a. Observe the prepared slide under the microscope, beginning with the scanning lens and then proceeding to higher magnification levels. Locate the nucleus in several cells. Locate the cytoplasm and the plasma membrane. On a sheet of paper, make a drawing of a few cells, and label the observed parts.

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Experiment Microscopy and the Metric System

8. View a plant cell:

a. Observe the slide under the microscope, beginning with the scanning lens and then proceeding to higher magnification levels. Locate the cell wall and the nucleus in several cells. Make a drawing of a few cells and label the observed parts.

b. Count a column of cells, stacked end-to-end, across the field of vision under high-power magnification.

c. F. Based on the field of vision measurement you calculated above, compute the average length of one cell in the column of cells with this formula:

μm average length of cell = μm diameter of field of view ÷ total number of cells in the column.

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Experiment Microscopy and the Metric System

d. What differences were noted between the animal cells and the plant cells?

e. How do the differences dictate the form of the organism?

Discussion A. What is the purpose of staining cells before viewing them under a microscope?

B. What type of microscope would you use to view the following organisms? There may be more than one correct response for each.

Strep throat culture Mitochondria in an animal cell

Structure of a bird feather Chloroplasts in a leaf cell

Earthworm digestive system Enterococcus bacteria

DNA structure in the nucleus Spores from a mushroom

Cells from plant leaf Herpes simplex

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Experiment Microscopy and the Metric System

C. Summarize the capabilities of each of the microscopes listed in Table 2-3 below.

Table 3: Summary of Microscope Capabilities

Type of Microscope Type of Detection Beam Magnification Range, metric

Examples of Viewable Specimens

Dissecting

Compound light

Scanning electron

Transmission electron

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Laboratory summary 1. What have you learned from doing this laboratory?

2. Why is the information presented in this laboratory intrinsic to all future studies in biology?

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Basic Chemistry for Investigating Living Systems Margaret Vorndam, M.S. Version 42-0030-00-01

Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.

Experiment Summary:

Students will study the four major macromolecules and examine their function in living organisms. Students will use colorimetry and Biuret reagent to detect the presence of proteins, Iodine to detect starches, Benedict’s reagent to detect sugars, and Sudan III to detect the presence of lipids in cells.

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ExpErimEnt

Objectives ● To explain how colorimetry can be used to qualitatively detect cellular chemical components

● To chemically differentiate between proteins, sugars, starches, and lipids

● To identify the roles of molecular components in living systems

● To comprehend the value of using a systematic approach to research

● To describe why hypotheses, controls, standards, and quality control are important in scientific research

● To suggest scenarios where colorimetry can be applied to medical and industrial applications

Estimated Time Required to Complete the Experiment: 4 to 8 hours total.

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Experiment Basic Chemistry for Investigating Living Systems

materials MATERiAlS

FRoM lABEl oR BoX/BAg QTy iTEM DESCRiPTioN Student Provides 1 Cutting board 1 Distilled water 1 Stove or microwave oven 1 Masking tape 1 Oven mitts or potholders 1 Vegetable oil 1 Plastic wrap or aluminum foil 1 Sharp knife 1 Potato 1 Aluminum Foil 12 x 12cm 5 Small jars, rinsed with distilled water 1 Onion 1 Pan for water bath 1 Tap water 1 Egg or prepared egg white available from grocer From LabPaq 1 Aluminum Foil - 6"x 6" 2 Beaker, 50 mL, plastic 1 Cylinder, 50 mL - Graduated, Plastic

1

Dissection-kit with 7-tools - including the following: Bent Probe, Dropping Pipet, Probe, Ruler in pocket, Scalpel with 2 Blades - Note blades are in the pocket, Scissors, Tweezers

1 Filter Paper 12.5-cm 1 Goggles-Safety

1 IKI Indicator, 2.1% - 13 mL in Glass Vial in Bubble Bag 1 Mortar and pestle, ceramic

1 Pencil, marking 10 Pipet, Long Thin Stem 1 Pipet, Empty Short Stem 6 Test Tube, 13 x 100 mm in Bubble Bag

1 Starch Solution, 1% Stabilized - 60 mL in Dropper Bottle 1 Rack, Test Tube, 6 x 13 mm 1 Thermometer-in-cardboard-tube

1 a-Amylase Powder - 0.10 g in Dropper Bottle - Add 10 mL Distilled Water

1 Biuret Reagent - 4 mL in Pipet 1 Pipet, Graduated Jumbo (5 mL)

1 Benedict's Reagent - 15 mL in Dropper Bottle 1 Sudan III - 2 mL in Glass Vial 1 Glucose Solution, 20% 1 pH 7.0 Buffer (Yellow), 30 mL, in Dropper Bottle

Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq.

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Discussion and review Hints: For all exercises:

 Dedicate a long-stemmed (dropping) pipet or eye dropper for use with the same chemical in every instance of use to avoid the need to clean it. It is a good idea, however, to label it so that droppers are not inadvertently mixed between different chemicals. The pipet can be left in the solution when not in use to avoid contamination of the tip or stem.

 Place a piece of aluminum foil on the work surface before beginning to work. This will minimize the possibility of contaminating caps, dropping pipets, etc. when working with them.

Using laboratory analyses to test for the presence or absence of cellular components is important when assessing the state of human health. Blood test results for lipids, blood sugar, and many other components in blood help physicians assess our general health. In addition to human blood testing, chemical analyses of cells provide information about organisms and cell contents in many other applications.

All living material, whether it is mitochondria, cytoplasm, nuclear material, DNA, chloroplasts, or other type of cellular or intra-cellular component, consists of one of four major types of molecules:

1. starch and sugars (together referred to as carbohydrates) 2. proteins 3. fats (also referred to as lipids) 4. nucleic acids (including DNA and RNA)

Starches and sugars are carbohydrates because they share the same common molecular formula, CnH2nOn, where n is equal to the number of atoms of Carbon (chemical symbol is C), Hydrogen (H), and Oxygen (O). See the chart that follows.

In living systems, interconversion of carbohydrates and fats provide the energy storage and release equilibrium that allows cells, and thus the organism, to function. This interconversion is aided by enzymes which are proteins. When excess glucose, a carbohydrate, exists in the cellular environment, there are enzymatic mechanisms to shift the glucose to lipid form. When the glucose level in the cell is low, lipids are reconverted to glucose. If foodstuffs are consumed as lipids, (think of the grease on French fries) the enzymes in the liver and gall bladder aid in their breakdown to glucose.

Some lipids transfer across intestinal membranes into the bloodstream. HDL (high-density lipoproteins) can be moved through cell membranes and are also metabolized into glucose. LDL (low-density lipoproteins) are the bad lipids (think French fries again) and are implicated in diseases such as atherosclerosis as they tend to plate out onto the walls of blood vessels, causing vessel narrowing and ultimately stroke, heart attacks, and other cardiopulmonary diseases.

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Basic Cell Chemical Components

Cellular Chemical Component

Example Molecular Structure

High concentrations are found in...

Carbohydrates: Sugars

Sucrose

Honey, Beets, Sugar Cane, Milk, Fruits

 Carbohydrates:  Starches are combined units of sugars

Amylose

Wheat, Grain, Vegetables, Tubers

 Proteins

Leucine

Hair, Fingernails, Eggs, Enzymes, Muscle tissue (Meat)

 Lipids

Oleic Acid

Fat Tissue Vegetable Oils Ear Wax Cholesterols

One can think of the cycle of energy provided by food as follows:

Food (carbohydrates – proteins – lipids)

Ultimate breakdown and excretion as urea, CO2, and metabolic byproducts

digested by enzymes, absorbed into cells: glucose <> energy production or lipid storage through energy producing pathways such as the Krebs cycle and respiration

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So, chemical analyses are an important aspect of biology. This laboratory will introduce several basic analytical techniques used to determine the presence or absence of the four major cell molecules (starch, sugars, lipids, and proteins) through qualitative analyses.1 Qualitative analyses are used only to determine if a chemical is present. In quantitative analyses,2 a range of known chemical standards is used to also determine exactly how much of the chemical is present. In both types of analyses, a control sample is added that does not change or manipulate any of the test substance. The control sample is used to verify that everything is consistent from experiment to experiment and that any change in the test substance is causing the results observed.

Colorimetry is a type of analytical technique used to determine the presence or absence of a molecule such as lipids and sugars through qualitative analysis. It can also be used in quantitative determinations. The term colorimetry refers to the detection of a molecule by the color change observed in the prepared sample as the result of the presence of the molecule of interest. The molecule of interest is also referred to as the test substance. In colorimetry, a complexing reagent3 is added to the liquid sample and forms a color complex with the molecule for which it is specific. The presence of a molecule of interest is detected by a color change in the sample. There are a number of reagents, including those that we will use during this laboratory, which are specific to the detection of a given molecule. We will use the properties of reagent-molecule complexing to determine whether a molecule is present or absent.

In a medical laboratory, a spectrophotometer is one instrument that can be used to detect colorimetric complexes to determine how much test substance is present. A researcher can set the spectrophotometer to emit a specific wavelength, λ, of light that is optimally absorbed during the particular colorimetric reaction being conducted. The spectrophotometer emits the chosen wavelength and then detects how much of that light reaches a detector at the opposite end of the machine. A liquid sample is placed between the emission point and the detector, and the percent of the light that is adsorbed by the sample can then be measured. Standard solutions are used to create a standard curve at a number of known concentrations of the test substance to provide an absorption curve. Then, the absorption of the test substance can be compared to this standard curve to determine how much of the test substance is present in the sample of interest.

While a spectrophotometer will not be used in these exercises, it is important to understand how the colorimetric exercise can be extended in a laboratory situation to actually make measurement of the test substances. Here we will learn how to detect the qualitative presence of basic building- block molecules found in all living cells, proteins, carbohydrates (sugars and starches), and lipids, using basic colorimetric techniques.

1 Qualitative analysis is used to determine only whether a test substance is present or absent. 2 Quantitative analysis is used to determine whether a test substance is present or absent in a sample solution, and also how much is present. This analysis is typically done using standard solutions containing known amounts of the test substance of interest. The sample result is compared to the known standard results to determine the actual concentration in the sample. 3 Complexing reagents react with the test substance molecules. When one attaches to a molecule of the test substance, a color change occurs. Since the amount of color change in the sample solution is proportional to the amount of test substance present, a quantitative analysis using a spectrophotometer, plotted against known standard amounts, is possible. Spectrophotometers detect concentrations by measuring the absorbance of light at a pre-selected wavelength by the solution.

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A Simple Spectrophotometer

Light λ Emission Source @ 100%

Light Path →

Sample solution

absorbed 20% of light

Absorption Detector Detected @ 80%

Preparations Required Prior to Performing Lab Exercises

1% Albumen Solution Preparation, used in Exercises 1, 2, 3, 4: Place only the translucent (referred to as the “egg white”) part of an egg into a clean bowl. Avoid adding any white opaque-colored membrane material. Mix the egg white well with a fork to break up the strands of albumen, but do not beat it heavily. Using the graduated pipet, draw up 2 mL of the mixed egg white and place it into a small clean jar.

Drop 18 mL of the pH 7 buffer solution from the dropper bottle marked “pH 7” into the graduated cylinder. Pour the measured pH 7 buffer from the graduated cylinder into the jar containing the egg white. Stir well using the tip of the graduated pipet. Allow the precipitate to settle before drawing off the albumen solution from the top for use in the experiments. Cover the jar with a lid, plastic wrap, or aluminum foil until needed. Use the marker pencil to label the jar or a piece of masking tape placed on the jar as “1% albumen.” Replace the cap on the pH 7 buffer and reserve it for future labs. Clean the graduated cylinder and graduated pipet with distilled water for use in the next dilution. Calculation: Egg white contains ~10% albumen protein, so 2 mL egg white x 10% = 0.2 mL albumen. Thus, 0.2 mL ÷ 20 mL solution x 100 = 1% albumen solution.

Alpha-amylase Solution Preparation used in Exercises 1 and 2: Find the dropper bottle marked “alpha-amylase.” Carefully remove the bottle cap from the dropper bottle and then remove the dropping tip. This can be done by first placing a small clean piece of aluminum foil or plastic wrap over the tip. Then use your fingers or pliers to gently wiggle the tip upward to free it from the bottle. The foil or plastic will help prevent contaminating the tip with foreign substances. Place the removed tip on some foil or plastic until needed. Note the instructions on the dropper bottle and use the amount of water specified there for the following procedure.

Place an aliquot of distilled water into a clean beaker. Use the beaker to pour the exact mL of distilled water into the graduated cylinder. It helps if you use a long-stemmed pipet to control the water when the solution is close to the mL mark. Pour this aliquot of distilled water from the graduated cylinder into the dropper bottle containing pre-measured alpha-amylase powder.

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Carefully replace the dropping tip, still covered with foil or plastic. Remove the foil or plastic and replace the dropper bottle cap. Tighten it so that it pops the dropping tip into the bottle. Shake well and allow the amylase to dissolve before proceeding. Discard any distilled water remaining in the beaker.

Store the alpha-amylase dropper bottle solution in a refrigerator when not in use to reduce the rate of bacterial degradation. Calculation example for preparing a 1% solution: 0.10 g alpha-amylase powder is ~ equivalent to 0.10 mL when mixed in water, so 0.10 mL ÷ 10 mL x 100 = 1%.

0.4% Potato Starch Solution Preparation, used in Exercises 1, 2, 3, 4: Locate the dropper bottle containing the 1% potato starch solution. Shake well. Drip a small amount of the potato starch solution into a clean beaker. Use the graduated pipet to remove 5 mL of the solution from the beaker and place it in the clean graduated cylinder.

Pour distilled water into a clean beaker. Use the beaker to carefully add distilled water up to the 12.5-mL mark on the graduated cylinder containing the potato starch solution. It helps if you use a long-stemmed pipet to control the water when the solution is close to the 12.5-mL mark. Ensure that the bottom of the meniscus is right at 12.5 mL when the graduated cylinder is held at eye level. Place the solution from the graduated cylinder into a clean jar. Cover with a lid, aluminum foil, or plastic wrap until needed. Use the marker pencil to label the jar or a piece of masking tape placed on the jar as “0.4% potato starch.” Discard any distilled water that remains in the beaker.

Use the 0.4 % solution for experiments. Reserve the dropper bottle containing the 1% potato starch solution for future experiments. Store it in the refrigerator when it is not in use. Clean the graduated cylinder for future use.

Calculation: (1% x 5 mL potato starch solution) ÷ 12.5 mL water potato starch solution = 0.4% potato starch solution.

1% Glucose Solution Preparation, used in Exercise 3:

Retrieve the dropper bottle containing a 20% glucose solution. Calculation: 1g glucose ÷ 5 mL water = 0.20 g/mL x 100 = 20% glucose.

To make a 1% solution for use in this laboratory, dilute an aliquot of the 20% glucose solution in the dropper bottle, as follows:

 Drop a small amount of the 20% glucose solution into a clean beaker. Use a graduated pipet to transfer 1 mL of the glucose solution to a graduated cylinder.

 Pour a small amount of distilled water into a clean beaker. Use the beaker to add the distilled water to the graduated cylinder to the 20 mL mark. Use a long-stemmed pipet to add the water when close to the 20 mL mark to control accuracy. The meniscus should be right at

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20 mL when the graduated cylinder is held at eye level. Add the contents of the graduated cylinder to a clean jar. Cover the jar with plastic wrap or aluminum foil until needed. Use the marker pencil to label the jar or a piece of masking tape placed on the jar as “1% glucose.” Discard the distilled water that remains in the beaker.

Calculation: 20% glucose/mL water ÷ 20 mL water = 1% glucose/mL

Store the bottle in the refrigerator to limit biological growth or it may degrade rapidly due to airborne microbial introductions. Refrigeration will eventually slow this down.

Onion Juice Solution, used in Exercises 1, 2, 3, 4: This preparation may be conducted under a stove hood to minimize fumes. Using a clean cutting board, chop the onion into pieces. Further chop the onion pieces or use a mortar and pestle to macerate the onion until juice is visible. Collect the onion juice into a clean jar by drawing it up with a long-stemmed pipet and depositing it into the jar. Use the marker pencil to label the jar or a piece of masking tape placed on the jar as “onion juice.” Cover the jar with plastic wrap or aluminum foil until needed. Clean all utensils.

Potato Juice Solution, used in Exercises 1, 2, 3, 4: Using a clean cutting board, chop the potato into pieces. Further chop the potato pieces or use a mortar and pestle to macerate the potato until juice is visible. Collect the potato juice into a clean jar by drawing it up with a long-stemmed pipet and depositing it into the jar. Use the marker pencil to label the jar or a piece of masking tape placed on the jar as “potato juice.” Cover the jar with plastic wrap or aluminum foil until needed. Clean all utensils.

 Both of the above solutions can also be prepared in a blender or small food processor. You might need to add a small amount of distilled water to get this to work.

Water Bath: A hot water bath is required for some exercises. Steps for preparation of a water bath are outlined in the Appendix at the end of this manual.

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Exercise 1: Testing for the Presence of Proteins in Cells Search Key Terms: Protein, Biuret Reagent, enzyme, albumen, detection of protein.

Proteins are found in cell membranes, ribosomes, the endoplasmic reticulum, chromosomes, and in the cytoplasm of cells. Proteins function in cell-to-cell recognition, govern blood type, and control active transport into and out of a cell. Muscle fibers are high in protein, as are hair, nails and skin. Enzymes such as alpha-amylase, blood, antibodies, and hormones are made of proteins. Egg white is composed of water and the protein, albumen.

In this first exercise, we will use Biuret Reagent as a colorimetric indicator of the presence of protein in a number of samples. When protein molecules are present, Biuret Reagent reacts with the protein to form a violet color.

Protein + Biuret Reagent → Violet color

procedure 1. Record a hypothesis for each test tube sample treatment listed in Data Table 1 as to whether

it will or will not contain the test substance in this exercise which is protein.

Data Table 1: Biuret Reagent Test for ______________(Student to fill in)

Test Tube Contains Hypothesis: Contains Protein Yes or No?

Final Color is test substance present/absent?

1 Water 2 Albumen 3 Amylase

4 Potato Starch

5 Onion Juice 6 Potato Juice

2. Using the marker pencil, label six clean test tubes sequentially from #1 to #6. With the marker and a metric ruler, mark each of these test tubes at the 1-cm level from the bottom. Place the test tubes in the test tube rack.

3. Pour a small amount of distilled water into a clean beaker. Use this water in the following procedure and discard it when the steps are completed. Dedicate the glass dropper from the dissection kit for use with this beaker of distilled water.

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4. Treat the test tubes, as follows. See Photo 1 of color reactions at the lab’s end:

a. Test tube 1: Use the glass dropper and fill to 1-cm mark with distilled water from the beaker.

b. Test tube 2: Swirl or shake the albumen solution to mix it well; then use a long-stemmed pipet and fill to 1-cm mark with prepared 1% albumen solution.

c. Test tube 3: Use the dropper bottle containing alpha-amylase and fill to 1-cm mark with 1% alpha-amylase solution.

d. Test tube 4: Use a long-stemmed pipet and fill to 1-cm mark with 0.4% potato starch solution.

e. Test tube 5: Use a long-stemmed pipet and add a few drops of onion juice to the test tube. Fill to the 1-cm mark with distilled water.

f. Test tube 6: Use a long-stemmed pipet and add a few drops of potato juice to the test tube. Fill to the 1-cm mark with distilled water.

5. Next, cut off the tip of the end of pipet of Biuret Reagent. Add 5 drops of Biuret Reagent to each of the test tubes.

6. Mix contents of each test tube by holding the top of the test tube securely with one hand and flicking the test tube bottom with the fingers of your other hand several times.

7. Record the color of each test tube’s contents and results in Data Table 1 and answer the questions. When you have finished with this section, clean and dry test tubes for the next test.

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Questions A. What is the test substance?

B. Which test tube represents the control? Why?

C. Which test tube contained the most test substance?

D. Other than the control, which test tube contained the least test substance?

E. Did the results agree with your initial hypothesis in every case? Why or why not?

F. What are your conclusions about your results?

G. If the color change is not as you expected, what might be the reasons?

H. Add another 5 drops of Biuret Reagent to each test tube and stir as before. Do your results change?

Discussion

A. What is the purpose of this exercise?

B. Why is it important to clean droppers and equipment between chemical uses?

C. What other types of foods or substances contain high levels of protein?

D. Suggest a situation where you might use the Biuret Reagent colorimetric test.

E. What other types of analytical procedures detect the presence of proteins?

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Exercise 2: Testing for the Presence of Starch in Cells Search Key Terms: Carbohydrate, iodine detection of starch, starch.

Carbohydrates provide the fuel that drives cellular metabolism. Sugars and starches are both examples of carbohydrates. Sugars and starches share the same basic chemical formula, (CH2O) where the n indicates how many groups of the formula are joined together to form a chain of molecules. Sugar chains are short (saccharides) while starch chains are complex (polysaccharides). Separate colorimetric procedures can be used to detect the presence of either sugars or starches. We will begin with the starch reaction.

Starch + Iodine Indicator Solution à Blue-black Color

procedure 1. Record a hypothesis for each test tube sample treatment listed in Data Table 2 as to whether

it will or will not contain the test substance in this exercise which is starch.

Data Table 2: Iodine Solution Test for ______________(Student to fill in)

Test Tube Contains Hypothesis Contains Starch Yes or No?

Final Color is test substance present/absent?

1 Water 2 Albumen 3 Amylase 4 Potato Starch 5 Onion Juice 6 Potato Juice

2. Using the marker pencil, label six clean test tubes sequentially from #1 to #6. With the marker and a metric ruler, mark each of these test tubes at the 1-cm levels from the bottom. Place the test tubes in the test tube rack.

3. Pour a small amount of distilled water into a clean beaker. Dedicate the glass dropper to use with this distilled water in the following procedure. Discard the water when the steps are completed.

4. Treat test tubes, as follows and see Photo 2 of color reactions at the lab’s end.

a. Test tube 1: Use the glass dropper and fill to the 1-cm mark with distilled water.

b. Test tube 2: Swirl or shake the albumen solution to mix it well; then using a long-stemmed pipet, fill to the 1-cm mark with prepared 1% albumen solution.

c. Test tube 3: Using the dropper bottle containing alpha-amylase, fill to the 1-cm mark with 1% alpha-amylase solution.

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d. Test tube 4: Using a long-stemmed pipet, fill to the 1-cm mark with 0.4% potato starch solution.

e. Test tube 5: Using a long-stemmed pipet, add a few drops of onion juice to the test tube. Fill to the 1-cm mark with distilled water.

f. Test tube 6: Using a long-stemmed pipet, add a few drops of potato juice to the test tube. Fill to the 1-cm mark with distilled water.

5. Label one long-stemmed pipet with “iodine” to be dedicated for this use. Iodine stains, and the pipet will become stained after a few uses.

6. Add two drops of IKI iodine indicator solution to each test tube.

7. Mix contents of each test tube by holding the top of the test tube securely with one hand, and flicking the test tube bottom with the fingers of your other hand several times.

8. Record the color of each test tube’s contents and results in Table 2 then answer the questions. When you have finished with this section, clean and dry test tubes for next test.

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Questions A. What is the test substance?

B. Which test tube represents the control? Why?

C. Which test tube contained the most test substance?

D. Other than the control, which test tube contained the least test substance?

E. Did the results agree with your initial hypothesis in each case, why or why not?

F. What are your conclusions about the results?

G. If the color change is not as you expected it to be, what might be the reasons?

Discussion

A. What is the purpose of this exercise?

B. What other types of foods or substances contain high levels of starch?

C. Suggest a situation where you might use the iodine colorimetric test.

D. What other types of analytical procedures detect the presence of starch?

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Exercise 3: Testing for the Presence of Sugar in Cells Search Key Terms: Benedict’s reagent, sugar, monosaccharide, disaccharide.

Sugars and starches are both carbohydrate molecules. In this exercise, we will test for the presence of sugar. Recall that sugars and starches have the same chemical formula, but differ in the length of the chains that are joined together. Benedict’s reagent (copper sulfate) is used to detect the presence of reducing sugars. The reaction is:

Copper sulfate CuSO4 à Cu ++ + SO4

--

2Cu++ + reducing sugar (electron donor) à Cu+ Cu+ à Cu2O (red-brown precipitate)

Heat is applied to the sample to cause the reaction to occur more quickly. The color of the final reaction is dependent on the amount of reducing sugar present.

low à increasing amounts of reducing sugar à High green orange Red Brown

Not all sugars are reducing sugars, so this test is not a universal sugar-detector. However, it does work with all monosaccharides4 and some disaccharides5 such as glucose, fructose, lactose, and mannose, but not sucrose which is found in table sugar. The following exercise will compare the relative amount of the test substance in several samples.

procedure 1. Record a hypothesis for each test tube sample treatment listed in Data Table 3 as to whether

it will or will not contain the test substance in this exercise which is sugar.

Data Table 3: Benedict’s Reagent Test for ______________(Student to fill in)

Test Tube Contains Hypothesis Contains Sugar Yes or No?

Final Color is test substance present/absent?

1 Water 2 Glucose 3 Albumen

4 Potato Starch

5 Onion 6 Potato Juice

4 Monosaccharide sugars are not bonded to other sugar groups. The sugar is in its most elementary form. 5 Disaccharide sugars are two bonded monosaccharide sugar groups.

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2. Prepare a simmering water bath per the instructions in the Appendix.

3. Using the marker pencil, label six clean test tubes sequentially from #1 through #6. With the marker and a metric ruler, mark each of these test tubes at the 1/2 and 2-cm levels from the bottom. Place the test tubes in the test tube rack.

4. Treat test tubes, as follows and see Photo 3 of color reactions at the lab’s end:

a. Test tube 1: Fill to the ½ cm mark with distilled water using a dropper.

b. Test tube 2: Fill to the ½ cm mark with glucose solution using a long-stemmed pipet.

c. Test tube 3: Swirl or shake the albumen solution to mix it well; then, fill to the ½ cm mark with albumen solution using a long-stemmed pipet.

d. Test tube 4: Fill to the ½ cm mark with 0.4% potato starch solution using a long-stemmed pipet.

e. Test tube 5: Add a few drops of onion juice using long-stemmed pipet. Fill to the 1/2-cm mark with distilled water.

f. Test tube 6: Add a few drops of potato juice using a long-stemmed pipet. Fill to the 1/2-cm mark with distilled water.

5. Add Benedict’s reagent each to test tube to the 2-cm mark.

6. Place the test tubes in the test tube rack into the water bath and heat for 5 to 10 minutes.

7. Remove the rack containing test tubes from bath to a heat-proof surface.

8. Record the color of each test tube’s contents (refer to Photo 3 at the end of this lab) and results in Data Table 3 and answer questions. Then, clean and dry test tubes for next test.

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Questions A. What is the test substance?

B. Which test tube represents the control? Why?

C. Which test tube contained the most test substance?

D. Besides the control, which test tube contained the least test substance?

E. Did the results agree with your initial hypothesis in every case? Why or why not?

F. What are you conclusions about the results?

G. If the color change is not as expected, what might be the reasons?

Discussion

A. What is the purpose of this exercise?

B. Suggest a scenario where you might use the Benedict’s reagent colorimetric test.

C. How might one determine whether the potato or onion contains more sugar?

D. What other types of foods or substances contain high levels of sugar?

E. What other types of analytical procedures detect the presence of sugars?

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Exercise 4: Testing for the Presence of Lipids in Cells Search Key Terms: Lipid, steroid, vegetable oil, Sudan III.

Lipids store energy in cells and are components of cell membranes. Lipids are found in all cells, but especially in fat cells that compose adipose tissue. The storage of hormones is among one of the lipids’ roles. Steroids, waxes, and oils are also lipids. Lipids are not water-soluble; this is important to the success of this particular colorimetric analysis.

procedure In this procedure, Sudan III will be used to test for the presence of lipids. Sudan iii is a fat-soluble dye used for staining lipids such as triglycerides and lipoproteins.

1. Record a hypothesis for each sample treatment listed in Data Table 4 as to whether it will or will not contain the test substance in this exercise which is lipids.

Data Table 4: Lipid Test Results

Macromolecule Being Tested Hypothesis: Contains lipids, or not? Results from Test

Potato Starch onion Juice Vegetable oil Distilled Water Albumen Potato Juice

2. Place the filter paper from the LabPaq onto the piece of aluminum foil to prevent staining of the work surface. Use the pipet to place 6 separate drops of Sudan III onto different areas of the filter paper. Allow drops to dry. See figure below.

nOtE: The figure below shows only four areas, but this particular lab will have six areas which contain drops of Sudan III.

3. Use a pencil to trace an outline around each of the dried Sudan III drops.

4. Use the solutions prepared in earlier exercises to use for this lab. Use a dedicated pipet for each solution so there is no contamination between solutions.

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Placement of Sudan III on Filter Paper

5. Place one drop of potato juice, one drop of potato starch, one drop of albumen, one drop of distilled water, one drop of onion, and one drop of vegetable oil on separate drops of Sudan III.

6. Allow the drops to dry and then read the results.

7. Record the results in the table. Look for if the Sudan III stain stayed inside the drawn circle drawn with the pencil. If the stain dissolved in the substance and was carried outside the line, it indicates that the substance contains lipids.

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Color Reaction Photos Referenced in Laboratory

Photo 1: Color reactions of Biuret Reagent as an indicator of protein. Left to right: distilled water control (no protein) is nearly colorless, lavender/purple indicates high concentration

of protein, amber/purple indicates lesser concentrations of protein.

Photo 2: Color reactions of Iodine as anindicator of starch. Left to right: distilled

wter control (no starch) is amber, dark brown indicates low concentration of starch, black/ blue indicates high concentration of starch.

Photo 3: Color reactions of Benedict’s Reagent as an indicator

of simple sugars. Left to right: distilled water control (no sugar) is translucent blue, green indicates

low concentration of sugar, orange/ red indicates high concentration of

sugar.

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Experiment Basic Chemistry for Investigating Living Systems

Questions A. What is the test substance?

B. Which test tube represents the control?

C. Which test tube contained the most test substance?

D. Other than the control, which test tube contained the least test substance?

E. Did the results agree with your initial hypothesis in every case? Explain why or not.

F. If the color change is not as you expected it to be, what might be the reason(s)?

Discussion

A. What is the purpose of this exercise?

B. Explain the molecular basis as to why Sudan III can be used to detect the presence of lipids, but not sugar or proteins. Why can Biuret, Benedict's, and Iodine Reagents detect the presence of proteins, starches, and sugars, but not lipids?

C. What other types of foods or substances contain high levels of lipids?

D. What other types of analytical procedures detect the presence of lipids?

E. Fill in the summary Table 5 in the Lab Report Assistant section by noting which complexing reagents (dyes) can be used to determine which cellular components. Use + to indicate that the dye will detect the component, and – to indicate that it will not detect the component.

laboratory Summary

What have you learned from doing this laboratory?

Basic Chemistry for Investigating Living Systems Margaret Vorndam, M.S. Version 42-0030-00-01

Lab report Assistant This document is not meant to be a substitute for a formal laboratory report. The Lab Report Assistant is simply a summary of the experiment’s questions, diagrams if needed, and data tables that should be addressed in a formal lab report. The intent is to facilitate students’ writing of lab reports by providing this information in an editable file which can be sent to an instructor.

Observations Data Table 1: Biuret Reagent Test for ______________(Student to fill in)

Test Tube Contains Hypothesis: Contains Protein Yes or No?

Final Color is test substance present/absent?

1 Water

2 Albumen

3 Amylase

4 Potato Starch

5 Onion Juice 6 Potato Juice

Data Table 2: Iodine Solution Test for ______________(Student to fill in)

Test Tube Contains Hypothesis Contains Starch Yes or No?

Final Color is test substance present/absent?

1 Water 2 Albumen 3 Amylase

4 Potato Starch

5 Onion Juice 6 Potato Juice

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Data Table 3: Benedict’s Reagent Test for ______________(Student to fill in)

Test Tube Contains Hypothesis Contains Sugar Yes or No?

Final Color is test substance present/absent?

1 Water

2 Glucose

3 Albumen

4 Potato Starch

5 Onion 6 Potato Juice

Data Table 4: lipid Test Results

Macromolecule Being Tested Hypothesis: Contains lipids, or not? Results from Test

Potato Starch onion Juice Vegetable oil Distilled Water Albumen Potato Juice

Data Table 5: Testing the Chemical Composition of Cells Indicate a + mark if the reagent reacts with test substance. Indicate a - mark if the reagent does NOT react with test substance. Test Substance Biuret Solution iodine Stain Benedict’s Reagent Sudan iii Protein (Albumen)

Sugar Starch Lipid

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Experiment Basic Chemistry for Investigating Living Systems

Exercise 1: Testing for the Presence of Proteins in Cells Questions A. What is the test substance?

B. Which test tube represents the control? Why?

C. Which test tube contained the most test substance?

D. Other than the control, which test tube contained the least test substance?

E. Did the results agree with your initial hypothesis in every case? Why or why not?

F. What are your conclusions about your results?

G. If the color change is not as you expected, what might be the reasons?

H. Add another 5 drops of Biuret Reagent to each test tube and stir as before. Do your results change?

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Discussion

A. What is the purpose of this exercise?

B. Why is it important to clean droppers and equipment between chemical uses?

C. What other types of foods or substances contain high levels of protein?

D. Suggest a situation where you might use the Biuret Reagent colorimetric test.

E. What other types of analytical procedures detect the presence of proteins?

Exercise 2: Testing for the Presence of Starch in Cells Questions A. What is the test substance?

B. Which test tube represents the control? Why?

C. Which test tube contained the most test substance?

D. Other than the control, which test tube contained the least test substance?

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Experiment Basic Chemistry for Investigating Living Systems

E. Did the results agree with your initial hypothesis in each case, why or why not?

F. What are your conclusions about the results?

G. If the color change is not as you expected it to be, what might be the reasons?

Discussion

A. What is the purpose of this exercise?

B. What other types of foods or substances contain high levels of starch?

C. Suggest a situation where you might use the iodine colorimetric test.

D. What other types of analytical procedures detect the presence of starch?

Exercise 3: Testing for the Presence of Sugar in Cells Questions A. What is the test substance?

B. Which test tube represents the control? Why?

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C. Which test tube contained the most test substance?

D. Besides the control, which test tube contained the least test substance?

E. Did the results agree with your initial hypothesis in every case? Why or why not?

F. What are you conclusions about the results?

G. If the color change is not as expected, what might be the reasons?

Discussion

A. What is the purpose of this exercise?

B. Suggest a scenario where you might use the Benedict’s reagent colorimetric test.

C. How might one determine whether the potato or onion contains more sugar?

D. What other types of foods or substances contain high levels of sugar?

E. What other types of analytical procedures detect the presence of sugars?

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Exercise 4: Testing for the Presence of Lipids in Cells Questions A. What is the test substance?

B. Which test tube represents the control?

C. Which test tube contained the most test substance?

D. Other than the control, which test tube contained the least test substance?

E. Did the results agree with your initial hypothesis in every case? Explain why or not.

F. If the color change is not as you expected it to be, what might be the reason(s)?

Discussion

A. What is the purpose of this exercise?

B. Explain the molecular basis as to why Sudan III can be used to detect the presence of lipids, but not sugar or proteins. Why can Biuret, Benedict's, and Iodine Reagents detect the presence of proteins, starches, and sugars, but not lipids?

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C. What other types of foods or substances contain high levels of lipids?

D. What other types of analytical procedures detect the presence of lipids?

E. Fill in the summary Table 5 in the Lab Report Assistant section by noting which complexing reagents (dyes) can be used to determine which cellular components. Use + to indicate that the dye will detect the component, and – to indicate that it will not detect the component.

laboratory Summary

What have you learned from doing this laboratory?

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Experiment Basic Chemistry for Investigating Living Systems

Cell Structure and Function: Cell Types and Transport Margaret Vorndam, M.S. Version 42-0037-00-01

Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.

Experiment Summary:

Students will explore the differences between prokaryotic and eukaryotic cells. They will view prepared slides of Anabaena and Amoeba to look at cell structure and relate these structures to function. Students will study the differences between plant and animal cells and observe Elodea microscopically. They will test diffusion through both solids and liquids to compare rates. Students will compare rates of transport through a differentially permeable membrane using dialysis tubing and solutions of starch, glucose, and Iodine. They will analyze the results with Benedict’s reagent.

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ExpErimEnt

Objectives ● To explain the differences between prokaryotic and eukaryotic cells

● To know whether an organism is prokaryotic or eukaryotic upon observation

● To explain the differences between plant and animal cells, and relate the differences to structure and function

● To explain how metabolic raw materials are moved into cells and how waste products are moved out

Estimated Time Required to Complete the Experiment: 4 to 8 hours total.

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Experiment Cell Structure and Function: Cell Types and Transport

materials

MATERIALS FROM: lABEl oR BoX/BAG: QTy ITEM DESCRIPTION:

Student Provides 1 Distilled water 1 Tap water 1 Glass jar, 2-cup size minimum 1 Measuring cup 1 Oven mitts or potholders 2 Rubber bands 1 Stove or microwave oven 1 Food coloring 1 Spoon 1 Clear bowl, 4-cup size

1 Microscope 1 Watch with minute hand 1 Refrigerator

1

Elodea (Anacharis), or other thin leaf water plant, purchased from an aquarium shop and kept in clean water. Supply of Elodea and other aquatic plants in aquarium shops is unpredictable. It may be purchased from an online aquatic plant shop, but shipping costs extra and can take up to a week by UPS ground.

1 Clear storage container with flat bottom and lid, round or square, ~ 6” diameter

1 Powdered gelatin, 1/4 oz such as unflavored Knox®, 1 envelope or 2½ teaspoons

From LabPaq 1 Cylinder, 50 mL - Graduated, Plastic

1

Dissection-kit with 7-tools - including the following: Bent Probe, Dropping Pipette, Probe, Ruler in pocket, Scalpel with 2 Blades - Note blades are in the pocket, Scissors, Tweezers

1 Goggles-Safety

1 IKI Indicator, 2.1% - 13 mL in Glass Vial in Bubble Bag

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Experiment Cell Structure and Function: Cell Types and Transport

1 Pencil, marking 1 Thermometer-in-cardboard-tube

10 Pipette, Long Thin Stem 1 Rod, Stirring rod - Glass

1 Slide, blank (10 pcs) 1 Slide, Cover Slip Cube (20 pcs)

1 Starch Solution, 1% Stabilized - 60 mL in Dropper Bottle 1 Test tube cleaning brush

1 Test Tube(1), 25 x 150 mm in Bubble Bag

1 Benedict's Reagent - 8 mL in Dropper Bottle 1 Glucose Solution, 20%

1 Dialysis Tubing 6 inches

Slide box 1 Slide - Amoeba-Whole Mount 1 Slide - Anabaena

Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq.

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Experiment Cell Structure and Function: Cell Types and Transport

Discussion and review Preparations Required Prior to Performing Lab Exercises

Exercise 3 requires the use of a semi-solid gelatin plate which must be made several hours prior to conducting that exercise. To prepare the plate, pour ½ cup of cold tap water into a 4-cup bowl, sprinkle the gelatin over the surface, and allow it to stand for 10 minutes. Stir and then add 1½ cups of boiling tap water and stir again until the mixture is clear. Do not add sugar. Pour the gelatin into the clear storage container to a depth of about one centimeter. Cover with a lid and refrigerate until ready for use. Bring gelatin to room temperature about half an hour before beginning Exercise 3. See the instructions for preparing a hot water bath in the Appendix for use in Exercise 4.

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Experiment Cell Structure and Function: Cell Types and Transport

Exercise 1: Prokaryotes and Eukaryotes –Differences in Cell Structure and Function

Search Key Terms: Prokaryote, eukaryote, Amoeba proteus, Anabaena, Eukarya, Cyanobacteria, blue-green algae, biological cell types.

Chemistry and chemical processes govern the functions of biological systems. Simple atoms, such as carbon (C), hydrogen (H), and oxygen (O), bond to form organic molecules such as proteins, lipids, and carbohydrates (sugars and starches). These molecular components in turn combine to form the organelles and other structures that make up the living cell. Some cells live singly in the environment, as do bacteria, cyanobacteria, blue-green algae, and some protists, while other cells are organized into colonies. Specialization of cell organization causes formations of tissues, organs, and systems in more complex organisms. Cellular organic macromolecules may also be bonded to iron (Fe), phosphorus (P) and other atoms that allow cells to transport oxygen in blood, carry information to the brain, and form bone structure.

Let us explore this wondrous world of biological beings by beginning with the simple cells of the prokarya first. Divisions Bacteria (previously called Monera) and Archaea contain organisms that are referred to as prokaryotic. In Greek, pro = before, and karyon = nucleus. Prokaryotes are structurally simple organisms, and they are represented among the oldest fossils found on Earth. Despite their simple structure, prokaryotes are capable of living and metabolizing to produce their own energy in the most hostile of environments. They can be anaerobic (metabolizing in the absence of oxygen) or aerobic (metabolizing in the presence of oxygen), or both (facultative anaerobes). Some types of prokaryotes contain photosynthesizing pigments and can produce oxygen, such as the blue-green algae Anabaena. In fact, evolutionary thinking attributes the first forms of life to this cell category which is believed to have produced the first oxygen in the young Earth atmosphere and enabled the evolution of other life forms that use oxygen.

Typical Prokaryote Cell Typical Eukaryote Cell

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Experiment Cell Structure and Function: Cell Types and Transport

Division Eukarya - from Greek, eu = new, and karyon = nucleus - includes all other life forms. Eukaryotes are found in the Kingdoms Protista, Fungi, Animalia, and Plantae. Evolutionists think that eukaryotes developed from prokaryotic cells after conditions became favorable for their existence.

This line of thinking is reinforced by the parallel function of the similar organs between these two cell types. The prokaryotes have a protective capsule or slime coat, a cell wall, and a plasma membrane that protect them from the external environment. In eukaryotes this protection is conveyed by the cell membrane. Prokaryotes share similarities with eukaryotes in possessing cytoplasm and ribosomes, but the nucleoid is a precursor to the nucleus of the eukaryotic cell. It is not as well defined a structure as is a true nucleus. Prokaryotes and some simple eukaryotes are capable of forming endospores to protect themselves from hostile environments. The endospore releases the organism when conditions are again favorable for survival. We will begin this experiment by focusing on the similarities and differences between these two cell types.

procedure 1. Before beginning, set up a data table similar to Data Table 1: Cell Structure, in the Lab Report

Assistant section.

2. Observe the Anabaena and Amoeba prepared slides under the low power setting of the microscope. Then increase the power by switching objective lens until cell contents are visible.

3. Draw a representative cell of each organism into the Lab Report Assistant section.

4. On your drawings, label the cell structures from Data Table 1: Cell Structure for each organism you view. Not every structure listed may be visible. Check those structures that are locatable on the slide.

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Exercise 2: Eukaryotes – Plant and Animal Cell Structure and Function Search Key Terms: Plant cell, animal cell, elodea (Anacharis), Amoeba proteus, eukaryotic.

Within the Eukaryotes of the Kingdom Animalia and Plantae, we find that cells also differ, but not as extremely as do eukaryote and prokaryote cells. They have a structured nucleus that is generally confined inside of the nuclear membrane, except during nuclear division.

Plant and animal cells are surrounded by a cell membrane and share organelles such as mitochondria, ribosomes, cytoplasm, vacuoles, and other structures not as readily visible under a compound light microscope.

However, differences are striking. For instance, the plants are generally rigid and not pliable when touched like animals. Plant cells are surrounded by cell walls that hold the cell and provide a structure for the plant. Animal cells lack cell walls. The addition of a wall also effects how plants and animals reproduce and grow. Plants also contain chlorophyll-filled chloroplasts which enable them to photosynthesize. Only a very few single-celled animals in the Kingdom Protista have this ability.

In this exercise, Ameoba proteus will be used to show the features of an animal cell. While the Ameoba is a member of Kingdom Protista, it is serving only as an example of an animal-like eukaryotic cell. Now, we will observe some of the more obvious differences between plant and animal-like cells.

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Experiment Cell Structure and Function: Cell Types and Transport

procedure 1. Prepare a wet mount of a young leaf from an elodea plant (see picture). Young leaves will be

found at the tip of the plant’s stem. Do not allow the leaf to dry out. Place a drop of water on the slide and lay the leaf on the slide with the top of the leaf facing up.

Elodea, Anacharis sp.

2. Place a cover slip over the leaf and drop the slip at an angle onto the leaf and water to avoid trapping air bubbles under the cover slip.

3. Observe the leaf on the slide by lowering the low power objective lens to its lowest position. Then focus on the leaf by raising the low power lens until the leaf surface is in focus. Slowly move the slide until the side edge or end walls of the leaf surface can be seen. Adjust light for a clear view.

4. Switch to high power and examine a few cells. Observe the chloroplasts around the inner walls of the cells. The central vacuole - a membrane-bound, fluid-filled sac in the center of the cell - appears to force the chloroplasts to the cell wall. However, if the microscope focus is adjusted, the even distribution of the chloroplasts on the surface of the cell should also be apparent. Are the chloroplasts moving? This motion is due to the movement of the invisible cytoplasm within the cell. This movement is called cytoplasmic streaming.

5. Find the cell nucleus. If it is visible, it will appear as a very light gray lump in the cell.

6. Make a drawing of one or two cells in the Lab Report Assistant section and label the structures that are visible. These structures may include the cell wall, the plasma membrane, chloroplasts, the central vacuole, the nucleus, mitochondria, and ribosomes.

7. Now, again observe the prepared slide of the amoeba under the low power setting of the microscope and then increase the power until cell contents are visible.

8. Compare the drawing of the amoeba cell from Exercise 1 to the drawing of the plant cell.

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Experiment Cell Structure and Function: Cell Types and Transport

Exercise 3: Transport of Materials through the Cell Membrane – An Example of Passive Transport Search Key Terms: Biological cell membrane, cell membrane diffusion, diffusion, cell membrane passive transport.

Cells must exchange gases, liquids, and solids with the external environment in order to survive. Cells must have a source of materials that provide fuel to drive cell function. All cells are surrounded by a plasma membrane that protects the cell from the outside environment and that must also selectively pass material through to the cell interior for cell processes to occur. The property of the cell membrane that allows some environmental constituents through, while preventing the passage of others, is called selective permeability.

The process by which molecules move from an area of high concentration to mix into an area of low molecular concentration is called diffusion. This process is dependent on temperature since molecules move or vibrate faster when they are warmer. Diffusion also varies with molecular packing in the cell membrane and cytoplasm. In this exercise we will investigate the effect of two medium densities and temperature, on diffusion. This particular exercise is an example of passive transport since no mediation by the cell membrane occurs.

procedure Part I: Semi-solid

1. Before beginning, set up a data table similar to Data Table 2: Diffusion Rate Comparison in the Lab Report Assistant section.

2. Read through the exercise, then propose and record a hypothesis about what you believe will occur.

3. Remove the container of gelatin previously prepared from the refrigerator and allow it to come to room temperature. This takes about 30 minutes.

4. Center the container of gelatin on a flat metric ruler. Choose a measuring point on the ruler that is located just below the approximate center of the container.

5. Squeeze one drop of food coloring onto the gelatin in container above the chosen metric ruler mark. Immediately note the start time, T0.

6. Allow the container to sit at room temperature until the food coloring has spread away from the center (see following photos). Note the final time, T1, and ruler measurement in mm for the radius from where the dye was initially spotted to the endpoint where the food coloring dye has diffused. This experiment may take several hours, but should be checked every 15 minutes until the coloring drop does not appreciably spread farther.

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Experiment Cell Structure and Function: Cell Types and Transport

7.

Container with gelatin and dye drop at beginning of procedure at Time T0

Container with gelatin and diffused dye at end of procedure at Time T1

Record the beginning and end times and mm measurements in Data Table 2.

Part II: Liquid

1. Before beginning, set up a data table similar to Data Table 2: Diffusion Rate Comparison, in the Lab Report Assistant section.

2. Before you begin, read through the exercise, then propose and record a hypothesis about what you believe will occur.

3. Place room temperature tap water in a clean container to a depth of one cm.

4. Center the container on a flat metric ruler. Protect it from vibration or movement so that the water is completely still.

5. Squeeze two drops of food coloring into the container water above a chosen mm line on the ruler. Note the beginning time, T0.

6. After 10 minutes (T1), read the mm distance that the food coloring has traveled from the beginning mm line. Note the final time and distance in Data Table 2.

Example calculations for Table 2: If To = 9:30 am and T1 = 9:45 am, then 9:45 am - 9:30 am = 15 minutes and 15 minutes ÷ 60 minutes/hour = 0.25 hour. If color moved 25 mm, the Rate of Diffusion = 25 mm ÷ 0.25 hour = 100 mm/hour.

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Experiment Cell Structure and Function: Cell Types and Transport

Exercise 4: Transport of Materials through Cell Membranes – An Example of Selective and Active Transport Search Key Terms: Cell Membrane Active Transport, differentially permeable membrane.

A cell is surrounded by a plasma membrane. The plasma membrane specifically selects which molecule will pass into or out of the cell, thus protecting cellular homeostasis.1 The plasma membrane is said to be differentially permeable because it discriminates between the components that are permitted to pass.

This exercise is a demonstration that loosely mimics active and selective transport in a cellular context. However, it is not a true example of active transport but serves as a demonstration of how transport can operate to discriminate among molecules. The dialysis tubing represents the cell membrane which prevents large molecules from passing through the membrane. In living systems, there are chemical interactions occurring between the molecules and the cell membrane that control molecular passage. Unlike this dialysis tubing exercise, the cell membrane can select larger molecules over smaller molecules for transport into or out of the cell.

The cell membrane is composed of a phospholipid bilayer interspersed with proteins and various carbohydrates. The phospholipid is made up of the subunits glycerol and fatty acids. In this particular lipid the glycerol molecule has a phosphate group attached to it. The glycerol molecule is a water loving molecule and will interact with the water in the cytoplasm. A molecule that loves water is called hydrophilic. The fatty acid component of the phospholipid molecule is water fearing so it will not interact with water, it is said to be hydrophobic. The glycerol (heads) of the phospholipids form an outer barrier for the phospholipids fatty acid (tails) much like two pieces of bread make a sandwich. In this analogy the filling of the sandwich would be represented by the fatty acids.

Glycerol heads

Fatty acid tails

Fatty acid tails

Glycerol heads

1 Homeostasis is the property of an open system, especially living organisms, to regulate its internal environment to maintain a stable, constant condition, by means of multiple dynamic equilibrium adjustments, controlled by interrelated regulation mechanisms.

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Experiment Cell Structure and Function: Cell Types and Transport

procedure 1. Before beginning, set up a data table similar to Data Table 3: Diffusion through a Differentiallly

Permeable Membrane in the Lab Report Assistant section.

2. Read through Exercise 4 and record a hypothesis about what the outcome will be.

3. Dialysis membrane tubing2 is nearly transparent and inflexible when dry. To prepare the tubing for this exercise, trim the dialysis tubing to about 12 cm to 16 cm long. Soak it in distilled water in a clean glass jar until it becomes pliable, approximately 10 minutes.

4. Set up a water bath at simmering (just below 100°C) temperature. See Appendix for help here.

5. Securely tie off one end of the membrane tubing with a rubber band to ensure that added contents will not leak. Test this by adding a small amount of distilled water to the bag to ensure that the tied end does not leak. Why is this critical to the experiment? Empty out and discard the water before proceeding.

6. Add all of the 20% glucose solution to the membrane tubing bag.

7. Add 4 droppers-full of potato starch solution to the same bag.

8. While holding the open end of the membrane tubing bag, knead the outside of the bag to mix the contents together. Rinse the outside of the bag with tap water. Record the color of the bag’s contents in Data Table 3.

9. Use the graduated cylinder to measure and place about 150 mL of distilled water into a clean glass jar.

10. Mix droppers full of IKI solution in the water in the jar until the water is the color of tea or amber. Record the color of the jar’s contents in Data Table 3.

11. Place the dialysis tubing bag containing the sugar/starch mixture into the jar of iodine water. Secure the open end of the membrane bag to the outside of the jar with a rubber band so that it is closed and so the contents cannot spill into the jar.

12. Allow the beaker and the membrane bag to stand until a decided color change is noted or up to one hour. If no change occurs after one hour, discontinue the experiment. Record the color of the water and the membrane bag contents in Data Table 3. Remove the dialysis tubing bag, but save the jar of water. Rinse the contents of the dialysis bag into a sink and dispose of the dialysis tubing in the trash.

13. Use the metric ruler and marker pencil to mark a test tube at 1 cm and 3 cm above the bottom. Then place it in a test tube rack.

2 Dialysis tubing is used in the treatment of kidney disease where filtering of impurities from the blood is necessary to protect the patient from toxic buildup of waste materials.

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Experiment Cell Structure and Function: Cell Types and Transport

14. Use a long-stemmed pipette to fill the test tube to the lower 1-cm mark with some of the water from the glass jar.

15. Fill the test tube to the 3-cm mark with Benedict’s reagent.

16. Place this test tube in a test tube rack and place the rack in a simmering water bath (~100°C). Make sure that the hot water does not flow into the test tube. Heat the test tube for 10 minutes.

17. Remove the test tube and the test tube rack from the water bath and note any color in Data Table 3.

Laboratory summary

What have you learned from doing this laboratory?

Now that you have completed this lab make sure you read the lab for next week. This will help you plan your time better. Take some time and highlight anything you will need to prepare in advance. As you read the lab write out a hypothesis for each exercise.

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Experiment Cell Structure and Function: Cell Types and Transport

Cell Structure and Function: Cell Types and Transport Margaret Vorndam, M.S. Version 42-0037-00-01

Lab report Assistant This document is not meant to be a substitute for a formal laboratory report. The Lab Report Assistant is simply a summary of the experiment’s questions, diagrams if needed, and data tables that should be addressed in a formal lab report. The intent is to facilitate students’ writing of lab reports by providing this information in an editable file which can be sent to an instructor.

Observations

Data Table 1: Cell Structures Prokaryote Cell Structure

Organism name:_______

Eukaryotic Cell Structure

Organism name:___ _______

Flagella Cell wall

Capsule or slime layer Nucleoid

Plasma membrane Cytoplasm

Ribosome Thylakoid

Endospore

Pseudopodia Cell membrane

Nucleus Cytoplasm

Contractile vacuole Food vacuole

Endospore

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Experiment Cell Structure and Function: Cell Types and Transport

Data Table 2: Diffusion Rate Comparison

Medium Beginning Time, To End Time, T1

length of Time in hours

= T1 – To

Movement of color in mm

Rate of diffusion in mm/hour

Semisolid (Gelatin)

Liquid (Water)

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Experiment Cell Structure and Function: Cell Types and Transport

Data Table 3: Diffusion through a Differentially Permeable Membrane At Beginning of Exercise At End of Exercise

What are its contents?

What it its initial color?

What is its final color?

Benedict’s test color

What are its final contents?

Dialysis Tubing NA

Beaker Water

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Experiment Cell Structure and Function: Cell Types and Transport

Exercise 1: Prokaryotes and Eukaryotes - Differences in Cell Structure and Function results 1. What differences are observed between the two cells that were drawn?

2. List the specific cell part that each cell type possesses to: Prokaryote Eukaryote

Escape from hazardous conditions?

Regulate water content?

Store food?

Reproduce?

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Experiment Cell Structure and Function: Cell Types and Transport

Questions A. With which cell type, prokaryote or eukaryote, are you most familiar?

B. Which cell type would be most successful in extreme environments? Why?

C. Because the Prokaryote bacteria are generally 3 µm or less in length, they may not exhibit specific structures under a compound light microscope. What type of microscope would be a better choice for viewing bacteria?

D. Research amoebas online and give a few examples including where they are found and what energy source they use.

E. Discuss whether cyanobacteria and amoebas are beneficial or detrimental to humans and to the environment in general.

Exercise 2: Eukaryotes – Plant and Animal Cell Structure and Function results 1. What differences are observed between the two cells that were drawn?

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Experiment Cell Structure and Function: Cell Types and Transport

2. List the cell part that each cell type possesses to:

Questions A. With which cell type, plant or animal, are you most familiar?

B. Which cell type would be most successful in extreme environments? Why?

Plant Animal

Regulate water content?

Produce energy to power cell function?

Store food?

Reproduce?

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Experiment Cell Structure and Function: Cell Types and Transport

Table 1: Comparison of Structure and Function: Animal and Plant Cells Animal Cells Plant Cells

Differences:

Similarities:

C. Outline the general differences between plant and animal cells in Table 1. Then outline the similarities.

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Experiment Cell Structure and Function: Cell Types and Transport

Exercise 3: Transport of Materials through Cell Membranes – An Example of Passive Transport Questions A. Which medium exhibited the fastest rate of diffusion?

B. What might be reasons to explain the differences in the rate of diffusion?

C. Graph the two sets of results from Table 2 on the same graph using a computer spreadsheet program and include this graph in your report. Why is simultaneous graphing of the results useful here? What type of graph would best demonstrate rates of diffusion in gelatin versus liquid? Hint: The graph should show a continuity from To to T1 for both mediums.

Discussion A. Were your two hypotheses supported or refuted?

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Experiment Cell Structure and Function: Cell Types and Transport

B. How might the rate of diffusion be increased? Decreased?

C. How might this be tested?

D. Why is this exercise be an example of passive transport?

E. Why must the spread of the coloring in the gelatin be monitored? For example, why can it not just be left for 10 hours before the measurement is taken?

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Experiment Cell Structure and Function: Cell Types and Transport

F. What would be a graphic method to detect when the rate of diffusion in the gelatin was at maximum and was beginning to slow?

G. Construct a table that compares your results to the average of your classmates’ results, if available. Indicate how many classmates’ results are represented in the average (N=?). Include it in your report. Are results similar? If not, what might explain the differences in outcomes?

Exercise 4: Transport of Materials through Cell Membranes – An Example of Active Transport Questions A. What are the test substances?

B. Why did a color change occur in the jar water? What does the color change indicate?

C. Why did a color change occur in the dialysis bag? What does the color change indicate?

D. What does the Benedict’s reagent test detect? What does the test of the jar water indicate?

E. What DID NOT diffuse across the dialysis bag membrane? Why?

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Experiment Cell Structure and Function: Cell Types and Transport

Discussion A. Was your hypothesis supported or refuted?

B. How might the dialysis membrane tubing be similar to a plasma membrane?

C. What type of transport mechanism does this exercise simulate?

D. What can be concluded from this exercise?

Laboratory summary What have you learned from doing this laboratory?

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Experiment Cell Structure and Function: Cell Types and Transport

Cell Structure and Function: Tonicity and pH Margaret E. Vorndam, M.S. Version 42-0038-00-01

Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.

Experiment Summary:

Students will study how cells maintain homeostasis and how osmosis is used to regulate internal salt concentrations when placed in hypotonic, hypertonic, and isotonic solutions. Students will place potatoes and Elodea in various concentrations of salt and observe the effects on tonicity and turgor pressure. Students will also learn about pH and buffers and test the buffering effect of different solutions to the addition of an acid. Students will graph the results to assist in analyzing the effectiveness of the buffered systems.

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ExpErimEnt

Objectives ● To explain how cells control homeostasis (tonicity, turgor, pH) for their interior environments

● To understand how buffering environments in living systems preserve homeostasis

● To construct a graph demonstrating the progression of results

Time Allocation: Four to eight hours total.

Safety Issues: Specifically review cutting, visual, and chemical safety procedures.

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Experiment Cell Structure and Function: Tonicity and pH

materials MATERiAlS

FROM: QTy ITEM DESCRIPTION:

Student Provides 1 Aluminum foil or plastic wrap 1 Cutting board and knife 1 Fork 1 Bowl, small 1 Distilled water 1 Spoon 1 Potato, cut into two thin 15x7-cm strips

1

Table salt, non-iodized (NaCl). Although iodized table salt, which contains iodine as a dietary additive, will likely work here, scientists use the purest form of chemical available. Purchased non-iodized table salt may be used just as regular iodized table salt is used in cooking and table use.

1 Egg or prepared egg white from grocer 1 Microscope

1

Elodea (Anacharis), or other thin leaf water plant, purchased from an aquarium shop and kept in clean water. If elodea cannot be located, purchase any other thin-leaved aquarium plant, such as a sword plant.

From labPaq 2 Beaker, 50 mL, plastic 1 Cylinder, 50 mL - Graduated, Plastic

1 Dissection-kit with 7-tools - including the following: Bent Probe, Dropping Pipette, Probe, Ruler in pocket, Scalpel with 2 Blades - Note blades are in the pocket, Scissors, Tweezers

1 Microscope Slides with Cover Slips 1 Goggles-Safety

1 Gloves, Disposable (1 pair) 1 Pencil, marking 1 Rod, Stirring rod - Glass 1 Digital scale 1 Slide - Cover Glass - Cover Slip Cube 5 Test Tube, 13 x 100 mm (1 pc) 1 Test-tube-rack-6x13-mm

1 Test tube cleaning brush 1 Pipette, Graduated Jumbo (5 mL) 1 Hydrochloric Acid, 0.1 N - 6.5 mL in Pipette 1 pH 7.0 Buffer (Yellow), 30 mL, in Dropper Bottle 1 pH Test Strips, Wide Range with Color Scale - in Bag 2”x 3”

Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq.

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Experiment Cell Structure and Function: Tonicity and pH

Discussion and review Living cells protect cellular functions by regulating their internal environments. Since metabolic processes such as energy production, growth, reproduction, etc., can occur only if immediate conditions are favorable, cells’ capability to modify their environments can be critical to the organism’s survival. In this series of exercises, we will examine the cell’s response to external conditions and how cells regulate the internal environment to compensate for those conditions.

Tonicity describes the osmotic pressure gradient set up across a cell membrane due to the concentration1 of a solute2 on the outside of a cell as compared to the concentration of the solute inside of a cell. This osmotic pressure gradient exists only if the solute has a molecular structure that prevents it from crossing the cell membrane. Note that tonicity does not actually involve the movement of solvent, but only describes the state of the osmotic pressure gradient conditions that exist for the cell.

Preparations Required Prior to Performing Lab Exercises:

For Exercises 1 and 2, prepare a 10% sodium chloride (NaCl) solution as follows:

1. With the marker pencil, label the side of a clean beaker “NaCl” (salt, sodium chloride).

2. Use the scale to weigh 1 gram of table salt. Put the salt into the beaker.

3. Measure 10 mL of distilled water into the graduated cylinder.

4. Pour the 10 mL of distilled water into the beaker containing the table salt.

5. Use the stirring rod to mix until the salt grains are completely dissolved.

6. Cover the beaker with aluminum foil or plastic wrap and set aside until used.

Hints: If a dropping pipette or eye dropper is dedicated for use with only one chemical, it will not need to be cleaned. However, you must label it to ensure it is not accidentally used with a different chemical by mistake!

1 The chemical symbol that indicates concentration of a chemical is shown in brackets. For instance, the concentration of salt dissolved in water in this exercise can be written as [NaCl] = 10% aqueous. 2 A solute is a substance that is dissolved in or combined with a solvent. A phase change from solid to liquid is generally, but not always, involved. Example: If salt is dissolved in water, salt would be the solute.

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Experiment Cell Structure and Function: Tonicity and pH

Exercise 1: The Case of the Limp Vegetable Search Key Terms: Tonicity, isotonic, hypotonic, hypertonic, diffusion, differentially permeable membrane, solute, osmotic pressure gradient, osmosis.

The cell’s environmental osmotic pressure gradient is defined as hypertonic, isotonic, or hypotonic, depending on whether the gradient is toward the outside of the cell membrane, toward the inside of the cell membrane, or in equilibrium on each side of the cell membrane.

For example, if a solute such as salt is dissolved in water and a cell is placed in this solution, an osmotic gradient will exist because the salt content of the cell’s cytoplasm is not equal to the salt content in the water solution into which the cell was placed. The physical existence of this gradient is described by using the terms hypertonic, isotonic, or hypotonic. Because the osmotic pressure gradient exists, the cell will respond by attempting to neutralize it by diluting the salt or other solute between the inner and outer environment. Osmosis is the process that occurs to neutralize the osmotic pressure gradient between each side of the cell membrane.

Tonicity of the Cell

Cell Environment Type Movement Appearance

Hypertonic gradient Water moves in the direction that will create equivalent conditions both inside and outside of the cell. ---------------------------- In a hypertonic environment, water exits from the cell in an attempt to dilute the solute to the outside of the cell.

Cell shrivels as it loses water.

isotonic gradient Water moves in the direction that will create equivalent conditions both inside and outside of the cell.

-----------------------------

In an isotonic environment, water and solute are equally present, so a state of equilibrium exists.

No effect. Water moves equally in both directions.

Hypotonic gradient Water moves in the direction that will create equivalent conditions both inside and outside of the cell.

------------------------------ In a hypotonic environment, water enters into the cell in an attempt to dilute the solute on the inside of the cell.

Cell expands as it gains water.

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Experiment Cell Structure and Function: Tonicity and pH

Osmosis is defined as the movement of water across a differentially permeable membrane. For cells then:

● When the concentration of water and solute is equal both inside and outside of the cell membrane, the cell is said to be in an isotonic (iso = equal in Greek) solution or environment and the movement of solvent is equal in both directions, out of the cell and into the cell.

● However, if the concentration of solute is lower on the outside of the cell than on the inside of the cell, the cell environment is hypotonic. In this case, the cell attempts to bring itself into a state of equilibrium, so water is absorbed into the cell in an effort to dilute the solute and the cell swells.

● Where there is less water and more solute on the outside of the cell membrane and more water/less solute on the inside, the cell is in a hypertonic environment. The cell attempts to rid itself of water so that its environment will be equal. So, water moves out of the cell.

The following exercise demonstrates these differences. In this first exercise, we will use the reaction of a potato placed in water and in a salt solution and observe cellular reactions to the solutions.

procedure 1. Read through this exercise and record a hypothesis for each test tube sample treatment below

as to what might be expected to occur in the potato cells in a water environment versus a saltwater environment.

2. Mark two test tubes sequentially as #1 and #2.

3. Place the test tubes upright into the test tube rack and treat the test tubes as follows:

a. Test tube 1: Fill with distilled water.

b. Test tube 2: Fill with the prepared 10% sodium chloride (NaCl) solution.

4. Use the cutting board and knife to peel a potato. Then slice two thin strips of potato from the whole peeled potato. The strips should measure about 1.5 cm wide by 7 cm long and have no peel on them. If necessary, trim the side of the potato strip so that it will slide into the test tube opening.

5. Use the tweezers to place one potato strip in each test tube, and allow the test tubes to sit for approximately one hour.

6. Remove potato strips from the test tubes and keep track of which strip came from which test tube. Record observations in Data Table 1.

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Experiment Cell Structure and Function: Tonicity and pH

Data Table 1: Comparison of States of Tonicity

Test Tube Solute/Solvent

Appearance of Potato Strip

Type of Tonicity Gradient: Is the potato strip in a Hypertonic, Hypotonic, or Isotonic at the beginning of this exercise?

1

2

Questions A. How does each potato strip look? Which one is limp and which one is crisp?

B. How can the difference be explained?

C. What caused the change in appearance of each strip?

Discussion A. Did the exercise support or refute the initial hypothesis? Explain why.

B. Explain why vegetables stored in a refrigerator over time become limp.

C. In this exercise, two of the three types of tonicity gradients were observed. Suggest an experiment to provide an example of the third type of tonicity gradient in action.

D. What kinds of cell environmental conditions might affect tonicity in cells?

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Experiment Cell Structure and Function: Tonicity and pH

Exercise 2: Turgor Search Key Terms: Turgor, plant cell, central vacuole.

The existence of high osmotic pressure within a plant cell is more specifically referred to as turgor pressure. In Latin, turgire means to be swollen. Plant cells are surrounded by cell walls. When a hypotonic gradient exists in a plant cell, water will move into the cell, but the cell expansion is limited by the cell wall.

When the plant cell is turgid, the central vacuole gains water and compress the cell contents against the plasma membrane/cell wall. This is similar to an inflated balloon in a box full of Styrofoam® peanuts. When a plant cell is viewed under a microscope, differences in cell component arrangements are evident depending on the amount of turgidity in the cell, for example, how large the central vacuole is at the time. This exercise will demonstrate the difference.

Leaves of a live water plant, elodea, will be placed in a water environment and a saltwater environment. Observation of cellular changes in these two environments will indicate the effect that the adjusting central vacuole has on cell contents.

procedure 1. Read through this exercise and record a hypothesis for each sample treatment below as to

what might be expected to occur in the plant cells in a water environment versus a salt water environment.

2. Remove a young leaf from the elodea sprig. Young leaves are found toward the terminal end where new leaves are produced.

3. Using tweezers, place the elodea leaf on a microscope slide.

4. Place a drop of distilled water on the leaf and then cover it with a microscope slide cover slip to keep it from drying out.

5. Observe the prepared slide of the elodea leaf under the microscope. This represents the leaf cells in a hypotonic state.

6. Prepare another slide of an elodea leaf, but use the 10% NaCl solution to mount the leaf instead of distilled water.

7. Allow the leaf in 10% NaCl solution to sit for approximately 5 minutes. Observe the leaf in NaCl solution under the microscope. This represents the leaf cells in a hypertonic state.

8. Make a drawing of the leaf showing the arrangement of the internal cell components that are viewable. Label the drawn cell parts that are identified in the drawing. The central vacuole and chloroplasts should be present.

9. Record observations in Data Table 2.

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Experiment Cell Structure and Function: Tonicity and pH

Data Table 2: Comparison of Turgor Pressure States in Elodea Leaf Cells

Solute/Solvent

Scientific Term for Condition:

Is the leaf in a Hypertonic, Hypotonic, or Isotonic at the beginning of this exercise?

Appearance of Cells at End of Procedure

Water 10% Sodium Chloride, NaCl solution

Questions A. How can the differences in appearance of the cells in the two solutions be explained?

B. What caused the appearance of the cell to be altered by using 10% NaCl solution instead of water?

C. In what direction did the water move in the hypotonic solution? Into the cell. In the hypertonic solution?

D. Plasmolysis refers to the movement of water out of the cell, thus causing the central vacuole to become smaller. The volume of the entire cell is reduced and the plasma membrane may be visible, separated from the cell wall, because it pulls away from the cell wall with the reduction in volume. In which turgor state would this be most likely to occur?

Discussion A. Did this exercise support or refute the initial hypothesis? Explain.

B. Where might an example of this turgor pressure adjustment be observed happening in nature or around your home?

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Experiment Cell Structure and Function: Tonicity and pH

Exercise 3: Buffering Effect and pH Search Key Terms: Buffer, buffering capacity, pH, acid, base.

Buffered aspirin is acetylsalicylic acid that has an antacid added to protect the stomach linings of people who have problems with heartburn caused by stomach acid. Aspirin inhibits the Cox enzyme that protects the stomach lining, so people prone to heartburn experience pain as a result of taking aspirin. The added buffer “neutralizes” stomach acid, thus minimizing the erosion of the unprotected lining.

The cells of living organisms are naturally buffered to withstand some exposure to changes in the pH. For example, acid rain is caused by industrial smoke stacks, power plants, and automobiles that inject the gases carbon monoxide (CO), carbon dioxide (CO2), sulfur oxides (SOx)

3, and nitrogen oxides (NOx) into the atmosphere. These gases react with atmospheric water to form carbonic acid (H2CO3), sulfuric acid (H2SO4), and nitric acid (HNO3) which all lower the pH of rainfall from the typical pH of 5 to 6, to a much lower pH. During rainfall these acids precipitate onto vegetation, and at some point the acids overwhelm the natural buffering capacity of the leaves and cause considerable damage to foliage. In the United States, environmental regulations now mandate that power plants install air scrubbers that remove smoke stack gases and that automobiles be equipped with emission-control devices. Industrial emissions are governed similarly; however, acid rain remains a problem.

An acid can be defined as a substance that produces hydrogen ions, H+, in solution. A base can be defined as a substance that produces hydroxide ions, OH-, in solution. Both are present to varying amounts in any aqueous solution.

pH is a scale of measurement used to indicate the alkalinity or acidity of solutions. The scale is based on the concentration of hydrogen (H+) ions in the solution. The concentrations of H+ ions and hydroxide (OH-) ions have a predictable proportionate relationship.

3 The use of the x in chemical formulas refers to the presence of the compound in more than one chemical form. For instance, SOx can refer to a mixture of SO2, sulfur dioxide, and SO3 and SO4 ions. The same is true for NOx.

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Experiment Cell Structure and Function: Tonicity and pH

pH Typical Solutions at pH

Ba si c – Al ka lin

e 14 Liquid drain cleaner containing lye, sodium hydroxide 13 Bleach, Draino®

12 Detergent water, mineral lime 11 Ammonia in water 10 Antacid 9 Baking soda 8 [OH-] > [H+] Ocean water, egg white

Neutral 7 [H+] = [OH-] Distilled water

Ac id

ic

6 [H+] > [OH-] Saliva, urine, milk 5 Drinking water, rain water 4 Tomato juice, orange juice 3 Lemon juice, vinegar 2 Stomach acid, cola 1 Battery acid, sulfuric acid 0 Hydrochloric acid

pH ranges from 0 to 14. pH 7 are neutral, where [H+] = [OH-] (the brackets indicate concentration of the ions). Below pH 7, [H+] > (is greater than) [OH-], and above pH 7 [H+] < (is less than) [OH-]. Solutions with pH < 7 are termed “acidic” and solutions with pH > are termed “basic.” Orange juice, which contains ascorbic acid, is approximately pH 4.5. Household ammonia has an approximate pH of 11. Saliva pH is in the range of 5 to 7.

The pH scale is not linear. It is logarithmic. A pH change from 7 to 8 would indicate a tenfold increase in [OH-], while a pH change from 7 to 9 is a hundredfold increase in [OH-] ion. The equilibrium equation is:

H+ + OH- → H2O

Living cells can withstand some pH change in the environment because they do have some buffering capacity. Unfortunately, in the case of acid rain, the pH change can overwhelm the plant cells’ ability to buffer against it. Damage to foliage occurs and is widespread in the areas where acid rain is a problem. In environments in the Northeastern United States and Europe where soils are derived from volcanic rock which is already an acidic substrate with little buffering capacity, acid rain has killed off many organisms in biotic communities and sterilized entire lakes. Where soils are formed from limestone deposits left from old inland sea invertebrate-derived calcareous deposits, vegetation may be affected, but aquatic systems are protected by the carbonates dissolved in the water. Limestone is calcium carbonate, CaCO3, which creates a basic solution when dissolved in water. When the acid rain contacts the limestone, the basic carbonates neutralize the acid in the rainwater. A graphic example of this reaction would be produced by adding vinegar (acetic acid) to baking soda (sodium bicarbonate).

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Experiment Cell Structure and Function: Tonicity and pH

This exercise will demonstrate the differences between buffered and unbuffered living systems. Three solutions - water, a chemically buffered solution, and egg albumen in a chemically buffered solution - will be compared for their capability to buffer the effects of acid.

A note about pH indicators: pH indicators change color based on the amount of H+ or OH- that is present in a solution. pH indicator solutions change to specific colors at certain pHs. As a result, paper that has been impregnated with an indicator solution can be used to provide a general reading of the pH of a solution into which it is dipped. The color that the paper turns can be compared to a color scale to determine the approximate pH of the solution. pH papers, depending on the indicators impregnated into them, can be wide spectrum indicating pH of solutions from 0 to 14, or they can be narrower and selective for a specific pH range. This exercise uses a wide- spectrum pH paper. Compare the color of the test strips to the pH color chart included with them to read the pH levels.

procedure 1. Read through this exercise and record a hypothesis for each sample treatment below as to

what pH changes might be expected to occur in response to the addition of acid.

2. Prepare a 1% buffered albumen protein solution:

a. If using a fresh egg, carefully crack the egg without breaking the yolk, separate the white into a clean bowl, and discard the yolk.

b. If using prepared egg white (albumen), place it in a clean bowl.

c. Rapidly beat the egg white with a fork for one minute to break up the albumen.

d. Use the graduated pipette to draw up 1 mL of the beaten egg albumen.

e. Place the egg albumen contents of the pipette into the graduated cylinder.

f. Add 9 mL of the pH 7 buffer solution to bring the total albumen and buffer solution to the 10-mL level in the graduated cylinder. Mix together or shake well.

g. Cover the graduated cylinder with aluminum foil or plastic food wrap and set aside.

Calculation: Egg white contains ~10% albumen protein, so 1 mL egg white x 10%/100% = 0.1 mL albumen. 0.1 mL albumen ÷ 10 mL solution x 100 = 1% albumen solution.

3. Mark three test tubes sequentially as #1, #2, and #3 with the marker pencil. Place the test tubes in a test tube rack and treat test tubes as follows:

a. Test tube 1: Fill ½-full with distilled water.

b. Test tube 2: Fill ½-full with pH 7 buffer solution.

c. Test tube 3: Fill ½-full with buffered albumen solution.

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Experiment Cell Structure and Function: Tonicity and pH

4. Remove 3 pH test strips from the book and cut them in half, lengthwise, creating 6 half-sized strips.

5. Mark with a pencil two ends of six pieces of pH indicator paper as #1.

6. Mark with a pencil two ends of six pieces of pH indicator paper as #2.

7. Mark with a pencil two ends of six pieces of pH indicator paper as #3

8. While holding the marked end of the pH paper with a tweezers, dip the opposite tip of one of each labeled pH paper into the correspondingly numbered test tube, and then withdraw it.

9. Determine the pH of each test tube’s solution by comparing the color of the dipped test strip to the pH color scale. Record the beginning pH of the solution for each test tube in Data Table 3.

Data Table 3: Initial and Ending pH Comparison for Test Tube Solutions Test Tube Contents Beginning pH Ending pH Total drops of 0.1 N HCl added

1 Water

2 Buffer (inorganic) solution)

3 Buffered protein Albumen (organic) solution

10. Add five drops of 0.1N HCl (hydrochloric acid) to each test tube.

11. Carefully mix the contents of each test tube by flicking or swirling.

12. Use second set of labeled pieces pH papers (#1, #2, and #3) to determine the “ending pH” of each test tube’s contents again and record these pH readings in Data Table 3 below.

13. Mark two clean 50-mL beakers with #2 and #3 respectively with the marker pencil. Pour the

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Experiment Cell Structure and Function: Tonicity and pH

contents of test tubes #2 and #3 into the appropriately marked beaker. Record into Data Table 4 the ending pH determined for test tubes #2 and #3 from Data Table 3.

note: Use a fresh piece of pH paper for each step in the remainder of the experiment.

14. Add another 5 drops of 0.1 N HCL to each beaker and carefully swirl to mix. Check the pH and record it in Data Table 4.

Data Table 4: Results of Acid Addition to Buffered Solutions Total Drops of HCl Added Beaker #2 pH Beaker #3 pH

0 5 - initial addition while in test tube 10 - after solution is placed in beaker and 5 more drops of HCl are added 15

20

25

30

35

40

45

50

15. Continue to carefully add 5 drops of 0.1N HCl, check pH, and record it in Data Table 4 nine more times so that a total of 50 drops have been added to each solution.

16. Set up a graph similar to Figure 1: Graph of pH Change in Response to Buffering in the Lab Report Assistant section. Graph results in Figure 1. Use a pencil to graph beaker #2 results, and use a pen to graph beaker #3 results.

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Experiment Cell Structure and Function: Tonicity and pH

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Experiment Cell Structure and Function: Tonicity and pH

Questions A. What was the initial pH of the water in test tube #1?

B. What was the pH of the water after the addition of acid?

C. What was the initial pH of test tube solutions #2 and #3?

D. What was the pH after adding 5 drops of acid in each of test tubes #2 and #3?

E. When did the pH appreciably change?

F. What caused this dramatic shift in pH?

Discussion A. Did this exercise support or refute the initial hypothesis? Explain.

B. Why did this pattern occur?

C. What was the difference between solutions #2 and #3?

D. What difference was observed in the way the two solutions reacted to the addition of acid?

E. Why is the buffering capacity of organic substances so important to living organisms, including humans?

Laboratory summary What did you learn from doing this laboratory?

Now that you have completed this lab, make sure you read the lab for next week. This will help you plan your time better. Take some time and highlight anything you will need to prepare in advance. As you read the lab, write out a hypothesis for each exercise.

Cell Structure and Function: Tonicity and pH Margaret E. Vorndam, M.S. Version 42-0038-00-01

Lab report Assistant This document is not meant to be a substitute for a formal laboratory report. The Lab Report Assistant is simply a summary of the experiment’s questions, diagrams if needed, and data tables that should be addressed in a formal lab report. The intent is to facilitate students’ writing of lab reports by providing this information in an editable file which can be sent to an instructor.

Observations

Data Table 2: Comparison of Turgor Pressure States in Elodea leaf Cells

Solute/Solvent

Scientific Term for Condition:

Is the leaf in a Hypertonic, Hypotonic, or Isotonic at the beginning of this exercise?

Appearance of Cells at End of Procedure

Water

10% Sodium Chloride, NaCl solution

Data Table 1: Comparison of States of Tonicity

Test Tube Solute/Solvent

Appearance of Potato Strip

Type of Tonicity Gradient: Is the potato strip in a Hypertonic, Hypotonic, or Isotonic at the beginning of this exercise?

1

2

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Experiment Cell Structure and Function: Tonicity and pH

Data Table 4: Results of Acid Addition to Buffered Solutions Total Drops of HCl Added Beaker #2 pH Beaker #3 pH

0 5 - initial addition while in test tube 10 - after solution is placed in beaker and 5 more drops of HCl are added 15

20

25

30

35

40

45

50

Data Table 3: Initial and Ending pH Comparison for Test Tube Solutions Test Tube Contents Beginning pH Ending pH Total drops of 0.1 N HCl added

1 Water

2 Buffer (inorganic) solution)

3 Buffered protein Albumen (organic) solution

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Experiment Cell Structure and Function: Tonicity and pH

Figure 1: Graph of pH Change in Response to Buffering

pH 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

0 5 10 15 20 25 30 35 40 45 50 Drops of HCl

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Experiment Cell Structure and Function: Tonicity and pH

Exercise 1: The Case of the Limp Vegetable Questions A. How does each potato strip look? Which one is limp and which one is crisp?

B. How can the difference be explained?

C. What caused the change in appearance of each strip?

Discussion A. Did the exercise support or refute the initial hypothesis? Explain why.

B. Explain why vegetables stored in a refrigerator over time become limp.

C. In this exercise, two of the three types of tonicity gradients were observed. Suggest an experiment to provide an example of the third type of tonicity gradient in action.

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Experiment Cell Structure and Function: Tonicity and pH

D. What kinds of cell environmental conditions might affect tonicity in cells?

Exercise 2: Turgor Questions A. How can the differences in appearance of the cells in the two solutions be explained?

B. What caused the appearance of the cell to be altered by using 10% NaCl solution instead of water?

C. In what direction did the water move in the hypotonic solution? Into the cell. In the hypertonic solution?

D. Plasmolysis refers to the movement of water out of the cell, thus causing the central vacuole to become smaller. The volume of the entire cell is reduced and the plasma membrane may be visible, separated from the cell wall, because it pulls away from the cell wall with the reduction in volume. In which turgor state would this be most likely to occur?

Discussion A. Did this exercise support or refute the initial hypothesis? Explain.

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Experiment Cell Structure and Function: Tonicity and pH

B. Where might an example of this turgor pressure adjustment be observed happening in nature or around your home?

Exercise 3: Buffering Effect and pH Questions

A. What was the initial pH of the water in test tube #1?

B. What was the pH of the water after the addition of acid?

C. What was the initial pH of test tube solutions #2 and #3?

D. What was the pH after adding 5 drops of acid in each of test tubes #2 and #3?

E. When did the pH appreciably change?

F. What caused this dramatic shift in pH?

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Experiment Cell Structure and Function: Tonicity and pH

Discussion A. Did this exercise support or refute the initial hypothesis? Explain.

B. Why did this pattern occur?

C. What was the difference between solutions #2 and #3?

D. What difference was observed in the way the two solutions reacted to the addition of acid?

E. Why is the buffering capacity of organic substances so important to living organisms, including humans?

Laboratory summary What did you learn from doing this laboratory?

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Experiment Cell Structure and Function: Tonicity and pH

Enzymes Margaret Vorndam, M.S. Version 42-0053-00-01

Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.

Experiment Summary:

Students will learn the importance of enzymes and how they are used in biological systems. They will explore enzyme activity rates as a function on environmental conditions such as pH, temperature, and concentration of enzyme and substrate.

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ExpErimEnt

Objectives ● To recognize an enzyme-facilitated reaction

● To explain how enzyme activity can be affected by environmental conditions

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Experiment Enzymes

materials

MATERIALS FROM: QTy ITEM DESCRIPTION: Student Provides 1 Distilled water 1 Food-grade plastic wrap or aluminum foil 1 Masking tape 1 Refrigerator or ice water bath at 0°C 1 Stove 1 Timer or clock with second hand

1 1 1 1

Pot or pan for hot water baths Hot water baths, one at 100°C and one at 37°C Trivet or wooden cutting board Trash bag

From LabPaq 1 Beaker, 50 mL, plastic 1 Metric ruler (may be found in dissection kit) 1 IKI Indicator, 2.1% - 13 mL in Glass Vial in Bubble Bag 1 Pencil, marking 15 Pipette, Long Thin Stem 1 Starch Solution, 1% Stabilized - 60 mL in Dropper Bottle 5 Test Tube, 13 x 100 mm 1 Test-tube-rack-6x13-mm 1 Thermometer-in-cardboard-tube

1 1 1 1

Well-Plate-96 Test-tube-clamp-holder Apron Gloves packages-1 pair

1 a-Amylase Powder - 0.12 g in Dropper Bottle - Add 12 mL Distilled Water

1 pH 11.5 Buffer - 2 mL in Pipette (Caution: Contains NaOH, which is a caustic lye) 1 pH 3.5 Buffer - 2 mL in Pipette 1 pH 5.0 Buffer - 2 mL in Pipette 1 pH 6.8 Buffer - 2 mL in Pipette

Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq.

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Experiment Enzymes

Discussion and review Enzymes are catalysts1 that aid metabolic and digestive processes in biological systems. Enzymes mediate reactions that could not normally occur under existing cellular conditions of pH, temperature, and the prevailing concentration of substrate. If the breakdown of molecules had to occur without enzymes, far more energy would be required to drive the building and breakdown of food molecules. Enzymes are proteins, but not all proteins are enzymes. For instance, skin and hair are mostly made of protein, but are not enzymes.

Enzymes are specific in their facilitative activities, and each enzyme is associated with the reaction of a particular substrate molecule in the cell. Example digestive enzymes and the molecular reactions that they facilitate are listed below.

Digestive Enzymes Location in body Reaction that it Facilitates Alpha-amylase Saliva Conversion of starch to sugar Chymosyn (rennin) Infant stomachs Digests milk Lipase Saliva, activated in stomach Fat digestion Lysozyme Saliva Kills bacteria in mouth

Pepsin Saliva, activated in stomach Breakdown of proteins to smaller molecules. Example, plant cell walls Trypsin Intestine Protein digestion

An enzyme lowers the energy of activation that would ordinarily be required for a chemical reaction to occur. Figure 1 demonstrates this reaction.

Figure 1: Comparison of Energy Required for Reaction to Proceed

1 “A substance usually present in small amounts relative to the reactants that modifies and especially, increases the rate of chemical reaction without being consumed in the process.” The American Heritage Dictionary, Houghton Mifflin Company, Boston, Mass. 1985. ISBN 0-395-32943-4.

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Experiment Enzymes

An enzyme is three-dimensional, and its shape accommodates hook-up with the specific substrate that it catalyzes, much like two puzzle pieces. Enzymes can catalyze many substrate reactions before they wear out, and may catalyze thousands to millions of reactions per minute. Enzymes may be either degradative, breaking down their substrates into smaller molecules, or may assist synthesis of molecules, building larger products from substrates. There are enzymes that facilitate digestion, cellular metabolism, cellular breakdown, muscle contractions, healing, and virtually every other function of living systems.

An example of an enzyme-facilitated reaction is that of alpha-amylase enzyme which is found in saliva. When you are chewing bread, amylase in saliva mixes with the bread and begins the process of converting the bread starch into sugar. If you chew bread long enough, it will begin to taste sweet.

Enzymes function within a narrow set of environmental conditions in a cell or within an organ. If temperature or pH changes appreciably, an alteration in the enzyme’s structure can occur which prevents it from functioning or, conversely, may enable its functioning. For instance, pepsin is found in saliva, but does not begin to act on proteins until the acidic pH of the stomach enables it.

In these laboratory exercises, we will look at enzyme activity rates as a function of environmental conditions: pH, temperature, and concentration of enzyme and substrate. Alpha-amylase will be used to demonstrate how enzyme-facilitated reactions may be affected by changing environmental conditions, much like they would in a living system. Alpha-amylase facilitates the conversion of starch to sugar in living beings, including humans. IKI, iodine potassium iodide, colorimetrically reacts with starch to produce a black or dark blue color. Because sugar does not react with the IKI solution, as the alpha-amylase breaks the starch molecule apart to form a sugar, the IKI color will not turn black or blue-black, but instead will remain amber. This will indicate that the starch is no longer present, but has been converted to sugar. This colorimetric test will be used in the following exercises to determine reactivity.

Figure 2: Color reactions of iodine as an indicator of starch using a well plate. Left: Blue/Black color=starch

is present. Right: Amber color=no starch.

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Experiment Enzymes

Shades between amber and blue black indicate that there is some starch present, and not all of it has been digested to sugar. Note: The iodine indicator solution only functions as an indicator of the presence of starch. It is not involved in the actual reaction that occurs between starch and the alpha-amylase enzyme.

Preparations Required Prior to Performing Lab Exercises:

1% Alpha-amylase Solution Preparation, used in all exercises: Carefully remove the cap from the dropper bottle marked as alpha-amylase. Note the label instructions as to how much water, in mL, to add to the dropper bottle to make a 1% solution. Then remove the dropping tip by first placing a small clean piece of aluminum foil or plastic wrap over the tip. Use your fingers or pliers to gently wiggle the tip upward to free it from the bottle. The foil or plastic will help to prevent contaminating the tip with foreign substances. Place the removed tip on a square of aluminum foil or plastic wrap.

Place some distilled water into a clean beaker. Use the beaker to pour exactly the amount specified on the dropper bottle label of mL distilled water into the graduated cylinder. It helps if you use a long-stemmed pipette to control the water when the solution is close to the mL mark. Pour this mL of distilled water from the graduated cylinder into the dropper bottle containing pre-measured alpha-amylase powder. Replace the still covered dropping tip into the bottle. Tighten it so that it pops the dropping tip into the bottle. Shake well and allow the amylase to dissolve before proceeding. Remove the foil or plastic and replace the dropper bottle cap. Discard any distilled water remaining in the beaker.

Store the alpha-amylase dropper bottle solution in a refrigerator when not in use to reduce the rate of bacterial degradation.

Calculation Example: 0.12 g alpha-amylase powder is ~ equivalent to 0.12 mL when mixed in water, so 0.12 mL ÷ 12 mL x 100 = 1%. The bottle label may differ from this.

Water Bath: All exercises require heating test tubes in water baths. Review the Appendix instructions on how to construct and use water baths.

Since 100°C is at boiling temperature (100°C = 212°F) at STP, this temperature can be approximated by keeping the water bath just at simmering (very slow boil) on the stove. Before boiling the water, immerse a test tube rack into the water. The test tube rack will hold the test tubes in the water bath during this experiment. Use the test tube clamp to retrieve the test tubes from, and place the test tubes in, the water bath. Note that water may boil at a lower temperature at high altitudes, but the temperature reached at simmering should be sufficient for the purposes of this exercise.

For the 37°C water bath, use the Celsius thermometer to monitor the water temperature. Maintain the temperature at ± 3°C on the thermometer by alternately sliding the pan on and off of the heat or by adding warm or cool water to the bath as necessary to keep it at 37°C. A larger pan with more water will help to stabilize the temperature for a longer period of time.

For 0°C water bath, recall that 0°C = 32°F. Since water freezes at 32°F, an ice water bath will approximate 0°C. Place ice in a one-cup container, and add water. Because ice is present, the water will be at 0°C as long as the ice has not melted completely.

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Experiment Enzymes

● Since 100°C is at boiling temperature (100°C = 212°F), this temperature can be approximated closely by keeping the water bath just at simmering/very slow boil on the stove.

● For the 37°C water bath, use the Celsius thermometer to monitor the water temperature. Do this calculation:

37°C is equal to __?__ °F **

The temperature conversion equation for °C to °F is:

C = (5/9) x ° F – 32 or F = (9/5) x ° C + 32

**The answer is posted at end of this exercise; try to compute before checking it.

96-well microplate: Place masking tape strips along the short and long sides of the microplate. Along the short side, number five columns from 1 to 5. These numbers will correlate to your test tubes in the following exercises.

Along the long side, number the columns 0, 5, 10, 15, 20, 25, and 30 to represent minutes. At 30 minutes, make a line. When filling the microplate with iodine indicator drops per the directions below, only fill to the 30-minute column until it is determined that testing must proceed for a longer period of time. Having a well plate column numbered as 60 and another as 90 will save time if the reaction must be run longer.

Figure 3: Attach tape to the sides of the well plate and number the

columns and rows.

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Experiment Enzymes

Iodine Pipette: Label one long-stemmed pipette with “iodine” to be dedicated for this use in all exercises. Iodine stains and the pipette will become stained after a few uses. Use a square of aluminum foil or plastic wrap to protect the work surface from iodine stains.

Safety Warnings:

● Heating safety – Use care when handling of hot water. Use hand protection and eye protection. Protect work surface with heatproof base, such as a trivet or wooden cutting board.

● Visual safety – Some solutions may cause damage if they get into eyes. Hot items emit steam. Wear safety glasses.

● Chemical safety – iodine indicator solution – wear apron and rubber gloves.

● Cover work surface with something, such as a plastic garbage bag.

The exercise requires the use of iodine indicator solution. Be careful when using the indicator solution as spills can permanently stain clothing and other items.

Practice using a disposable, long-stemmed dropping pipet before beginning this exercise. Tap water may be used with the microplate to practice adding drops of solution into the microplate wells. Rinse the dropping pipet and microplate with distilled water, and remove excess water before proceeding.

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Exercise 1: Enzymes and Temperature Search Key Terms: Enzyme activity, alpha-amylase, enzyme, and temperature.

Enzyme activity is influenced by temperature. You will test the alpha-amylase enzyme activity on starch under three temperature environments: 0°C, 37°C, and at the boiling temperature of water, 100°C at STP.

procedure Before you begin, read through the exercise then propose and record a hypothesis about what you believe will occur.

1. Set up three water baths at 0°C, 37°C, and 100°C. If you are unable to use the three water baths simultaneously, perform the exercise at one temperature, then repeat the exercise at the other temperatures and compare your final results when all three tests have been completed. This will, of course, take more time.

2. Mark the 96-well microplate as previously directed. You will use only the first three columns of the microplate, marked 1, 2, and 3, for this exercise. These three columns will coincide with test tube numbers.

3. Mark and dedicate separate long-stemmed pipettes to use with the alpha-amylase, the IKI solution, and the starch solution. Practice filling and carefully dispensing drops of distilled water from a clean pipette until you become proficient at releasing only one drop at a time. This will help to avoid wasting IKI solution.

4. Place one drop of the IKI solution into each microplate well of the columns marked 1, 2, and 3 and for the time period rows from 0 to 30 minutes. If you mistakenly release 2 or 3 drops, you can still use the microplate without cleaning it out.

5. Cover the microplate with plastic wrap until needed to prevent contamination.

6. Label three test tubes as #1, #2, and #3.

7. With the marker pencil and metric ruler, mark each of the three test tubes at 1 cm and 6 cm from the bottom. Place the test tubes in the test tube rack.

8. Use the dedicated alpha-amylase pipette to fill each test tube to the 1-cm mark with alpha-amylase solution.

9. Mark three additional long-stemmed pipettes #1, #2, and #3. You will use these with the correspondingly numbered test tubes to collect samples. Insert the pipettes into their related test tubes to prevent them from becoming contaminated and leave them in the test tubes when not in use. However, you may want to place the pipette for the 100°C water bath on a piece of aluminum foil next to the hot water bath since the heat may melt the pipette if left in the test tube.

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10. Begin the incubation: Place test tube #1 in the 0°C ice water bath, test tube #2 in the 37°C water bath, and test tube #3 in the 100°C water bath. Begin timing for 5 minutes.

11. After 5 minutes, add 1% starch solution to each test tube to the 6-cm mark. Remove the pipettes from the test tubes and hold them while you add the starch. Reinsert the pipettes into the test tubes after the starch has been added to the 6-cm mark.

12. Mix the contents in each test tube by stirring with its dedicated pipette. Immediately after mixing, transfer two drops of its solution into the microplate well row “0” for zero minutes, and the columns marked 1, 2, and 3, which correspond to the test tubes labeled #1, #2, and #3, respectively. Immediately view the initial response after the two drops are added to each row and record the color of the wells in Data Table 1. Ignore any other color change that may happen after the initial response. Return the pipette to its corresponding test tube.

13. Return each test tube to the same water bath in which it was originally placed and begin timing the experiment. Stir the test tubes with the pipettes occasionally.

14. After 5 minutes, transfer drops to the well plate:

a. Use test tube #1’s dedicated pipette to transfer two drops of its solution into the microplate well for the column marked #1 in the row that corresponds to the minutes after the amylase was added to the test tubes (for example, row 5 for 5 minutes).

b. Immediately view the initial response and record the color of the well in Data Table 1. Ignore any color change that may happen after the initial response.

c. Squeeze excess solution from the pipette back into the test tube, stir the test tube, and replace the pipette.

d. Repeat steps 14a through 14c for test tubes #2 and #3, transferring the drops into each column of the row that is marked with the same number as the test tube.

15. Continue repeating steps 14a through 14d every 5 minutes for a total of 30 minutes or until the drops from the test tube solutions turn amber when mixed with IKI solution, whichever comes first.

16. Discontinue testing a test tube when the response of mixing it with the IKI solution is amber (-). However, you must continue timing the other test tubes for up to 30 minutes or until they also exhibit the amber color upon testing. Ignore any color change that occurs after your initial observation.

17. After 30 minutes, if test tube #1 and/or #3 has not changed to amber when tested, place the unchanged test tube(s) into the 37°C water bath prepared for test tube #2.

18. Allow the test tube(s) to sit in the 37°C water bath for another 30 minutes and then test for the presence of starch by placing one drop of IKI indicator solution and two drops of test tube contents in the related 60-minute well row. Record your initial color observation in Data Table 1.

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Experiment Enzymes

Data Table 1: Effect of Temperature on Amylase Enzyme Conversion of Starch to Sugar Time, minutes

Well/Test Tube 1 0°C

Well/Test Tube 2 37°C

Well/Test Tube 3 Boiling (100°C at STP)

0 5 10 15 20 25 30 Place test tubes that still contain starch in 37°C water bath. Continue timing for 30 minutes. 60

Add 1 cm of alpha-amylase solution to test tubes that still contain starch. Place in 37°C water bath. Continue timing for 30 minutes.

90

19. If one or both of test tubes #1 and #3 still contains starch after 60 minutes, add another 1 cm of alpha-amylase solution into it, mix and allow it to sit in the 37°C water bath for another 30 minutes. Retest for starch in the 90-minute well row by placing one drop of IKI solution and two drops of test tube contents. Record your results in Data Table 1.

20. Clean and dry the well plate, the pipettes used in the test tubes and the test tubes for use in the next exercise.

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Questions A. Which color indicated that starch was still present in the test tube?

Which color indicated that the starch was no longer present?

B. When the starch “disappeared” as indicated by the color change, what happened to it?

C. What is the variable in this exercise?

What could be considered the control treatment?

D. Based on the results recorded in Data Table 1, which temperature(s) is/are optimal for enzyme conversion activity?

Which temperature facilitated the conversion first?

Second?

E. Did other temperatures facilitate enzyme activity at all?

Why or why not?

F. Why was the water bath temperature of 37°C chosen as the “mid-range” temperature? Hint: What is the oF equivalent of this oC temperature?

G. Why was it important to immediately observe the color of the test tube contents/iodine in the microplate well?

H. Why didn’t you add the iodine indicator directly to the test tubes?

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Experiment Enzymes

I. What happened when you incubated the test tube(s) that still contained amylase/starch at 37°C?

Did both test tubes indicate the disappearance of starch after the incubation?

If not, why?

J. What happened after you added 1 cm of alpha-amylase solution to the test tube(s) that still contained starch, and incubated them at 37°C?

Why did this occur after the second addition of amylase, but not before?

K. Construct a graph that presents your findings from Table 1 with time on the x-axis, and relative concentration of starch on the y-axis (++ is highest, +, or – is lowest). Include the graph in the report.

Discussion A. Was the initial hypothesis supported or refuted? Explain.

B. What have you learned from this experiment?

C. What practical applications could this knowledge have?

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Experiment Enzymes

Exercise 2: Enzymes and Substrate Concentration Search Key Terms: Enzyme activity, enzymes, and concentration.

Enzymes can be reused many times to facilitate the conversion of substances before they are denatured. This exercise will demonstrate the effects of substrate concentration on enzyme reactivity.

procedure Before you begin, read through the exercise then propose and record a hypothesis about what you believe will occur.

1. Set up a water bath at 37°C.

2. Label the microplate wells as previously directed. Columns 1 through 5 will correspond to the five test tubes used in this experiment.

3. Use the IKI long-stemmed pipet to place one drop of the IKI solution into each well of the microplate columns marked 1 through 5 and for the six time period rows from 5 to 30 minutes. Cover with plastic wrap and set aside.

4. Mark five test tubes sequentially from #1 to #5.

5. With the marker pencil and metric ruler measure and mark each test tube at 1/2 cm above its bottom.

6. Further mark the test tubes as follows:

a. Mark test tube #1 at 2 cm above the bottom.

b. Mark test tube #2 at 3 cm above the bottom.

c. Mark test tube #3 at 4 cm above the bottom.

d. Mark test tube #4 at 5 cm above the bottom.

e. Mark test tube #5 at 6 cm above the bottom .

7. Fill each test tube to the 1/2-cm mark with alpha-amylase solution.

8. Place the test tubes in a test tube rack and into a 37°C water bath for 5 minutes.

9. Mark the bulbs of five clean, long-stemmed pipets sequentially from #1 to #5, place the pipets in the test tubes corresponding to their numbers, and leave the pipets in the test tubes when not in use.

10. After five minutes, remove the test tube rack from the water bath. Add 1% starch solution to each test tube’s second-cm mark from the bottom per the above diagram.

11. Mix the contents in each test tube by stirring with its dedicated pipette. Immediately after mixing, transfer two drops of its solution into the microplate well row “0” for zero minutes,

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Experiment Enzymes

and the columns marked 1, 2, 3, 4, and 5, which correspond to the test tubes labeled #1, #2, #3, #4, and #5, respectively.

12. Immediately note the color of each mixture, after the two drops are added to each row and then return the pipette to its corresponding test tube.

13. Record the initial response color of the wells in Data Table 2.

14. Return each test tube to the water bath and begin timing the experiment. Occasionally stir the test tubes with the pipettes.

15. After 5 minutes, transfer drops to the well plate:

a. Use test tube #1’s dedicated pipette to transfer two drops of its solution into the microplate well for the column marked #1 in the row that corresponds to the minutes after the amylase was added to the test tubes (for example, row 5 for 5 minutes).

b. Immediately note the response and record the initial color change in Data Table 2.

c. Squeeze excess solution from the pipette back into the test tube, stir the test tube, and replace the pipette inside of it.

d. Repeat steps 15a through 15c for test tubes #2, #3, #4, and #5, transferring the drops into each column of the row that is marked with the same number as the test tube.

Data Table 2: The Effect of Concentration on Amylase Enzyme Conversion of Starch to Sugar

Time, minutes Test Tube #1

Test Tube #2

Test Tube #3

Test Tube #4

Test Tube #5

Concentration of Amylase: Fill in % per Test tube #1 example →

0.5 cm ÷ 2 cm, or 1:4 = 25% ______% ______% ______% ______%

0 5 10 15 20 25 30 If test tube(s) still contains starch, let sit for 30 minutes and retest. 60

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16. Continue repeating steps 15a through 15d every 5 minutes for a total of 30 minutes or until all test tube solutions test color is amber, whichever comes first.

17. Discontinue testing a test tube when its test color in the microplate well is amber (-). However, you must continue timing the other test tubes for up to 30 minutes or until they also exhibit the amber color upon testing. Ignore any color change that occurs after your initial observation.

18. While waiting, record the concentration of alpha-amylase to starch present in each test tube in Data Table 2. Test tube #1 calculation has been completed for you.

19. If any test tube solution has not changed to amber after 30 minutes of testing, return the test tube(s) to the water bath for another 30 minutes, then test 2 drops of the solution(s) with one drop of IKI indicator solution in the 60-minute well row and record your results in Data Table 2.

20. When finished, clean and dry the well plate, the pipets used in the test tubes, and the test tubes for use in the next exercise.

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Questions A. What is the variable in this exercise?

B. Which test tube has the highest concentration of alpha-amylase in the starch?

C. How could this experiment be improved?

D. Construct a graph that presents your findings in Table 2 with time on the x-axis, and relative concentration of starch on the y-axis (++ is highest, +, or – is lowest). Use five different colors to represent the concentration of amylase. Include it in the report.

Discussion A. Was your initial hypothesis supported or refuted as to which test tube starch contents would convert to sugar first?

B. What have you learned from this experiment?

C. What practical applications could this knowledge have?

D. Describe another way to do this experiment that would test the same concept.

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Exercise 3: Enzymes and pH Search Key Terms: Enzyme activity, enzymes, and pH.

Enzymatic activity is critically affected by the pH of the environment in which the enzymes are found. In this activity, we explore what pH is optimal for the alpha-amylase to convert starch to sugar. We will test how alpha-amylase functions at four pH levels: pH 3.5, 5, 6.8, and 11.5.

CAUTION! pH 11.5 in a buffer solution can only be obtained by using a very alkaline powder, lye, also known as sodium hydroxide. Lye is used in oven cleaners and is extremely caustic! Handle the bottle containing the pH 11.5 buffer and the other bottles with extreme care. If you spill any buffer on your skin or work surface, flush it immediately with water to avoid chemical burns.

procedure Before you begin, read through the exercise then propose and record a hypothesis about what you believe will occur.

1. Set up a 37°C water bath.

2. Label the microplate wells as previously directed, except only column numbers 1 through 4 are needed to correspond to the four test tubes used in this exercise.

3. Use the labeled long-stemmed pipette to place one drop of the IKI solution into each well of the microplate columns marked 1 through 4 and for the six time period rows from 5 to 30 minutes. Cover with plastic wrap and set aside.

4. Mark four test tubes sequentially from #1 to #4.

5. With the marking pencil and metric ruler, measure and mark each test tube at 1 cm, 2 cm, and 4 cm from the bottom. Place in the test tube rack.

6. Carefully add enough pH 3.5 buffer to test tube #1 to reach the bottom 1-cm mark.

7. Likewise, add the pH 5 buffer to test tube #2, the pH 6.8 buffer to test tube #3, and the pH 11.5 buffer to test tube #4 to the bottom 1-cm mark.

8. Add enough alpha-amylase solution to each test tube containing different pH solutions to reach the 2-cm mark for each test tube.

9. Place all four test tubes in the test tube rack into the 37°C water bath.

10. Mark the bulbs of four clean, long-stemmed pipettes sequentially from #1 to #4, place the pipettes in their correspondingly numbered test tubes and stir the tubes’ contents.

11. After 5 minutes, remove the rack from the water bath and add starch solution to fill each test tube to the 4-cm mark. Mix well by stirring with the long-stem pipette.

12. Replace the test tubes into the water bath and begin timing for 30 minutes.

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13. Mix the contents in each test tube by stirring with its dedicated pipette. Immediately after mixing, transfer two drops of its solution into the microplate well row “0” for zero minutes, and the columns marked 1, 2, 3, and 4 which correspond to the test tubes labeled #1, #2, #3, and #4, respectively.

14. Immediately observe the color of the wells in Data Table 3 after the two drops are added to each row and then return the pipette to its corresponding test tube.

15. Record the initial observed color of each mixture in Data Table 3.

16. Return each test tube to the same water bath in which it was originally placed and begin timing the experiment. Stir the test tubes with the pipettes occasionally.

17. After 5 minutes, transfer drops to the well plate:

a. Use test tube #1’s dedicated pipette to transfer two drops of its solution into the microplate well for the column marked #1 in the row that corresponds to the minutes after the amylase was added to the test tubes (for example, row 5 for 5 minutes).

b. Immediately record the color of the well in Data Table 3.

c. Squeeze excess solution from the pipette back into the test tube, stir the test tube, and replace the pipette inside of it.

d. Repeat steps 17a through 17c for test tubes #2, #3, and #4, transferring the drops into each column of the row that is marked with the same number as the test tube.

Data Table 3: The Effect of pH on Amylase Enzyme Conversion of Starch to Sugar Time, in minutes Test Tube #1 Test Tube #2 Test Tube #3 Test Tube #4 pH ( #1 example →) Student to fill in → pH 3.5 pH ________ pH ______ pH _______

0 5 10 15 20 25 30 If test tube(s) still contains starch, let sit for 30 minutes and retest. 60

18. Continue repeating steps 17a through 17d every 5 minutes for a total of 30 minutes or until all test tube solutions test color is amber, whichever comes first.

19. Discontinue testing a test tube when its test color in the microplate well is amber (-). However, you must continue timing the other test tubes for up to 30 minutes or until they also exhibit the amber color upon testing. Ignore any color change that occurs after your initial observation.

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Experiment Enzymes

20. If no color change is observed after 30 minutes, wait 30 minutes more and retest in the 60-minute well plate row.

21. When finished, clean and dry the well plate, the pipettes used in the test tubes, and the test tubes for use with future exercises.

** Answer to earlier calculation question: 37°C is equal to 98.6 °F

Questions A. What is the variable in this experiment?

B. In which test tube(s) did the amylase convert starch to sugar?

Why might you expect this outcome?

C. Did any test tubes NOT exhibit a color change?

Why?

Discussion A. Did the results support or refute your hypothesis as to which test tube starch contents would convert to sugar first?

Laboratory summary A. What other conditions that may affect the action of enzymes?

B. How might you propose to test this?

C. What have you learned from this laboratory?

D. What practical applications might this knowledge have?

Enzymes Margaret Vorndam, M.S. Version 42-0053-00-01

Lab report Assistant This document is not meant to be a substitute for a formal laboratory report. The Lab Report Assistant is simply a summary of the experiment’s questions, diagrams if needed, and data tables that should be addressed in a formal lab report. The intent is to facilitate students’ writing of lab reports by providing this information in an editable file which can be sent to an instructor.

Observations note: The observed well colors should be recorded according to the following symbols. Collect data until the well plate yields an amber color. Then, discontinue testing that test tube’s contents.

++ = Black or blue-black, Starch present + = Light Blue or Light blue-black, Some starch present - = Amber, Orange, or Red, No starch present

Data Table 1: Effect of Temperature on Amylase Enzyme Conversion of Starch to Sugar Time, minutes

Well/Test Tube 1 0°C

Well/Test Tube 2 37°C

Well/Test Tube 3 Boiling (100°C at STP)

0 5 10 15 20 25 30 Place test tubes that still contain starch in 37°C water bath. Continue timing for 30 minutes.

60

Add 1 cm of alpha-amylase solution to test tubes that still contain starch. Place in 37°C water bath. Continue timing for 30 minutes.

90

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Experiment Enzymes

Data Table 2: The Effect of Concentration on Amylase Enzyme Conversion of Starch to Sugar

Time in minutes Test Tube #1 Test Tube #2 Test Tube #3

Test Tube #4

Test Tube #5

Concentration of Amylase: Fill in % per Test tube #1 example →

0.5 cm ÷ 2 cm, or 1:4 = 25% ______% ______% ______% ______%

0 5 10 15 20 25 30 If test tube(s) still contains starch, let sit for 30 minutes and retest. 60

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Experiment Enzymes

Data Table 3: The Effect of pH on Amylase Enzyme Conversion of Starch to Sugar Time, in minutes Test Tube #1 Test Tube #2 Test Tube #3 Test Tube #4 pH ( #1 example →) Student to fill in → pH 3.5 pH ________ pH ______ pH _______

0 5 10 15 20 25 30 If test tube(s) still contains starch, let sit for 30 minutes and retest. 60

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Experiment Enzymes

Exercise 1: Enzymes and Temperature Questions A. Which color indicated that starch was still present in the test tube?

Which color indicated that the starch was no longer present?

B. When the starch “disappeared” as indicated by the color change, what happened to it?

C. What is the variable in this exercise?

What could be considered the control treatment?

D. Based on the results recorded in Data Table 1, which temperature(s) is/are optimal for enzyme conversion activity?

Which temperature facilitated the conversion first?

Second?

E. Did other temperatures facilitate enzyme activity at all?

Why or why not?

F. Why was the water bath temperature of 37°C chosen as the “mid-range” temperature? Hint: What is the oF equivalent of this oC temperature?

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Experiment Enzymes

G. Why was it important to immediately observe the color of the test tube contents/iodine in the microplate well?

H. Why didn’t you add the iodine indicator directly to the test tubes?

I. What happened when you incubated the test tube(s) that still contained amylase/starch at 37°C?

Did both test tubes indicate the disappearance of starch after the incubation?

If not, why?

J. What happened after you added 1 cm of alpha-amylase solution to the test tube(s) that still contained starch, and incubated them at 37°C?

Why did this occur after the second addition of amylase, but not before?

K. Construct a graph that presents your findings from Table 1 with time on the x-axis, and relative concentration of starch on the y-axis (++ is highest, +, or – is lowest). Include the graph in the report.

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Experiment Enzymes

Discussion A. Was the initial hypothesis supported or refuted? Explain.

B. What have you learned from this experiment?

C. What practical applications could this knowledge have?

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Experiment Enzymes

Exercise 2: Enzymes and Substrate Concentration Questions A. What is the variable in this exercise?

B. Which test tube has the highest concentration of alpha-amylase in the starch?

C. How could this experiment be improved?

D. Construct a graph that presents your findings in Table 2 with time on the x-axis, and relative concentration of starch on the y-axis (++ is highest, +, or – is lowest). Use five different colors to represent the concentration of amylase. Include it in the report.

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Experiment Enzymes

Discussion A. Was your initial hypothesis supported or refuted as to which test tube starch contents would convert to sugar first?

B. What have you learned from this experiment?

C. What practical applications could this knowledge have?

D. Describe another way to do this experiment that would test the same concept.

Exercise 3: Enzymes and pH Questions A. What is the variable in this experiment?

B. In which test tube(s) did the amylase convert starch to sugar?

Why might you expect this outcome?

C. Did any test tubes NOT exhibit a color change?

Why?

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Experiment Enzymes

Discussion A. Did the results support or refute your hypothesis as to which test tube starch contents would convert to sugar first?

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Experiment Enzymes

Laboratory summary A. What other conditions that may affect the action of enzymes?

B. How might you propose to test this?

C. What have you learned from this laboratory?

D. What practical applications might this knowledge have?

Copyright © 2008 by Margaret E. Vorndam, 1413 County Road 671, Rye, CO 81069

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Experiment Enzymes

Photosynthesis and Respiration Margaret E. Vorndam, M.S. Version 42-0103-00-01

Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.

Experiment Summary:

Students will learn how photosynthesis occurs using paper chromatography to separate out chlorophyll and other pigments from spinach leaves. Students will use Elodea to measure the production of oxygen into a respirometer and use Bromothymol blue to measure the production of carbon dioxide gas during respiration.

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ExpErimEnt

Objectives ● To explain how plants convert sunlight into energy through photosynthesis

● To describe the role of respiration as complimentary to the photosynthetic process

Time Allocation: Four to eight hours total.

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Experiment Photosynthesis and Respiration

materials MATERiAlS

FROM: QTy ITEM DESCRIPTION: Student Provides 1 150 watt light 1 Small, glass baby food-size jar 1 Larger jar to contain small jar 1 Toothpick or other 1 - 2” long stick 1 Bar straw 1 Tap water 1 Paper clip 1 Pencil 1 Plastic food wrap 1 Distilled water 1 Mortar & pestle or chopping knife 1 Cutting board or food processor/blender 1 Scissors 1 Timing watch or clock 1 Large jar 1 Paper towels or aluminum foil 1 Rubber bands 2 Spinach leaves 1 Vinegar 1 Baking soda (sodium bicarbonate, NaHCO3) 1 Elodea, Anacharis, or other thin-leaved aquatic plant with ends cut back to expose fresh stem tissue 1 Acetone, small amount from hardware store or uncolored nail polish remover containing acetone as first ingredient From labPaq 1 Cylinder, 50 mL - Graduated, Plastic

1 Beaker, plastic, 100 mL

1 Dissection-kit with 7-tools - including the following: Bent Probe,

Dropping Pipet, Probe, Ruler in pocket, Scalpel with 2 Blades - Note blades are in the pocket, Scissors, Tweezers

1 Respirometer Assembly; bent glass tube in #4 stopper 1 Rod, Stirring rod - Glass 1 Digital scale

1 Test Tube (1), 25 x 150 mm in Bubble Bag 1 Test-tube-rack-12x13-mm

1 Test tube cleaning brush 1 Well-Plate-96

1 Pipet, Graduated Jumbo (5 mL)

1 Bromthymol Blue, 0.01% Adjusted with NaHCO3 - 30 mL in Dropper Bottle 1 Filter Paper 9.0 cm

Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq.

Safety Issues: Specifically review Cutting, Glass, Heating, and Chemical safety procedures.

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Experiment Photosynthesis and Respiration

Discussion and review Respiration and photosynthesis are the two major sources for energy in plants and animals. During respiration, plants and animals produce energy for metabolic processes by breaking down foods (carbohydrates, lipids, and proteins) that have been stored in their bodies or ingested. Carbon dioxide gas, CO2, is one end product of respiration. Other sources of CO2 in the environment come from the combustion of fossil fuels, the decomposition or organic matter, and natural chemical reactions.

Plants counteract the overall buildup of CO2 in the atmosphere through their energy-producing photosynthetic cycle. Plants absorb photon energy produced by the sun and radiated to earth. The sun-derived energy excites electrons in the chlorophyll-containing thylakoid membranes of plant chloroplast stroma,1 and the electrons begin a cascade of reactions that drive the cycles that cause photosynthesis. Plants use CO2 as a primary source of carbon from which to build glucose and other sugars during photosynthesis. But they also give off Oxygen, O2, as a by-product in this process. The irony is that plants are not poisoned by too much CO2 in the environment, but animals can die of oxygen deprivation if CO2 levels become too high, and O2 levels drop.

The Energy Cycle: Our atmosphere is 0.03% CO2, and 21% O2. The remaining atmospheric component is mainly nitrogen. Water vapor, H2O, is also present in the atmosphere and is derived from lakes, streams, oceans, plants, and animals. Oceans absorb an appreciable amount of CO2 along with plants to maintain a relatively steady state of O2 production via photosynthesis that counters the CO2 produced during respiration by plants and animals.

2

Photosynthesizing plants on Earth convert less than 2% of incoming solar radiation to food in the form of proteins, carbohydrates and fats. Animals acquire this energy by eating plants, either directly, or by consuming other animals that eat plants.

The process of photosynthesis relies on the ready capability of chlorophyll to gain and lose electrons. When sunlight strikes the leaf, packets of energy called photons excite the electrons of the chlorophyll found in the chloroplasts of plant cells. The captured energy enables the production of carbohydrates (CH2O)n, water (H2O), and, very importantly, oxygen (O2) from the carbon dioxide (CO2) and water (H2O). The photosynthetic reaction can be summarized as:

Sunlight energy + CO2 + 2H2O Ú (CH2O)n +H2O + O2

where n indicates the number of joined molecular units that form the carbohydrate.

1 We can see individual chloroplasts when viewing plant cells under a light microscope. Each chloroplast contains many bodies that are double-membraned. The inner environment of these membrane-surrounded bodies is filled with stroma, an enzyme-rich fluid in which thylakoids are located. Under an electron microscope, one is able to see that thylakoids are stacked, much like coins. The membranes of the thylakoids contain chlorophyll and other pigments that can absorb the solar radiation energy that powers the processes that result in the production of carbohydrates and oxygen. Do an online search on the terms thylakoid, stroma and chloroplasts to learn more. 2 However, many scientists are concerned that this balance is being disturbed by the Greenhouse Effect. You can find more information about the Greenhouse Effect online.

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Experiment Photosynthesis and Respiration

Chlorophyll a and b are the primary photosynthesizing pigments, but other pigment families such as carotenoids and xanthophylls also aid in the process. Not all of these pigments are found in all photosynthesizing plants. However, eukaryotic plants generally contain chlorophyll a (blue-green in color) or b (yellow-green in color) and carotenoids and/or xanthophylls (yellow or orange in color). Carotenoids - think carrots - become noticeable in autumn when the greenish chlorophylls decline and leaves take on the hues of the longer-lived oranges, yellows and reds of the various pigments. In this exercise, we will examine the energy processes responsible for life

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Experiment Photosynthesis and Respiration

Exercise 1: Chlorophyll Pigments Search Key Terms: Paper chromatography, chromatography, chlorophyll, xanthophyll, carotenoids, photosynthesis, Rf value.

This exercise demonstrates the presence of various photosynthesizing pigments in plants. Paper chromatography is a way of separating pigment molecules that are differentially soluble in a solvent. Pigments move at different rates as the solvent moves up a strip of absorbent paper. This separation occurs due to the differing polarity3 and size of the pigment molecule.

Paper chromatography is used in modern-day biochemical laboratories to separate components of chemical mixtures, much as you will be doing in this laboratory exercise using chlorophyll. After the chemicals have been separated via chromatography, the solvent is dried off and the paper can be cut apart to obtain the different molecular fractions of the original mixture. Nowadays, gas chromatographs (GC) or high-performance liquid chromatographs (HPLC) can be used to analyze and prepare similar fractions obtained from mixtures.

In this exercise, you will use acetone, an organic solvent, as the solute. The chlorophyll pigments will dissolve in the acetone and can then be separated as the acetone diffuses up the paper strip. You will be able to observe several separated fractions of the chlorophyll after the acetone dries off of the paper strip. You should be able to view chlorophyll b, a yellow-green pigment, followed by chlorophyll a, a blue-green pigment close to the spinach leaf extract on a test strip. Next, xanthophylls, may be found as a light yellow to grayish strip of color carotenoids, the topmost layer of color, appear as a dull orange-yellow color above the xanthophylls.

procedure Before you begin, read through the exercise, then propose and record a hypothesis about what you believe will occur.

1. Use a mortar and pestle or a knife and cutting board to mash the spinach leaves. The pieces should not be recognizable.

2. Place the mashed spinach leaves in the small jar.

3. Pour enough acetone solvent into the jar to cover the mashed spinach leaves.

4. Use the glass stir rod to slurry the mashed leaves with the acetone so that no clumps of leaf material are evident.

5. Cover the opening of the jar with plastic wrap and fasten it on with a rubber band.

6. Fill the larger jar with enough hot tap water to surround the leaf/acetone content in the small jar.

3 Polarity is a term that refers to the distribution of the charges associated with a molecule’s shape.

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Experiment Photosynthesis and Respiration

7. Ensure that the water remains hot during the steeping period. Aluminum foil or plastic wrap fastened with a rubber band over the top of the jar or a plate placed on it will help to preserve the heat longer. Replace the water in the outer jar with hot tap water if it cools appreciably.

8. Allow the leaf material/acetone mixture to steep for 30 to 60 minutes and agitate the mixture occasionally during the steeping period.

9. Prepare the paper chromatograph equipment while the spinach leaves are steeping:

a. Use the test tube rack, set on its side, to support the large test tube. Fasten rubber bands near the top and the bottom of this assembly to keep the test tube affixed to the rack.

b. With a pencil and metric ruler, draw a rectangular 10 x 1 cm strip on a piece of round filter paper and cut it out with scissors. Place it inside of the test tube to ensure it will fit inside the tube.

c. Place a toothpick or similar across the top of the test tube so that it can rest centered over the tube’s opening. Suspend the paper clip from the middle of the toothpick to hang down inside the test tube.

d. Remove the paper strip. Use a pencil to make a centered mark on the paper strip where it meets the bottom of the suspended paper clip.

e. Use the dissecting needle to make a small hole approximately ¼ inch below the pencil mark. Insert the paper clip through this hole to suspend the paper strip from the top of the test tube. Your objective is to suspend the paper strip inside of the test tube without it touching the bottom or insides of the walls. If the strip touches the sides, trim a small amount off one side until it will swing freely within the test tube without touching the sides. If it touches the bottom, do not cut it. Raise it by inserting the paper clip farther down the paper strip.

f. With the marker pencil, mark the outside of the test tube at the bottom point of the suspended paper strip. Then remove and set aside the toothpick, paper clip, and paper strip assembly.

10. After the spinach leaf and acetone have steeped at least 30 minutes, draw up the colored acetone/leaf extract into a glass dropper without drawing up any solid leaf matter.

11. To avoid getting any solution extract on the sidewalls, Insert the dropper vertically into the center of the test tube opening and drip the contents into the bottom of the test tube. Fill the test tube with the extract to just above the mark on the outside of the test tube by about ¼ inch.

12. Replace the filter paper strip-paper clip-toothpick assembly so that the filter paper extends into the acetone extract at the bottom of the test tube, but does not touch the bottom. The sides of the filter paper strip should not contact the sides of the test tube.

13. Cover the test tube opening with a piece of plastic wrap or aluminum foil to minimize evaporation of the acetone.

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14. Allow the test tube to sit for up to an hour or until the acetone solvent front, appearing as a wet area, travels to the top of the filter paper strip.

15. Remove the filter paper strip from the test tube. Use a pencil to mark the topmost level of the solvent front on the paper chromatogram. Place the filter paper on a piece of wax paper or aluminum foil to allow the acetone to evaporate. It should leave the chlorophyll fractions spread out on the filter paper strip.

16. Identify the areas of pigments that you observe on the filter paper. Record the colors that you observe in Data Table 1.

17. Clean equipment in preparation for the next experiment.

Data Table 1: Rf Value Comparison for Chlorophyll Pigments

Chlorophyll Pigment

Observed Color

Distance to top of pigment, mm Rf

other Students’ Rf Average

# of Rf Results Averaged from other Students (N)

Top of Solvent Front NA

Carotenoids

Xanthophylls

Chlorophyll a

Chlorophyll b

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Experiment Photosynthesis and Respiration

Questions: A. Identify the areas of pigments that you observe on the filter paper. Are they located in the order that you expected?

If not, why not?

B. Compare your paper strip results to the results of the radicchio paper strips below. Are the same pigments present in radicchio leaves?

If not, which chlorophyll pigments are missing?

How might you have guessed that this might be the case?

Discussion A. Was the initial hypothesis supported or refuted? Explain.

B. If you do not see separate bands of color on the filter paper, what might you conclude?

C. What might explain why the separation of the colors occurred?

D. How do your values compare to your classmates’ values for the same pigments on Table 1 and in your graph? If they are not similar, what may be the reason?

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Exercise 2: Conversion of Light Energy to Food by Plants Search Key Terms: Photosynthesis, leaf structure, gross oxygen production, respiration.

During photosynthesis, energy from sunlight facilitates the conversion of carbon dioxide and water into plant carbohydrate and oxygen. Oxygen is subsequently expelled through the leaves.

Photosynthesis: 6 H2O + 6CO2 → C6H12O6 + 6 O2 ↑ (given off as gas)

In this exercise, we will measure the rate of gross oxygen production as a by-product of photosynthesis.

procedure Before you begin, read through the exercise then propose and record a hypothesis about what you believe will occur.

NOTE: This exercise works best with a sprig of Elodea, a freshwater plant usually found in aquarium stores. If Elodea is unavailable, substitute any other leafy, freshwater aquariumplant. If no freshwater aquarium plants are available, substitute fresh leaves from plants growing in streams or lakes or fresh leaves from lettuce, philodendrons, or similar plants. All live plants will exhibit photosynthesis and respiration, but the reaction will proceed more rapidly with fresh Elodea or similar broad leaf aquatic plant life.

1. Your respirometer comes with the bent glass tube inserted in the rubber stopper. This assembly is then inserted in the large test tube. Attach the respirometer tube upright to the test tube rack or other solid object using rubber bands.

2. Make a 3% sodium bicarbonate solution by first using your scale to measure out 1.5 grams of sodium bicarbonate. Place the 1.5 g of the sodium bicarbonate into a beaker. Add 50 mL of distilled water. Stir with the glass stir rod to completely dissolve the sodium bicarbonate into the water.

3. Cut off about ½ inch of the Elodea plant’s stem end that is opposite the leaf tip growth. Place a generous quantity of Elodea into the respirometer tube with Elodea stem ends pointing up toward the open end of the test tube.

4. Fill the test tube with the 3% sodium bicarbonate solution. Tap the tube to free any and all air bubbles that are trapped by the Elodea leaves.

5. Insert the rubber stopper with the bent tube into the top of the test tube until it is snug, but not tight. You may need to adjust the level of the solution in the test tube so that the solution extends a bit into the horizontal tube past the bend above the rubber stopper. Note: the sodium bicarbonate solution does not need to touch the bottom of the rubber stopper; some

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air space is okay. Important: DO NOT touch or readjust the rubber stopper once you have done the next step!

6. Use the marker pencil and mark the starting position of the solution that has extended into the bent tube on the bent tube.

7. Direct a 150-watt light at the respirometer, but first place a glass jar of tap water between the respirometer and the light source to absorb the light’s heat. NOTE: do not exceed the wattage rating of the lamp.

8. When the solution in the bent tube begins moving toward the tube’s end, begin to time the experiment.

9. After ten minutes, turn off the light, and mark the finish point of the solution front on the bent tube. Measure the distance the solution has moved from the starting mark and record this in Data Table 2. This figure is the “net photosynthesis rate.”

Data Table 2: Gross Photosynthesis in Elodea Rates Distance of travel, mm

Net Photosynthesis Rate over 10 minutes = mm of movement from marked beginning point on bent tube to end point after 10 minutes

Respiration Rate over 10 minutes = mm of movement from marked end point on bent tube toward beginning point after 10 minutes

Gross Photosynthesis over 10 minutes (= mm Net Photosynthesis + mm Respiration)*

Rate of Photosynthesis over one hour (= Gross Photosynthesis ÷ 10 minutes x 60 minutes)

10. Carefully wrap aluminum foil around your respirometer containing the Elodea to exclude all light. Be careful and do NOT disturb the rubber stopper position.

11. Wait ten minutes and then mark the finish point where the solution has moved back toward the bend and the rubber stopper. Record your results in Data Table 2. This figure is the “respiration rate.”

12. When finished, place the Elodea in a container of water to prevent it from drying out; pour the sodium bicarbonate solution down the sink; clean the glass tube and rubber stopper, removing all marks; and rinse the test tube with distilled water.

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Experiment Photosynthesis and Respiration

Questions A. What molecule was produced by the Elodea while it was exposed to light?

B. Which organelle(s) in the plant cell is responsible for this reaction?

C. From where was this molecule derived?

D. What was the purpose of the light?

E. What molecule was produced by the Elodea while it was wrapped in aluminum foil?

F. Which organelle was responsible for this reaction?

G. Compare your Rate of Photosynthesis to the average rates reported by other students in Data Table 3, if possible. How similar was your result to theirs?

Data Table 3: Comparison of Photosynthetic Rate

My Photosynthetic Rate Result Average of Other Students’ Photosynthetic Rate

Results N =______ (number of data averaged)

H. Name at least three experimental variables that could explain why your outcome might vary from that of other students or from your own, if you repeated the experiment.

I. How could one determine how much oxygen is produced by the plant in one hour?

1. From geometry, recall that Volume = πr2h, where r = inside radius of a circle, or here the inside of the tube, and h = length of the movement, which means π x radius squared x height. Since the radius is ½ of the inside diameter, this equation is equivalent to = 3.1416 x (inside diameter of the bent tube in mm ÷ 2)2 x height (equivalent to the distance that the solution traveled) = volume in mm3.

2. To convert mm3 volume to uL (typically the volume specification used at

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this level of measurement), use the conversion factor of 1 mm3 = 1 uL. Do the calculation to determine the volume of oxygen that is produced by the plant in one hour.

3. How long would the plant need to photosynthesize to produce one liter of O2?

Discussion A. Did this experiment support or refute your initial hypothesis. Explain.

B. Chlorophyll gives plant leaves their green color, (or red, in the case of radicchio, since it lacks a pigment that is typically present in green leaves). Research light and color, and hypothesize whether chlorophyll absorbs green light to any appreciable extent. Propose an experiment similar to the one that you just performed that could be used to support your hypothesis.

C. There is current controversy as to whether the atmosphere is warming due to the burning of fossil fuels and the destruction of the rainforests (the Greenhouse Effect). How does photosynthesis and respiration relate to this controversy?

D. Why are the tropics particularly important to oxygen production?

Therefore, why should you support actions that reduce the cutting of rainforests?

E. While this exercise has addressed oxygen production, what other important products result from photosynthesis? Hint: Beside the production of oxygen, why are all animals dependent on plants?

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Exercise 3: Respiration in Plants Search Key Terms: Respiration, fermentation, carbonic acid and water, Bromothymol blue.

The complement of photosynthesis is respiration. Respiration in both plants and animals drives cellular function. Respiration requires carbohydrate (CH2O)n, such as glucose (C6H12O6) and oxygen (O2). It releases energy in the form of adenosine triphosphate (ATP) for cell functioning and carbon dioxide (CO2) as a gas byproduct. ATP is essential to the cell since none of its various energy requiring processes such as active transport, cytoplasmic streaming, cell division, muscle contraction, etc., can proceed without its presence.

Photosynthesis: 6 H2O + 6CO2 → C6H12O6 + 6 O2 ↑ (given off as gas)

Respiration: C6H12O6 + 6 O2 → 6 H2O + 6CO2 ↑ (some given off as gas)

During respiration, carbohydrates are broken down. If this reaction is incomplete, it is referred to as fermentation, and the end product is an alcohol and CO2. Yeast is an organism that can facilitate this incomplete reaction nicely. We appreciate the by-products of incomplete respiration (fermentation) that yeast provides when we enjoy the fine wines and brews of the world and leavened products such as bread and cheeses. Fermentation also refers to the breakdown of carbon-containing compounds in the cells of our bodies under low oxygen conditions as a way to produce energy. This anaerobic process is common in athletes, in particular.

Carbon dioxide (CO2) dissolved in water (H2O) forms carbonic acid (H2CO3). Carbonic acid is a weak acid and, in solution, is part of equilibrium between carbonic acid and hydrogen ion, H+, and bicarbonate ion, HCO3

-. The two reactions are

CO2 + H2O D H2CO3 (carbonic acid, weak acid)

H2CO3 D H + + HCO3

- (bicarbonate ion, weak base)

During photosynthesis a plant will use CO2 and H2O to produce O2 and carbohydrate. The removal of CO2 from the water by the plant will drive the equilibrium toward the left, as indicated by the longer arrow pointing left in the chemical equation below4. The carbonic acid dissociates and decreases in the solution to satisfy the imbalance caused by the removal of CO2 by the Elodea. This decreases the acidity of the solution and causes the solution pH to be closer to neutral pH.

4 A loose analogy to this process is osmosis. Recall that molecules attempt to spread themselves uniformly in a liquid mixture. In this chemical equation, the plant is removing CO2 during photosynthesis. So, the chemical equation attempts to balance itself by shifting the molecular (ionic) equilibrium to the left. When CO2 is produced in quantity, the chemical equation compensates by shifting to the right.

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Bromothymol blue is a pH indicator that is yellow in acidic solutions, green in neutral (pH 7) solutions and blue in basic solutions. In the above reaction a high level of the product of respiration, CO2, in water would force the reaction of water to the left, forming a weak acid with a green/yellow color due to the presence of the indicator Bromothymol blue. If CO2 was removed from the water as could happen during photosynthesis, the reaction would be forced to the right toward a neutral to basic state. The Bromothymol blue indicator color in the water would then be green/blue depending on the concentration level of CO2 in the water.

Bromothymol blue (Yellow)

H2CO3 (weak acid)

Bromothymol blue (Blue)

CO2 + H2O (more basic)

acid (H+) alkaline (OH-)

The addition of CO2 will cause the formation of a weak acid in the solution and the solution will change to yellow-orange or yellow. Then, add a plant, Elodea, to the solution, and time the reaction required for the plant to remove enough of the CO2 to force the above reaction back toward a neutral pH. The Bromothymol blue solution will indicate this reaction by a color change. Following this procedure, cover the Elodea in solution with aluminum foil and observe whether respiration is occurring. Finally, compare the rate of reaction that is facilitated by the plant to the rate which is not facilitated by the plant.

procedure 1. Read through this exercise; then construct and record a hypothesis as to what you believe will

occur.

2. Calibrate yourself for Bromothymol blue pH change observation. Place two drops of the Bromothymol blue pH indicator solution in each of two wells of the well plate. Use a clean dropping pipet to add a drop of vinegar (acid) to one of the Bromothymol blue wells. Add a grain of baking soda (basic) to the other Bromothymol blue well. Observe the color difference between the acid solution and the basic solution to aid you in observing the color changes in this experiment. Clean and store well plate.

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Experiment Photosynthesis and Respiration

3. Set up the respirometer as directed in Exercise 2 and connect the respirometer upright to the test tube holder or other solid object, as before, with rubber bands.

4. Fill the respirometer test tube with the Bromothymol blue pH indicator solution. If needed, add just enough distilled water so that when the stopper is in place in the test tube the Bromothymol blue solution extends a bit into the horizontal tube past the bend above the rubber stopper. A small air pocket between the stopper and the liquid is acceptable as long as the liquid extends slightly into the bent tube when the stopper is placed lightly into the test tube. Remove the stopper and bent tube and set aside.

5. Using the bar straw, gently breath bubbles into the Bromothymol-blue solution until it becomes a green-yellow or preferably an all-yellow color throughout the respirometer. Record this as the initial color in Data Table 4. Note: Ensure that you do not breathe out so hard that you splash the contents out of the tube. If you use a regular soda straw, you may have difficulty getting a color reaction to occur. Try to get some physical exercise before this step to increase your CO2 output.

Data Table 4: Comparison of Photosynthetic Rate

My Photosynthetic Rate Result Average of Other Students’ Photosynthetic Rate

Results N =______ (number of data averaged)

6. Hold the Elodea with a tweezers, stem side up. Rinse it in tap water and then rinse it well with distilled water. Do not touch the plant after it has been rinsed. Why?

7. Using the tweezers, place the Elodea, cut stem-end up, in the respirometer test tube. Lightly stopper the tube with the rubber stopper and bent tube. The colored solution should extend upward past the bend in the bent tube and into its horizontal portion. Do not disturb the stopper until the completion of the following steps.

8. Mark the beginning front of the Bromothymol blue liquid (still colored green-yellow or yellow) in the bent respirometer tube with the marker pencil.

9. Direct a 150-watt light at the respirometer, but first place a glass jar of tap water between the respirometer and the light source to absorb the light’s heat.

10. When the solution in the bent tube begins moving toward the tube’s end, turn off the light and record the beginning time of the movement as T0 (time zero).

11. When the rate of travel of the Bromothymol blue solution in the horizontal tube slows, turn the light back on until the liquid begins to move forward again. Continue to turn the light off and on until there is a color change in the Bromothymol blue solution. Record the end time in Data Table 4 when the color change occurs. This will be T1 (final time). The total time will be T1 – T0.

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12. Wrap the test tube containing the Elodea and Bromothymol blue solution in aluminum foil. Record the start time as T0. Allow the respirometer to sit until a color change in the liquid is observed. Periodically open then reclose the aluminum foil to observe the liquid’s color. Record the same readings as before. Terminate the experiment if no color change is observed after one hour. Record the final time of the color change as T1.

Now that you have completed this lab, make sure you read the lab for next week this will help you plan your time better. Take some time and highlight anything you will need to prepare in advance. As you read the lab, write out a hypothesis for each exercise.

Questions A. Why is the Elodea rinsed in distilled water?

B. What gas did you add to the Bromothymol blue solution?

How do you know? Respiration is our primary form of energy production.

C. Why did the color turn more green-yellow or yellow when you breathed into it?

D. Write an equation for the reaction that explains the color change.

E. What color change occurred after you placed the Elodea in the test tube and turned the light source on/off?

F. What gas is produced and responsible for this change?

G. What gas is being removed?

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H. What process is occurring in the first trial without the aluminum foil?

I. What did you observe when you repeated the exercise with the test tube containing the Elodea wrapped in aluminum foil?

J. What gas is produced?

What term could be applied to the second trial?

K. Which process took longer?

Why?

Discussion A. How did the time for color change vary between when the Elodea was present in the test tube (trial 1) versus when the Elodea was wrapped in aluminum foil? What might explain this?

B. Why do both plants and animals respire?

Would life in either group be possible without respiration?

C. Was the initial hypothesis supported or refuted? Explain.

D. Heating causes a Bromothymol blue indicator solution to change from yellow to blue very rapidly without a plant present. What explains this reaction? How did you control for heat in this exercise?

E. Compare your results to those of your classmates, if available, in Table 5. Were your results similar? What trends do you observe between the two trials?

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Data Table 5: Comparison of Photosynthetic Rate

My Result: Total time for color change,

T1 – T0, minutes

Average of Other Students’ Results: Total time for color change,

T1 – T0, minutes N =______ (number of data averaged)

Elodea, Bromothymol blue solution Elodea, Bromothymol blue solution in Foil

F. Explain why your data may have differed from that of other students. What variables could explain this difference?

G. How would you be able to check to ensure that it was the Elodea plant that facilitated the color change and not some other factor?

H. How do plants, such as Pinedrops, Pterospora andromedea, or the Ghost Plant, Monotropa uniflora, that are not photosynthetic, make their food?

Do they respire?

Laboratory summary What have you learned from doing this laboratory?

Photosynthesis and Respiration Margaret E. Vorndam, M.S. Version 42-0103-00-01

Lab report Assistant This document is not meant to be a substitute for a formal laboratory report. The Lab Report Assistant is simply a summary of the experiment’s questions, diagrams if needed, and data tables that should be addressed in a formal lab report. The intent is to facilitate students’ writing of lab reports by providing this information in an editable file which can be sent to an instructor.

Observations

Data Table 1: Rf Value Comparison for Chlorophyll Pigments

Chlorophyll Pigment

Observed Color

Distance to top of pigment, mm Rf

other Students’ Rf Average

# of Rf Results Averaged from other Students (N)

Top of Solvent Front NA

Carotenoids

Xanthophylls

Chlorophyll a

Chlorophyll b Distance to top of pigment, mm: Measure from where the top of the solvent extract level intersected the paper chromatograph to the topmost level of the color. Then measure this distance to the top of the solvent front and record.

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Data Table 3: Gross Photosynthesis in Elodea Rates Distance of travel, mm

Net Photosynthesis Rate over 10 minutes = mm of movement from marked beginning point on bent tube to end point after 10 minutes

Respiration Rate over 10 minutes = mm of movement from marked end point on bent tube toward beginning point after 10 minutes

Gross Photosynthesis over 10 minutes (= mm Net Photosynthesis + mm Respiration)*

Rate of Photosynthesis over one hour (= Gross Photosynthesis ÷ 10 minutes x 60 minutes)

*Was respiration occurring during photosynthesis? Add back the O2 that was consumed by plant respiration to obtain the total photosynthetic rate.

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Data Table 4: Comparison of Photosynthetic Rate

My Photosynthetic Rate Result Average of Other Students’ Photosynthetic Rate

Results N =______ (number of data averaged)

Data Table 4: Observation of pH Change in the Oxygen Cycle

Contents of Respirometer

Initial Color

T0, minutes

Final Color T1, minutes

Total time for color change, T1 – T0, minutes

Elodea, Bromothymol blue solution Elodea, Bromothymol blue solution in foil

Data Table 5: Comparison of Photosynthetic Rate

My Result: Total time for color change,

T1 – T0, minutes

Average of Other Students’ Results: Total time for color change,

T1 – T0, minutes N =______ (number of data averaged)

Elodea, Bromothymol blue solution Elodea, Bromothymol blue solution in Foil

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Experiment Photosynthesis and Respiration

Exercise 1: Chlorophyll Pigments results Rf = distance to top of pigment in mm ÷ distance to top of solvent front.

Rf Average = average of other students’ Rf values, if available.

1. Rf values are calculated ratios of how far a molecule moved up the paper chromatograph as compared to the length of the solvent front. These values are typically equivalent for multiple trials. Calculate your Rf values based on your paper strip results, and record them in Table 1. The Rf value of the solvent front will always = 1.0, since Rf is a ratio.

2. Construct a stacked bar graph to demonstrate how the Rf value and the chlorophyll pigment are related. The y-axis will represent the Rf scale from 0.0 to 1.0. On the bar graph, label the calculated Rf with the name of the associated chlorophyll pigment. When complete, the bar graph should closely resemble the actual chromatograph. Include a stacked bar graph for your results plus another stacked bar graph next to your graph with your classmates’ averaged values.

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Experiment Photosynthesis and Respiration

Questions: A. Identify the areas of pigments that you observe on the filter paper. Are they located in the order that you expected?

If not, why not?

B. Compare your paper strip results to the results of the radicchio paper strips below. Are the same pigments present in radicchio leaves?

If not, which chlorophyll pigments are missing?

How might you have guessed that this might be the case?

Example of Results obtained from Radicchio Paper Chromatograph

Radicchio Paper Chromatograph, dried and marked in preparation for Chlorophyll Pigment

Rf determinations

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Experiment Photosynthesis and Respiration

Discussion A. Was the initial hypothesis supported or refuted? Explain.

B. If you do not see separate bands of color on the filter paper, what might you conclude?

C. What might explain why the separation of the colors occurred?

D. How do your values compare to your classmates’ values for the same pigments on Table 1 and in your graph? If they are not similar, what may be the reason?

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Experiment Photosynthesis and Respiration

Exercise 2: Conversion of Light Energy to Food by Plants Questions A. What molecule was produced by the Elodea while it was exposed to light?

B. Which organelle(s) in the plant cell is responsible for this reaction?

C. From where was this molecule derived?

D. What was the purpose of the light?

E. What molecule was produced by the Elodea while it was wrapped in aluminum foil?

F. Which organelle was responsible for this reaction?

G. Set up a table similar to Data Table 3: Comparison of Photosynthietic Rate in the Lab Report Assistant section. Compare your Rate of Photosynthesis to the average rates reported by other students in Table 3, if possible. How similar was your result to theirs?

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H. Name at least three experimental variables that could explain why your outcome might vary from that of other students or from your own, if you repeated the experiment.

I. How could one determine how much oxygen is produced by the plant in one hour?

1. From geometry, recall that Volume = πr2h, where r = inside radius of a circle, or here the inside of the tube, and h = length of the movement, which means π x radius squared x height. Since the radius is ½ of the inside diameter, this equation is equivalent to = 3.1416 x (inside diameter of the bent tube in mm ÷ 2)2 x height (equivalent to the distance that the solution traveled) = volume in mm3.

2. To convert mm3 volume to uL (typically the volume specification used at this level of measurement), use the conversion factor of 1 mm3 = 1 uL. Do the calculation to determine the volume of oxygen that is produced by the plant in one hour.

3. How long would the plant need to photosynthesize to produce one liter of O2?

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Experiment Photosynthesis and Respiration

Discussion A. Did this experiment support or refute your initial hypothesis. Explain.

B. Chlorophyll gives plant leaves their green color, (or red, in the case of radicchio, since it lacks a pigment that is typically present in green leaves). Research light and color, and hypothesize whether chlorophyll absorbs green light to any appreciable extent. Propose an experiment similar to the one that you just performed that could be used to support your hypothesis.

C. There is current controversy as to whether the atmosphere is warming due to the burning of fossil fuels and the destruction of the rainforests (the Greenhouse Effect). How does photosynthesis and respiration relate to this controversy?

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Experiment Photosynthesis and Respiration

D. Why are the tropics particularly important to oxygen production?

Therefore, why should you support actions that reduce the cutting of rainforests?

E. While this exercise has addressed oxygen production, what other important products result from photosynthesis? Hint: Beside the production of oxygen, why are all animals dependent on plants?

Exercise 3: Respiration in Plants Questions A. Why is the Elodea rinsed in distilled water?

B. What gas did you add to the Bromothymol blue solution?

How do you know? Respiration is our primary form of energy production.

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C. Why did the color turn more green-yellow or yellow when you breathed into it?

D. Write an equation for the reaction that explains the color change.

E. What color change occurred after you placed the Elodea in the test tube and turned the light source on/off?

F. What gas is produced and responsible for this change?

G. What gas is being removed?

H. What process is occurring in the first trial without the aluminum foil?

I. What did you observe when you repeated the exercise with the test tube containing the Elodea wrapped in aluminum foil?

J. What gas is produced?

What term could be applied to the second trial?

K. Which process took longer?

Why?

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Discussion A. How did the time for color change vary between when the Elodea was present in the test tube (trial 1) versus when the Elodea was wrapped in aluminum foil? What might explain this?

B. Why do both plants and animals respire?

Would life in either group be possible without respiration?

C. Was the initial hypothesis supported or refuted? Explain.

D. Heating causes a Bromothymol blue indicator solution to change from yellow to blue very rapidly without a plant present. What explains this reaction? How did you control for heat in this exercise?

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Experiment Photosynthesis and Respiration

E. Set up a data table similar to Data Table 5: Comparison of Photosynthetic Rate in the Lab Report Assistant section. Compare your results to those of your classmates, if available, in Table 5. Were your results similar? What trends do you observe between the two trials?

F. Explain why your data may have differed from that of other students. What variables could explain this difference?

G. How would you be able to check to ensure that it was the Elodea plant that facilitated the color change and not some other factor?

H. How do plants, such as Pinedrops, Pterospora andromedea, or the Ghost Plant, Monotropa uniflora, that are not photosynthetic, make their food?

Do they respire?

Laboratory summary What have you learned from doing this laboratory?

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Experiment Photosynthesis and Respiration

Mitosis and Meiosis Margaret Vorndam, M.S. Version 42-0094-00-01

Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.

Experiment Summary:

Students will learn how to differentiate between mitosis and meiosis. They will use onion root tip and whitefish blastula slides to view the stages of mitosis and will model these stages using chromosome beads. Students will observe slides of ovaries and testes and will simulate meiosis using chromosome beads.

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ExpErimEnt

Objectives ● To explain how chromosomes recombine during sexual reproduction

● To understand how chromosomal sorting differs for meiosis and mitosis

● To name the various stages of mitosis and meiosis, and recognize cells in various stages of division

● To understand why crossing-over of genes in chromosomes can be positive or negative in survival of the species

Estimated Time Required to Complete the Experiment: 4 to 8 hours total.

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materials

MATERIALS FROM: lABEl oR BoX/BAG: QTy ITEM DESCRIPTION:

Student Provides 1 Scissors 1 Straw, cut into short pieces 1 Flat surface to represent cell 1 Transparent tape 1 Thread 1 Microscope

Chromosome Simulation Kit Bag

Chromosome Simulation Kit Bag 1

Chromosome Kit-BK - Centromeres - 4 Color Bead Set

Slide Box BK-2A Slide Box BK-2A 1 Slide - Onion Root Tip 1 Slide - Ovary 1 Slide - Testis 1 Slide - Whitefish Blastula

Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq.

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Experiment Mitosis and Meiosis

Discussion and review All living organisms grow through division or enlargement of cells. Living organisms also reproduce new organisms through asexual means by fission or mitosis, or through gamete production, followed by fertilization. In this experiment we will examine the processes responsible for organisms’ ability to grow and reproduce.

Cell division, also referred to as replication, is accomplished through the process of mitosis (Gk. mitos, thread + -osis). During cellular mitosis, cell chromosomes that contain the genetic code for the organism are replicated; the two chromosome sets are divided, the cell splits, and a set of chromosomes goes to each cell. Since no new genetic material is added during this process (discounting possible crossover mutations that may occur within the dividing cells), this process is referred to as asexual reproduction.

Organisms use this process of mitotic cellular replication to increase numbers or replace dead cells in cellular tissues and to repair damage from injuries. Simple organisms may exclusively use mitosis to reproduce exact copies of themselves. An example of this is a hydra, a Cnidarian that produces buds along its body which break away from the parent organism and grow into a new hydra. Other examples of mitotically facilitated reproduction include plant cuttings used to propagate new plants, potato tubers used to grow potatoes, and plants grown from bulbs. Cloning is another example of this type of process. In each example there is no reduction in the number of chromosomes or any exchange of genetic material with another like organism. Each new cell has the same number of chromosomes as did the original cell and is identical to the parent from which it was derived. Since no chromosomal division occurred in these mitotically divided cells, the total number of chromosomes is referred to as the 2n (diploid)1 number, where n equals the number of chromosome pairs in the cell.

In contrast, during sexual reproduction cellular replication also occurs, but an exchange of genetic material is necessary.2 In this process both meiosis (Gk. meiosis, diminution), a halving of the organism’s chromosome number, in combination with mitosis results in a recombination of the parent gametes in the new offspring. An organism that manifests traits of each parent is produced, containing genetic information that, in combination, is expressed differently than either parent alone. This exchange can convey an added capacity to the new organism/generation of organisms to respond to environmental conditions. Why?

1 Although the diploid number of chromosomes is the number that we commonly associate with mitosis, the student should be aware that some organisms do exist in polyploidy states, where more than one set of chromosomes are present. Terms such as triploid (3n), tetraploid (4n), etc. are associated with these organisms’ normal sets of chromosomes.

2 Some organisms carry both male and female reproductive parts and are able to self-pollinate. Humans must reproduce by the joining of sperm from a male and the egg from a female.

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Mitosis – The Life Cycle of a Cell: Cells are “born” when mitosis causes cells to split, creating two where there was one. Neither resulting cell is truly the originating or parent cell since both retain portions of the genetic material - organelles, cytoplasm, and outer membrane - from the original cell during the split. Like organisms, each cell has a finite longevity that is governed by its genetic material. After a period of existence genes within the cell that cause the cell to die are turned on, a process referred to as apoptosis. Why is this process of cell death necessary, and what benefit might it have to the organism? Until apoptosis occurs, active cells go through life cycles that are akin to the sequence presented in the life cycle figure located in Exercise 1.

Figure 1: Reproduction in Potato Plants

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Experiment Mitosis and Meiosis

Exercise 1: Mitosis in Animal and Plant Cells Search Key Terms: Whitefish blastula, onion root tip, mitosis, plant cell division, animal cell division, centrosome, microtubule.

Although the outcome of mitosis is the same for plant and animal cells, there are some differences in the process. We will examine the processes in an early embryonic stage of the whitefish embryo, an example of animal cells, and in onion root tips representing plant cells. Both cell types’ chromosomes are large and easy to observe under a microscope.

In plant mitosis, plants have the added task of dividing the cell wall to form new cell walls between the two cells. This step occurs during Telophase. Unlike animal cells, dividing plant cells do not form centrioles and asters. In plants, the centrosome is the “microtubule organizing center” in the cell and is responsible for spindle development. All other steps are similar to those outlined for animal cell mitosis.

procedure 1. Observe the prepared slide of the whitefish blastula under the microscope.

2. Draw cells that represent the various stages of mitosis: Interphase, Prophase, Metaphase, Anaphase, and Telophase.

3. For each drawing, label the parts of the cells that you can identify such as the nuclear membrane, chromosomes, cellular membrane, cytoplasm, spindle fibers, chromatids, cleavage furrow formation during Cytokinesis, and centrioles. It is unlikely that you will find all of these structures. Why?

4. Observe the slide of the onion root tip under the microscope.

5. Draw cells that represent the various stages of mitosis: Interphase, Prophase, Metaphase, Anaphase, and Telophase.

6. Label the parts of the cells that you can identify such as the nuclear membrane, chromosomes, cellular membrane, cytoplasm, spindle fibers, chromatids, cell wall formation region, and centrioles. It is unlikely that you will find all of these structures. Why?

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Experiment Mitosis and Meiosis

Mitotic Phase

Cell Activity

Chromosome Activity

Description of Activity

i n t e r p h a s e

8 0 % - 1 0 0 % 18-24 hours

B e f o r e I n t e r p h a s e 2n

A f t e r I n t e r p h a s e 2n

Cell nucleus contains nuclear material. Centrioles are paired outside of the nuclear membrane. Cell organelles replicate in cytosol (cytoplasm). The homologous pairs of chromosomes (one originally from mother-M and one from father-F replicate exact copies of themselves called sister chromatids joined to other sister chromatid at centromere. This process happens for every cell chromosome. Through this mitotic process, we will only follow the chromosome 16 pair. Individual balls of the chromosomes represent gene locations.

Prophase

2n

Cell’s nuclear membrane temporarily dissipates. Nucleolus, if present, disappears. Centrioles move to opposite poles of cell. Chromatid pairs (containing two sister chromatids) shorten and thicken.

Prometa-

phase 2n

The mitotic spindle begins to form in the former region of the nucleus. Microtubules appear in the vicinity of each centriole, forming an aster. The centromere of each pair of chromatids captures a spindle fiber as the fibers build between the opposite centriole poles. At this stage, the number of cell chromosomes can be counted since the chromatid pairs are distinct.

Metaphase

2n

Nuclear membrane gone. Centrioles are attached by spindle fibers to centromeres of chromatid pairs, now located along the equator (metaphase plate) of the cell.

Anaphase

2n

Sister chromatids split at the centromere. Each is drawn to an opposite centriole by the attached spindle fiber. Once at the centriole, like chromatids such as chromosome 16 again join to form homologous chromosome pairs. Each centriole now has a full compliment of chromosomes, as did the original cell at the beginning of Interphase, above. Cytokinesis (cytoplasm division) begins.

Telophase

In the cell nucleus of one cell – 2n

In the cell nucleus of the other cell – 2n

Nuclear membrane forms around homologous chromosome pairs. Nucleolus reappears, if present originally. Chromosomes lengthen and become threadlike in the nucleus. Cytoplasm cleavage continues until separation into two distinct, but exact, replicates of the original cell, result.

Each cell enters Interphase and prepares for its own mitotic episode as the cycle of cell division continues. Go back to top for next cycle.

Figure 2: Life Cycle of a Typical Cell

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Experiment Mitosis and Meiosis

Questions A. What is the purpose of mitosis?

B. What other term is commonly used in place of “mitosis”?

C. What is a blastula?

D. What are the differences in the mitotic processes between animal and plant cells?

E. Why might there be more mitotic division in an onion root tip than in other areas of the plant?

F. A summary of mitosis – fill in the correct answer. The nucleus in the undivided cell has the diploid (2n) number of chromosomes, and the nuclei in the two split cells also have _______ number of chromosomes. In mitosis, the chromosome number (check the correct choice) ___ stays the same or __ halves.

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Experiment Mitosis and Meiosis

Exercise 2: Meiosis in Animals Meiosis – The Production of Gametes: Sex cells called gametes are produced through meiosis, also called gametogenesis3 (= production of gametes) in animals and plants. Fertilization, the combining of gametes, can result in the formation of an embryo and ultimately, a new organism that shares genetic traits from both parent organisms.

In animals, eggs and sperm are the gametes produced as a result of meiosis during sexual reproduction. In animals, gametogenesis occurs in specific organs. In the female, eggs are produced through oogenesis4 in the ovary. In the male, sperm are produced through spermatogenesis4 in the testes.

In plants, gametes take the form of pollen grains produced in the anthers that contain the sperm and the egg that is produced in the ovary of a flower. In angiosperms where seeds are borne within a fruit that developed from the flower ovary, the male and female reproductive organs may be on the same plant. Some animals also bear both male and female organs on the same organism as earthworms do. These animals are referred to as hermaphrodites.

Because fertilization in both plants and animals results from the combining of the egg and the sperm into a new cell, the number of chromosomes must be halved to n (haploid) during the course of gametogenesis. Meiosis is the process that accomplishes this halving, or splitting of the chromosome pair into n chromosomes, from the typical cellular 2n number of chromosomes.

Meiosis occurs in two distinct steps: meiosis I and meiosis II. During meiosis I the primary 2n gamete-to-be cell (whether a spermatocyte or oocyte) divides, and the chromosome number is halved, from 2n to n. Figure 3 provides a diagram of the meiotic process.

During meiosis II, the two secondary n spermatocytes divide again, producing four n sperm. However, in the case of the two secondary n cells produced in oogenesis, only one becomes an egg. The other cell forms the first polar body. After meiosis II an n egg and two polar bodies result. Although one would have expected three, the other polar body disintegrates or continues to divide to form other polar bodies. If the egg is fertilized, the polar bodies function to retain and build cytoplasm for the egg. It must be stressed that the egg does not complete meiosis II unless a sperm fertilizes it. If not fertilized, the egg passes out of the body (animals) or eventually disintegrates (plants).

Note that there are many species where the egg will develop into an organism having n number of chromosomes. This phenomenon is known as parthenogenesis. Examples are the drones or male workers in colonies of bees, ants, and some termites. There are also examples where, after development, organisms may change sex depending on the environment in which they find

3 The terms, meiosis and gametogenesis, are equivalent. We will use them interchangeably throughout this laboratory, so the student should be aware that the terms refer to the same process.

4 Oogenesis and spermatogenesis are similar processes. These names refer to meiosis and gamete production within an ovary and testis, respectively.

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Experiment Mitosis and Meiosis

themselves. However, we will leave discussion of this topic to a genetics course. The n egg cell and the n sperm cell join, and the two nuclei fuse, bringing the new cell to the diploid (2n) state of chromosome number. But one-half of the chromosomes came from the sperm, and the other half came from the egg. In humans, the egg and sperm each have n = 23 chromosomes, but the new zygote formed by their union has the 2n = 46 chromosomes found in humans.

In this exercise, we will examine the gamete producing structures in animals. Although we will not be observing the equivalent process in plants, the steps are essentially the same. Remember that ovaries are the site of oogenesis (egg production) in female animal organisms, while testes produce the sperm via spermatogenesis.

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Experiment Mitosis and Meiosis

Meiosis I: Homologous chromosomes synapse and separate in sex gonads. 2n = chromosome paired with homologue chromosome. n = chromosome not paired with homologue chromosome

Meiotic Phase Cell Activity Chromosome Activity Description of Activity

interphase

1 cell 2n

Cell nucleus contains nuclear material. A centriole replicates, paired outside. The cell organelles replicate in cytoplasm. A homologue chromosome pair joined by centromere.

Prophase i

1 cell 2n x 2

The nuclear membrane and nucleolus, if present, disappears. A spindle forms from centrosome as centrioles move to cell poles. Synapsis occurs so that each original chromosome homologue is doubled. This results in the chromosome pair attached together, but containing four chromatids. A chromosome pair is referred to as “bivalent” as each now is doubled.

Metaphase i

1 cell

2n x 2

All cellular chromosomes (bivalent forms) line up randomly on metaphase plate. There is a 50/50 chance that the future daughter cells will receive either inherited male or female parent chromatid. (Chromosomes 16 and 5 are shown for demonstration purposes.) Spindle fibers are captured by centromeres of bivalent chromosomes.

Anaphase i

1 cell 2n x 2

Homologous chromosome pair separates and move to poles, pulled by spindle fibers. Note in this example that Female parent chromatid pair 5 at one centriole has joined Male parent chromatid pair 16, and that Male parent chromatid pair 5 at other centriole has joined that Female parent chromatid pair 16.

Telophase i

2 cells

n each

Nuclear membrane, nucleolus reappears. Spindle disappears. Cell separates into two cells, each containing a pair of chromatids. However, each pair of chromatids is identical.

At end of Telophase i

2 cells n each

Figure 3: Meiosis: The Production of Gametes in Preparation for Sexual Reproduction

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Experiment Mitosis and Meiosis

Meiosis II: Chromatids separate

Meiotic Phase Cell Activity Chromosome Activity Description of Activity

Prophase ii

2 cells, each going through meiosis II n each

x 2 Cell 1 Cell 2

In each cell resulting from Meiosis I: Centriole replicates. The nuclear membrane and nucleolus, if present, disappear. A spindle forms as the centrioles move to cell poles.

Metaphase ii

2 cells, each going through meiosis II n each

x 2 Cell 1

Cell 2

Each chromatid pair lines up at metaphase plate.

Anaphase ii 2 cells, each going through meiosis II n

Cell 1

Cell 2

The centromeres divide. Chromatid pairs separate and move to opposite poles of spindle.

Telophase ii

4 cells n

x 2

Cell 1a Cell 2a The spindle disappears. Nuclear membrane and nucleolus reappear, the cell splits.

Cell 1b Cell 2b At end of Telophase ii

4 cells n

Four sperm cells oR one egg cell and three polar bodies at n chromosomes each

Figure 3: Meiosis (continued)

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Experiment Mitosis and Meiosis

procedure 1. Examine a prepared slide of an ovary under the microscope. Low power will provide a broader

field of view.

2. Identify the numerous primary follicles. These will appear as small round structures. Each contains a primary oocyte, egg cell (Greek `ōon = egg).

3. Find the larger secondary follicles. These structures contain the secondary oocyte, or egg, that has undergone meiosis I. When ovulation occurs, the egg is released from the ovary for movement down the fallopian tube. If sperm are present in the fallopian tube, fertilization and meiosis II may result.

4. Examine a prepared slide of a testis under the microscope. Low power will provide a broader range of view.

5. Identify the numerous seminiferous tubules that contain meiotically dividing cells.

6. Go to high power, locate one tubule, and observe the mature tailed sperm present in the tubule.

Questions A. Which organ, the ovary or the testes, contained the greater number of gametes?

B. What may be a reason for this?

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Experiment Mitosis and Meiosis

Exercise 3: Simulating Meiosis procedure Refer to Figure 3 while doing the following exercise. We will be simulating meiosis using pop beads to reinforce the sequence of events.

1. Interphase:

a. Assemble the chromosomes: Count out eight each of the red and yellow pop beads (homologous chromosome pair colors, each representing a chromatid). The pop beads represent genes on the chromosome; each is composed of a three-nucleotide sequence, for instance, A-T-T, A-G-C, etc.

b. Locate four plastic centromeres, the small tubes containing magnets.

c. Connect a centromere in the middle of four red pop beads. Make two of these identical “chromatids” with two red beads on each side of each centromere. Each string of beads, connected at the centromere, is a chromatid.

d. Connect a centromere in the middle of four yellow pop beads. Make two of these identical “chromatids” with two yellow beads on each side of each centromere. This will result in two yellow pop bead chromatids, connected at their centromere, to form the chromosome.

e. Interphase for the single chromosome pair in the cell nucleus is complete. The beginning chromosome pair was a red and yellow strand, connected by their centromeres. You have just duplicated the chromosome pair, in anticipation of coming meiosis.

2. Prophase I:

a. Form a “tetrad” or foursome. The original chromosome pair is connected to the duplicated chromatids. All are connected at their centromeres.

b. The nuclear membrane of the cell disintegrates and the centrioles on the outside of the nuclear membrane move to opposite sides (poles) of the meiotic cell. Simulate the centrioles by placing two 1” pieces of a straw about two feet apart on a flat surface. Anchor them in place with tape.

c. Spindle fibers begin to form. Cut two pieces of thread about 2 feet long. Run one piece of thread through each of the centrioles (straw pieces). Tie one end of the thread from one centriole to the red chromosome centromeres; then tie a thread end from the other centriole to the yellow chromosome centromeres. Temporarily separate the tetrad to tie in the spindle fibers, but then reattach the tetrad arrangement.

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Experiment Mitosis and Meiosis

3. Anaphase I:

a. Grasp the spindle fibers on the outside of the opposite centriole, and pull the chromosomes to the centrioles. The red chromosomes should pull to one centriole pole, and the yellow chromosomes to the other pole.

b. NOTE that the original homologous red-yellow pair of chromosomes has now been split, with half of each pair being pulled to the opposite pole. This demonstrates the principle of independent segregation: For each homologous pair of chromosomes, the homologues separate from each other.

4. Telophase I:

a. All spindle fibers disintegrate. Untie the centromeres.

b. Nuclear membranes form around each separate set of chromosomes. The centrioles will replicate and be found as a pair just outside of the nuclear membrane.

5. Cytokinesis: Animal cells divide by forming a cleavage furrow and pinching off. Plant cells would divide by forming a cell plate, and splitting. Two cells result.

Note: Meiosis is halfway completed. Each cell contains two chromosomes, either a red pair or a yellow pair, the original number. However, these two cells are genetically different from the original cell because they contain different homologues, or alleles, at each gene locus. Recall that the original pair was a red and yellow matched strand.

6. Prophase II, in each cell resulting from Cytokinesis above:

a. Nuclear membrane disintegrates, and centrioles move to the opposite sides of the cell. Continue this exercise with one of the two cells, remembering that there is a second cell undergoing exactly the same steps. Use the green and blue beads to simulate the second cell’s activities.

b. Tape down the centrioles in an area that represents the cell, with the centrioles located about two feet apart.

c. Spindle fibers begin to form. Thread “spindle fibers” through the centriole, one per centriole.

d. Place the two chromosomes, attached at their centromere, in the center of the cell, between the two centrioles.

7. Metaphase II:

a. Tie one thread from one centriole to the centromere of one of the chromosomes. Tie the other thread from the other centriole to the centromere of the other chromosome.

b. Reattach the two chromosomes at the centromeres.

8. Anaphase II: Hold the ends of the spindle fibers on the outside of the centrioles, and pull the respective chromosome to the centriole with the spindle fiber.

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Experiment Mitosis and Meiosis

9. Telophase II: Untie the spindle fibers, as they are now disintegrating.

10. Cytokinesis: Animal cells now divide by forming a cleavage furrow and pinching off. Plant cells would divide by forming a cell plate and splitting.

Meiosis II is now complete. There are a total of four cells. Each cell contains one chromosome or n number of chromosomes. This has occurred for the formation of both the egg and the sperm separately.

11. Simulate fertilization by joining two n cells. Note that a 2n cell results with a pair of chromosomes, one from each n cell. That is what we began with at Interphase for both mitosis and meiosis. At this point, mitosis will increase the number of cells and a zygote will be formed, followed by the embryo stage.

12. Simulate Crossing-over (chiasma), a method by which recombination of genetic material occurs in chromosomes during meiosis. This commonly happens during Prophase I when segments of a chromatid may exchange places with identical segments on another non-sister chromatid.

We can simulate this by exchanging identical sections of one of our red pop bead chromatids with an identical section of another yellow pop bead chromatid. Why would gene recombination not occur if we exchanged a red chromosome section with an identical red chromosome section?

Now that you have completed this lab, make sure you read the lab for next week. This will help you plan your time better. Take some time and highlight anything you will need to prepare in advance. As you read the lab, write out a hypothesis for each exercise.

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Experiment Mitosis and Meiosis

Questions A. What does mitosis accomplish?

B. When and where does mitosis occur?

C. What does meiosis accomplish?

D. When and where does meiosis occur?

E. How do these two processes differ?

F. How do Prophase in Mitosis and Prophase I in Meiosis differ?

G. Does Meiosis occur anywhere else in the body beside within the gonads?

H. How does meiosis confer advantages to an organism when compared to asexual reproduction such as budding?

I. How might crossing over be a benefit to an organism?

J. How might crossing over be detrimental to an organism?

K. Crossing over can also occur during mitosis where chromosomes exchange segments. Is crossing over more advantageous to organism survival if it occurs during mitosis or meiosis? Explain.

Laboratory summary What have you learned from doing this laboratory?

Mitosis and Meiosis Margaret Vorndam, M.S. Version 42-0094-00-01

Lab report Assistant This document is not meant to be a substitute for a formal laboratory report. The Lab Report Assistant is simply a summary of the experiment’s questions, diagrams if needed, and data tables that should be addressed in a formal lab report. The intent is to facilitate students’ writing of lab reports by providing this information in an editable file which can be sent to an instructor.

Observations

Exercise 1: Mitosis in Animal and Plant Cells procedure 1. Observe the prepared slide of the whitefish blastula under the microscope.

2. Draw cells that represent the various stages of mitosis: Interphase, Prophase, Metaphase, Anaphase, and Telophase. Place your drawings here:

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Experiment Mitosis and Meiosis

3. For each drawing, label the parts of the cells that you can identify such as the nuclear membrane, chromosomes, cellular membrane, cytoplasm, spindle fibers, chromatids, cleavage furrow formation during Cytokinesis, and centrioles. It is unlikely that you will find all of these structures. Why?

4. Observe the slide of the onion root tip under the microscope.

5. Draw cells that represent the various stages of mitosis: Interphase, Prophase, Metaphase, Anaphase, and Telophase. Place your drawings here:

6. Label the parts of the cells that you can identify such as the nuclear membrane, chromosomes, cellular membrane, cytoplasm, spindle fibers, chromatids, cell wall formation region, and centrioles. It it is unlikely that you will find all of these structures. Why?

results For both the whitefish and the onion, what is the 2n number of chromosomes that were observed? Hint: separate chromosomes are easiest to view and count during Prometaphase.

Questions A. What is the purpose of mitosis?

B. What other term is commonly used in place of “mitosis”?

C. What is a blastula?

D. What are the differences in the mitotic processes between animal and plant cells?

E. Why might there be more mitotic division in an onion root tip than in other areas of the plant?

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Experiment Mitosis and Meiosis

F. A summary of mitosis – fill in the correct answer. The nucleus in the undivided cell has the diploid (2n) number of chromosomes, and the nuclei in the two split cells also have _______ number of chromosomes. In mitosis, the chromosome number (check the correct choice) ___ stays the same or __ halves.

Exercise 2: Meiosis in Animals Questions A. Which organ, the ovary or the testes, contained the greater number of gametes?

B. What may be a reason for this?

Exercise 3: Simulating Meiosis Questions A. What does mitosis accomplish?

B. When and where does mitosis occur?

C. What does meiosis accomplish?

D. When and where does meiosis occur?

E. How do these two processes differ?

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Experiment Mitosis and Meiosis

F. How do Prophase in Mitosis and Prophase I in Meiosis differ?

G. Does Meiosis occur anywhere else in the body beside within the gonads?

H. How does meiosis confer advantages to an organism when compared to asexual reproduction such as budding?

I. How might crossing over be a benefit to an organism?

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Experiment Mitosis and Meiosis

J. How might crossing over be detrimental to an organism?

K. Crossing over can also occur during mitosis where chromosomes exchange segments. Is crossing over more advantageous to organism survival if it occurs during mitosis or meiosis? Explain.

Laboratory summary What have you learned from doing this laboratory?

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Experiment Mitosis and Meiosis

Genetic Inheritance Margaret E. Vorndam, M.S. Version 42-0061-00-01

Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.

Experiment Summary:

Students will explore classic Mendelian Genetics using tobacco mosaic seeds grown on paper towels to view F2 generation seedlings. Students will use Punnett squares to determine the genotype of the monohybrid F1 generation. They will further explore classic dominant/recessive traits using a Punnett square to calculate both the F1 and F2 generations of a dihybrid cross. They will use chi square analysis to determine the statistical reliability of the results.

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ExpErimEnt

Objectives ● To show examples of inheritance from the perspective of Mendel’s laws

● To describe common types of genetic expressions resulting from inheritance

● To use a Punnett square to determine expected genetic outcomes

● To do Chi square analysis of progeny results as a way to determine frequency of alleles in offspring

Time Allocation: 4-8 hours total. Tobacco seeds will take up to 10 days to grow.

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Experiment Genetic Inheritance

materials

MATERIALS FROM: QTy ITEM DESCRIPTION: Student Provides 1 Paper towel 1 Source of water

1 Warm location for the seeds, either in a warm window or under an incandescent lamp 1 Roll of aluminum foil

From labPaq 1 Pair of tweezers (may be found in dissection kit) 1 Magnifier, dual 1 Petri dish, 90 mm Auxiliary items Bag 2-BK-2A 2 Seed, Tobacco seeds - Grn/Alb 50 in Vial

Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq.

Safety Issues: Specifically review Glass, Heating, and Chemical Safety Procedures.

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Experiment Genetic Inheritance

Discussion and review Advanced Preparation Required for F2 Seedling Production

Perform at least ten days before seedling data is required in this laboratory.

1. Cut a piece of paper towel to fit inside a Petri dish.

2. Place the paper towel inside the Petri dish and moisten with water. Do not oversaturate the paper towel (water should not pool in the Petri dish); just moisten the paper towel and squeeze out excess water.

3. Sprinkle all of the seeds onto the paper towel and then cover the Petri dish with the Petri dish lid.

4. Place seeds near a warm window with indirect light or place seeds under a warm incandescent lamp (with the lamp turned on).

a. Lay a piece of aluminum foil over the top of the Petri dish if it is under the light bulb or in direct sunlight to protect it from the direct light. In order to let a little light into the Petri dish, just cover the top of the Petri dish and allow some light to filter into the sides of the Petri dish.

5. Observe seeds for germination each day, and moisten the paper towel as needed. Do not let the paper towel dry out. Also, only add enough water to make the paper towel moist, not oversaturated.

6. There will be two types of F2 seedlings, green and yellow to very pale green/white which are albino (chlorotic). If the Petri dishes are kept in filtered light conditions, the green seedlings should quickly develop chlorophyll (green color) after they germinate.

7. Count, classify, and record the color of the seedlings in a data table as they germinate, for the albino seedlings die quickly. Remove the counted seedlings with tweezers to minimize confusion about which seedlings have been counted.

8. Keep the Petri dish covered with the plastic lid and limit exposure to room air as much as possible to avoid contamination.

Note: It will take 5–10 days for the seeds in this exercise to germinate.

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Experiment Genetic Inheritance

Discussion and review

Chromosome DNA (deoxyribonucleic acid) and cellular RNA (ribonucleic acid) provide the underlying code that engineers the phenotype and genotype of all organisms. RNA is essential to bacterial and viral existence, but it has an important role in the development of other organisms as well. In this lab we will specifically concentrate on DNA and its role in genetic inheritance.

Gregor Mendel initiated the study of genetics in the 1800s and laid the foundation underlying the genetic knowledge of today. He was able, without technology, to formulate the laws of genetics. He had only phenotypic (phenotype = what the organism looks like) information to develop his understanding as to how genes interact to create traits in individuals. Despite the technological limitations of his day, he initiated the idea of dominant and recessive allelic forms of genes.

Mendel proposed that a trait could be present in an individual with a genotype (genotype = the allelic combinations that result in the phenotype of an organism) of two dominant (AA) genes or with a genotype of a dominant and a recessive (Aa) gene. He also proposed that an individual exhibiting a recessive phenotype had a genotype of two recessive alleles (aa). Ultimately, he formulated the three laws of Mendelian genetics: the Law of Segregation, the Law of Independent Assortment, and the Law of Dominance. Note: an allele is one of a pair of genes or of multiple forms of a gene.

● The Law of Segregation states that the sperm of the father and the egg of the mother each hold only a single allele for a trait of the two alleles that the mother or father inherited from their parents.

● The law of independent Assortment states that the alleles separate during meiosis, independently of one another. The resulting gametes, especially the male’s sperm since there are so many produced, represent all possible allelic makeups of the parents. Recall that in the female only one of four cells ultimately develops into an egg. If a female only produces one egg per cycle, as do human females, there will be less chance (50%) that a given allele may be represented.

● The law of Dominance states that recessive genes will be present phenotypically only if both inherited alleles are recessive.

Today, biological research is focused on mapping the genomes (gene sequencing on chromosomes) of thousands of organisms. In agriculture, genetic engineering is used to increase the durability, productivity, and nutritional value of crops. Stem cell research will potentially provide cures for many diseases. This new research may lead to the production of replacement organs and treatment of disorders such as diabetes and cancer. Genomic research may cause major revisions of plant and animal kingdom taxonomies as researchers are able to reevaluate organism connections at the genetic level. Until recently, taxonomy had to focus simply on the phenotypic relationships of organisms. Now, scientists are proposing to use a type of genetic “bar code” to classify all organisms based on association of gene sequences.

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Experiment Genetic Inheritance

This laboratory will explore how DNA determines cell development and consequently influences the formation of tissue, organs and, ultimately, the organism. We will explore: a monohybrid cross1 where one gene pair influences the manifestation of a trait; a dihybrid cross1 where two gene pairs influence the manifestation of a trait; and then explore the use of statistical analyses to test our hypotheses, as do genetic scientists.

For purposes of nomenclature, it is common for geneticists to capitalize the letter that represents the dominant gene. Also, the first cross of a completely dominant organism with a completely recessive organism is commonly referred to as the Parent, or P cross, which results in the production of F1 (hybrid) progeny. If F1 organisms are crossed, they are referred to as the F1 parents of F2 progeny, and so on. This is the nomenclature that will be used in this laboratory.

Typically, the genes located on chromosomes are too small to see with a compound light microscope. However, there are some organisms like fruit flies (Drosophila sp.) that produce multiple copies of chromosomes in their salivary glands. The salivary gland cells in the larval stages of Drosophila contain large, multi-stranded polytene chromosomes. Polytene chromosomes result from repeated replication of the chromosomes during mitotic synapsis, but these chromosomes do not separate to the daughter cells. Instead, they remain connected through several divisions. As part of this behavior by the chromosomes, genes undergo “puffing” as the DNA uncoils and begins RNA synthesis.

Puffing is visible as bands in the chromosomes and is associated directly with known gene loci activation. The banding that is seen on the chromosomes is a result of gene activation at these points. The great number of non-separated chromosomes, in addition to the puffing of specific genes, allows these chromosomes to be viewed under a compound light microscope.

Monohybrid Genetic Crosses: Crosses resulting in hybrid offspring have been used to control the genetic makeup of vegetable and animal offspring for years. Most vegetable seeds that are purchased through seed companies are produced as hybrids, so that the genetic makeup, and thus the appearance of the plant and fruit, will be uniform for the entire crop2.

1 The term, “cross,” refers to the joining (mating) of gametes from a sperm and an egg resulting in the production of an offspring. If the genetics of the contributing parents are known, it is possible to predict the genetics of the resulting offspring. 2 Genotype refers to the actual alleles that the organism has. Phenotype refers to how the genotype is manifested in the organism. For instance, in the example of the tobacco plants, the genotype might be either GG or Gg to produce a phenotype of green color.

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Experiment Genetic Inheritance

Hybrid F1 seeds are produced by crossing a parent species organism with all dominant genes for a characteristic with a like parent species organism with all recessive genes for the same characteristic. All offspring will inherit one dominant gene and one recessive gene from the genetic characteristics of the parents. Recall that Mendel’s Law of Segregation states that “each organism contains two alleles for each trait and the alleles separate during the formation of gametes. Each gamete then contains only one allele for each trait. When fertilization occurs, the new organism has two alleles for each trait, one from each parent.”

In tobacco plants the genetic allele which contributes to the development of photosynthetic pigment is dominant, represented by G3, for green color. If a parent with two dominant genes has a genotype of GG, what will its phenotype be?

The recessive form of this tobacco gene, represented by g, lacks photosynthetic pigments, and thus, the plant is phenotypically white, or albino (lacking any pigment). A parent with two recessive genes will have a genotype of gg. Phenotypically, the plant will be albino. When a completely dominant4 green tobacco plant parent is crossed with a completely recessive4 albino tobacco plant parent, a hybrid form that contains both a dominant gene for photosynthetic pigment, G, and a recessive gene for no pigment, g, is produced in the F1 generation.

What is the only possible genotype of the F1 hybrid progeny? What is its phenotype?

A statistical tool, the Punnett square, can be used as a simple predictor of the genotype and allelic (genetic) frequency of a cross. If the genotype of each parent is known, then a square can be constructed based on the Law of Independent Assortment. Recall that in each parent, the pair of chromosomes containing the genetic material is separated during meiosis, so the sex gametes only have one gene each.

The Punnett square is based on the number of possible alleles (genes) that each parent can pass on to its offspring. If the parental gametes each contain a gene, one must account for the gene in each gamete produced by meiosis. The top and left sides of the square each represent either an egg or a sperm, the parental gamete, with the possible gene that it can carry, and the center of the square shows the possible combinations of offspring genes resulting from fertilization, based on what the gamete genes are. Note that the symbols for male and female do not influence the outcome of the cross, but they are included to indicate that the cross is accomplished through sexual reproduction.

3 The assignment of letters to an allele is arbitrary. Capitalization indicates a dominant expression, and a lower case letter indicates a recessive expression of the allele. 4 The genes in a completely dominant organism are both dominant, i.e., GG. In a completely recessive organism, the genes will both be recessive, i.e., gg.

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Experiment Genetic Inheritance

Check your guess about the F1 generation progeny against the Punnett square rendition of this cross:

Punnett Square of a Hybrid Cross – Expected Genetic Outcomes

Note that this example represents a typical outcome for a completely dominant organism mated with (crossed to) a completely recessive organism. The offspring will always be hybrid, inheriting a dominant and recessive allele genetically, and exhibiting the dominant form phenotypically.

The frequency of the phenotype will be 100% Green, or 1.0, since the recessive gene, g, is masked by the dominant gene, G. The phenotypic frequency can be expressed either as a percent or a ratio. In this example, the phenotypic ratio will be expressed as 100% Green (G): 0% albino (g), or, if using a ratio, 1 Green: 0 albino. These are both correct as they refer to quantifying parts of the whole result. If there were 200 progeny produced from the cross, 200 x 1 would be green, and none (200 x 0) would be albino.

When two hybrid F1 Gg progeny resulting from the above cross are mated, the F2 progeny which result also exhibit a typical phenotypic ratio of dominant green to recessive albino plants. This next exercise demonstrates what happens when a monohybrid cross between two F1 hybrid progeny is done.

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Experiment Genetic Inheritance

Exercise 1: F1 Hybrid Cross Search Key Words: Genetic cross, genetic inheritance, dominant and recessive genes, allele, hybrid inheritance, Punnett square.

In this exercise, we will examine the F2 outcome of an F1 hybrid cross of Gg x Gg. As with a cross between a completely dominant parent and a completely recessive parent, the outcome of the hybrid cross is predictable. We will observe the phenotype of resulting offspring.

procedure 1. Construct and record a

hypothesis about what the phenotypic outcome will be.

2. Record the F2 seedling color as suggested in the Preparation Steps.

3. Given the information on the hybrid cross and that the F1 parents are both Gg genetically, in the Data Table 1 Results, complete the Punnett square for F1 cross that can be used to predict the F2 progeny genetic outcome of a cross between the two F1 parents.

Data Table 1: Punnett Square for F1 Cross – Expected Genetic Outcomes

F1 Parent, genes: _________ (student to fill in the blanks)

alleles > alleles v

F1 Parent, genes : __________ (student fill in blank)

4. Based on the outcome of the Punnett square, record the frequency of green:white plants that will result in the F2 progeny. Record the expected number of green and white individuals that would result if 320 offspring were produced from the cross.

5. Record the actual data obtained from growing the F2 tobacco seeds in Data Table 2. Indicate the frequency (ratio) of the seedling colors that were observed.

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Experiment Genetic Inheritance

Data Table 2: Results of F1 Cross observed Phenotypes of F2 Progeny

# green plants = # white plants = Total # plants Petri Dish 1 > Petri Dish 2 > Total

Exercise 2: Dihybrid Genetic Crosses Search Key Terms: Dihybrid genetic cross, Dr. Barbara McClintock, Punnett Square, F2 progeny, corn genome.

The genetics of corn have been completely studied, thanks to the work of scientist and Nobel Prize-winner Dr. Barbara McClintock. The corn genome and Dr. McClintock’s research can be viewed by searching on the World Wide Web. Dihybrid crosses in corn are a good example that illustrates Mendel’s Law of Independent Assortment:

“Members of an allelic pair separate independently of members of another allelic pair. Therefore, all possible combinations of alleles can occur in the gametes.”

Scientists accomplish crosses between plants with pollen produced by the male flower from a plant with desired genetic characteristics. The pollen is collected and deposited on the female flower that has other desired genetic characteristics. It will ultimately unite with the egg from which the seed that grows the progeny plant is produced.

In the case of corn, the corn tassel represents the male flower, and the cob, encased in a protective covering of leaves, houses the female ovaries in which the seed is produced. Corn silk catches the falling pollen grain and provides the conduit through which the deposited pollen sperm unites with the egg. The seed that is produced represents the genetic makeup of the next generation of plants.

In this exercise, we will follow two phenotypic traits in corn. The cobs that are produced from the fertilization of the eggs contain all possible offspring allelic combinations in the kernels on each cob for the next generation. Each kernel on the cob represents a single progeny containing unique allelic makeup that can be represented in the genetic makeup of plants grown from it.

An analogous example would be a human couple that bears 100 children over the course of their lifetimes, with all births compacted into one event. Each child is genetically unique based on the

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Experiment Genetic Inheritance

genotype of each parent, produced from a single egg and a single sperm, and is analogous to a kernel on the cob.

We will follow through the genetic inheritance of corn kernels/plants, beginning with a completely dominant and completely recessive parent generation cross, P. In corn, the dominant gene for kernel color is P, for purple. The recessive yellow color gene is designated by p. The smooth kernel gene, S, is dominant over the wrinkled kernel, s. Refer to Figure 1 for this exercise.

Figure 1: Dihybrid Cross in Corn

procedure 1. Based on what you can conclude about its genetic makeup when told that the corn plant

parent cross (P) pictured in Figure 1 is between a completely dominant plant and a completely recessive plant

a. Construct and record a hypothesis about what the genetic makeup and the frequencies of the alleles for the F1 progeny plants in the dihybrid cross of corn will be.

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Experiment Genetic Inheritance

b. If these F1 progeny are mated, what will be the resulting allelic frequency for the F2 progeny? Record this hypothesis.

2. Based on the phenotype and genotype information for the P cross from Figure 1, record the genotypes and Expected Frequencies for P and F1 in Data Table 3. Hint: One of the cobs contains all ppss kernels. Which one?

Data Table 3: Dihybrid Cross in Corn – Results of P Cross P = purple, p = yellow S = smooth, s = wrinkled (student to fill in all blanks _______ ) Generation Expected Alleles Expected Alleles P > dominant x recessive ppss

F1 Progeny >

Frequency >

3. Construct a Punnett square for the P cross producing the F1 progeny. What is the only possible genetic outcome for the F1 progeny? What is the phenotypic outcome?

4. Using Data Table 3A, indicate the possible genetic outcomes for the F2 progeny. Hint: Because the Law of Segregation and the Law of Independent Assortment regarding how the alleles separate are in operation, make sure to account for ALL possible parent allelic combinations in the Punnett square. Unlike monohybrid crosses where only two allelic combinations are possible, there may be more than two combinations that must be accounted for by increasing the number of rows and columns in the Punnett square. The frequency of the outcomes for the Punnett square will be the Expected Frequency outcomes for the progeny alleles. One may actually determine how many total progeny outcomes will result from the F1 cross by using the formula 2n, where n = total number of alleles under consideration. Referring back to the tobacco seed cross where two alleles, G and g, were examined, 2n = 4, so four possible progeny genetic outcomes will be identified via the Punnett square obtained in Data Table 3A.

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Experiment Genetic Inheritance

Data Table 3A: Punnett Square for F1 Dihybrid Cross Expected genotypic outcomes (student to fill in)

Parent 1 F1 – can produce these gametes: ________ (student to fill in)

Parent 2 F1 – can produce these gametes: ________>

Shaded portion above represents the F2 progeny genotype and phenotype. Student to fill in.

5. Using the Punnett square data, record the phenotypes and the possible genotypes for each phenotype in the first two columns of Data Table 4. Note that the bottom row is begun as an example. Calculate the Expected Frequency for each phenotype and enter it in the third column.

Data Table 4: Dihybrid Cross in Corn – Results of F1 Cross in F2 Progeny Phenotype of Progeny (What they look like – word description)

Genetic Designations possible for this Phenotype

e.g., PPSS

Predicted Allelic Frequency

(Expected Ratio)

Number of Phenotype Counted:

(observed Number out of 100 counted)

Actual Allelic Frequency

(observed Ratio)*

Ex: Yellow, wrinkled ppss, ppss 1÷16= 0.06 4 4÷100= 0.04

* Actual Allelic Frequency (Observed Ratio) = Number of Phenotype Counted ÷ 100 total kernels counted

6. AFTER entering your Expected values in Data Table 4, count 100 of the kernels in the F2 progeny cob of Figure 1. Differentiate phenotypes as you count. Tabulate the results in the “Number of this Phenotype Total Counted (Observed)” column by your observed phenotype.

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7. Determine the frequency for the actual data, and record that Observed Frequency in the last column.

Questions A. How well do the predicted results match the actual results in Table 4?

B. Based on the Punnett Square predictions, can a statement be made as to whether your hypotheses are supported or rejected? Which and why?

C. Dihybrid F1 crosses result in a predictable F2 progeny phenotypic frequency that holds true universally. Based on the Expected outcome, what is it?

D. If your results are not as expected why might there be differences?

E. What applications might this type of genetic investigation have? How might the information be applied medically?

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Exercise 3: Chi-square and Hypothesis Testing Search Key Words: Chi square, goodness of fit, sex-linked gene, epistasis, incomplete dominance.

Besides using a Punnett square, we can statistically test an inheritance hypothesis for the expected results of these crosses and compare the results that we observed to the Expected ratio. We use a statistical tool, Chi square, commonly written as c2, to test Goodness of Fit of Observed data to Expected (hypothesized) data results when we do this analysis. Chi square is used in this instance to see if a genetic cross produces data that fits a predicted (“hypothesized”) ratio (frequency). It can definitely indicate how much the Observed numbers of corn phenotype individual counts vary from the Expected numbers of corn traits individual counts and gives us a quantitative measure of how closely our data match.

The formula used to calculate Chi square is:

c2 = Σ d 2

E where d is the deviation from the Expected result, and where e is the Expected result

procedure 1. Use the data recorded in Data Table 4 to complete Data Table 5.

Data Table 5: Χ2 Goodness of Fit Test for F2 Phenotypic Results from F1 Corn Cross

Phenotype Description of F2 Progeny from Table 4

observed Number from Table 4

observed Ratio from Table 4

Expected Ratio from Table 4

* Expected Number, calculated

** [observed No. – Exp. No.]2 ÷ Expected No.

e.g., Yellow, wrinkled

Σ Sum of column =

c2, Chi-square value *** >

* Expected Number, calculated = Σ Sum of Observed Number x Expected Ratio for that phenotype ** = (Observed number – Expected number, calculated) square ÷ Expected Number, calculated

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*** c2, Chi-square value = Σ Sum of (Observed number – Expected number, calculated) squared ÷ Expected Number, calculated

2. You will be working with statistics in this exercise make sure you refer to the Appendix for more information.

3. Add the figures in the Observed Number column and enter this sum below the column, to the right of Σ Sum of column =.

4. Multiply the Σ Sum of the Observed Number by the Expected Ratio in each row to determine the Expected Number, calculated, for each Phenotype. Record this number in the Expected Number, calculated column.

5. Calculate the results for the right-most column by using the equation provided below the table.

6. Add these results, and record the sum in the right-most bottom cell. Note that this figure represents the c2 value for your results.

7. To determine the probability that your actual results are close to your expected results, you must determine the Degrees of Freedom for your test. By definition, Degrees of Freedom = number of phenotypes (technically, treatments) – 1. The number of phenotypes that you have determined for this cross is 4, so the Degrees of Freedom equal 3 = (4 – 1).

8. Then, using the c2, Chi square value that you calculated, and with 3 Degrees of Freedom, look up the probability for the fit of your observed vs. expected results in the following c2 Table of Probabilities.

9. For purposes of determining the Goodness of Fit, a Probability of 1 (=100%) is a perfect fit where the Observed results are totally accurate. If, instead, the Goodness of Fit is zero, there is no agreement at all (0%). So, scientific methodology relies on the probability that the data is close enough. This is how the determination of Goodness of Fit shows if you have hypothesized well. Note that we will leave it to your Statistics class to explain the intricacies of using the c2 Goodness of Fit Test.

10. Use Table 6 to summarize your results.

c2 Table of Probabilities

good Fit Probability Poor Fit Probability Degrees of Freedom .90 .70 .60 .50 .20 .10 .05 .01

1 .02 .15 .31 .46 1.64 2.71 3.85 6.64 2 .21 .71 1.05 1.39 3.22 4.60 5.99 9.21 3 .58 1.42 1.85 2.37 4.64 6.25 7.82 11.34

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Experiment Genetic Inheritance

4 1.06 2.20 2.78 3.36 5.99 7.78 9.49 13.28

Now that you have completed this lab make sure you read the lab for next week. This will help you plan your time better. Take some time and highlight anything you will need to prepare in advance. As you read the lab write out a hypothesis for each exercise.

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Questions A. What can be concluded about your prediction of expected F2 progeny phenotypic outcome from the F1 cross? Was it close to the observed outcome?

B. How might the c2 test for fitness be used in other ways? Try it on the tobacco seedling F1 cross, for instance.

C. In a typical cross where a parent with a completely dominant trait is mated with a parent exhibiting a completely recessive trait, what is the expected genotypic outcome and allelic frequency for the F1 progeny?

Give an example.

D. In a typical cross where hybrid F1 parents are mated, what is the expected genotypic outcome and allelic frequency of the F2 progeny?

Give an example.

E. For the F2 progeny produced from a typical F1 hybrid mating, how many totally recessive individuals would be produced if the progeny total population is six offspring?

What if the progeny population was 20?

50?

1,000?

F. Excluding factors such as sex-linked genes, incomplete dominance or epistasis, etc., will the above cross results vary if different organisms are used, such as dogs or tulips? Why or why not?

G. How will factors such as sex-linked genes, incomplete dominance, or epistasis, etc., affect the expected outcomes that were investigated above?

Genetic Inheritance Margaret E. Vorndam, M.S. Version 42-0061-00-01

Lab report Assistant This document is not meant to be a substitute for a formal laboratory report. The Lab Report Assistant is simply a summary of the experiment’s questions, diagrams if needed, and data tables that should be addressed in a formal lab report. The intent is to facilitate students’ writing of lab reports by providing this information in an editable file which can be sent to an instructor.

Data Table 1: Punnett Square for F1 Cross – Expected Genetic Outcomes

F1 Parent, genes: _________ (student to fill in the blanks)

alleles > alleles v

F1Parent, genes:__________ (student fill in blank)

Data Table 2: Results of F1 Cross observed Phenotypes of F2 Progeny

# green plants = # white plants = Total # plants Petri Dish 1 > Petri Dish 2 > Total

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Experiment Genetic Inheritance

Data Table 3: Dihybrid Cross in Corn – Results of P Cross P = purple, p = yellow S = smooth, s = wrinkled (student to fill in all blanks _______ ) Generation Expected Alleles Expected Alleles P > dominant x recessive ppss

F1 Progeny >

Frequency >

Data Table 3A: Punnett Square for F1 Dihybrid Cross Expected genotypic outcomes (student to fill in)

Parent 1 F1 – can produce these gametes: ________ (student to fill in)

Parent 2 F1 – can produce these gametes: ________>

Shaded portion above represents the F2 progeny genotype and phenotype. Student to fill in.

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Data Table 4: Dihybrid Cross in Corn – Results of F1 Cross in F2 Progeny Phenotype of Progeny (What they look like – word description)

Genetic Designations possible for this Phenotype

e.g., PPSS

Predicted Allelic Frequency

(Expected Ratio)

Number of Phenotype Counted:

(observed Number out of 100 counted)

Actual Allelic Frequency

(observed Ratio)*

Ex: Yellow, wrinkled ppss, ppss 1÷16= 0.06 4 4÷100= 0.04

* Actual Allelic Frequency (Observed Ratio) = Number of Phenotype Counted ÷ 100 total kernels counted

Data Table 5: Χ2 Goodness of Fit Test for F2 Phenotypic Results from F1 Corn Cross

Phenotype Description of F2 Progeny from Table 4

observed Number from Table 4

observed Ratio from Table 4

Expected Ratio from Table 4

* Expected Number, calculated

** [observed No. – Exp. No.]2 ÷ Expected No.

e.g., Yellow, wrinkled

Σ Sum of column =

c2, Chi-square value *** >

* Expected Number, calculated = Σ Sum of Observed Number x Expected Ratio for that phenotype ** = (Observed number – Expected number, calculated) square ÷ Expected Number, calculated *** c2, Chi-square value = Σ Sum of (Observed number – Expected number, calculated) squared ÷ Expected Number, calculated

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Experiment Genetic Inheritance

Data Table 6: Summarization of c2 Good Fit Results for F1 Corn Cross c2 value from Table 5 =

Value at 3 Degrees of Freedom that is closest to c2 value =

What is the Fit Probability at the top of the column in which the value was found? What is the % of probability that the observed results match the expected results? (Multiply Fit Probability by 100)

Reading at the top of the Table, this Fit Probability indicates that the expected results hypothesis is a

good Fit Poor Fit

Circle the correct choice above

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Experiment Genetic Inheritance

Exercise 1: F1 Hybrid Cross A. Expected phenotypic ratio of green to white progeny: Calculation of Expected Ratio (Frequency) = = Total Number of (Color) Seedlings ÷ Total of All Seedlings.

B. If 320 F2 offspring resulted from this F1 cross, how many would be green?

White?

Discussion A. Did the results support or refute the hypothesis? Explain.

B. How similar are the observed to the expected results from the Punnett square?

C. If the results are not similar, how might the difference be explained?

D. Will a monohybrid F1 cross in corn yield the same ratio of expected phenotype in progeny as for the tobacco seedlings? Why or why not?

E. If available, compare your F2 seedling data to those of your classmates. Are the outcome ratios the same? Why might using a larger number of seedlings to determine this outcome be wise?

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Exercise 2: Dihybrid Genetic Crosses procedure 1. Based on what you can conclude about its genetic makeup when told that the corn plant

parent cross (P) pictured in Figure 2 is between a completely dominant plant and a completely recessive plant,

a. Construct and record a hypothesis about what the genetic makeup and the frequencies of the alleles for the F1 progeny plants in the dihybrid cross of corn will be. Record your hypothesis here:

b. If these F1 progeny are mated, what will be the resulting allelic frequency for the F2 progeny? Record this hypothesis here:

results A. What are the two hypotheses that you made about the allelic frequencies of progeny produced by the crosses:

P x P?

F1 x F1?

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B. Based on what you know about phenotypes and Figure 2, for the P generation, what is the corn plant genotype on each cob containing the P corn kernels? One is completely dominant, so its genotype is

One is completely recessive, so its genotype is .

C. Would it make a difference in the outcome of this cross if the genotype of one parent is PPss and the other is ppSS?

D. From the phenotype of the kernels on each P generation cob what would the predicted genotype of any F1 plant be?

E. Given the 2n equation predict how many different genetic outcomes will be possible from an F1 cross resulting in the F2 generation in a dihybrid corn cross.

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F. If a F2 corn cob resulting from this F1 cross contained 563 seeds, how many of the seeds would you expect to look like the F1 parent?

Questions A. How well do the predicted results match the actual results in Table 4?

B. Based on the Punnett Square predictions, can a statement be made as to whether your hypotheses are supported or rejected? Which and why?

C. Dihybrid F1 crosses result in a predictable F2 progeny phenotypic frequency that holds true universally. Based on the Expected outcome, what is it?

D. If your results are not as expected why might there be differences?

E. What applications might this type of genetic investigation have? How might the information be applied medically?

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Exercise 3: Chi-square and Hypothesis Testing Questions A. What can be concluded about your prediction of expected F2 progeny phenotypic outcome from the F1 cross? Was it close to the observed outcome?

B. How might the c2 test for fitness be used in other ways? Try it on the tobacco seedling F1 cross, for instance.

C. In a typical cross where a parent with a completely dominant trait is mated with a parent exhibiting a completely recessive trait, what is the expected genotypic outcome and allelic frequency for the F1 progeny?

Give an example.

D. In a typical cross where hybrid F1 parents are mated, what is the expected genotypic outcome and allelic frequency of the F2 progeny?

Give an example.

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E. For the F2 progeny produced from a typical F1 hybrid mating, how many totally recessive individuals would be produced if the progeny total population is six offspring?

What if the progeny population was 20?

50?

1,000?

F. Excluding factors such as sex-linked genes, incomplete dominance or epistasis, etc., will the above cross results vary if different organisms are used, such as dogs or tulips? Why or why not?

G. How will factors such as sex-linked genes, incomplete dominance, or epistasis, etc., affect the expected outcomes that were investigated above?

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Experiment Genetic Inheritance

Human Genetics Margaret E. Vorndam, M.S. Version 42-0068-00-01

Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.

Experiment Summary:

Students will use common traits such as attached ear lobes and tongue rolling to analyze inheritance patterns. Students will learn about chromosomes research a human genetic disease.

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ExpErimEnt

Objectives ● To explain what commonly recognized traits are present in humans

● To develop simple genetic maps/charts

● To outline the causes of genetic disorders

● To do a simple research information search

Time Allocation: Four hours total. Exercises may be done at different times.

Safety issues: None

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Experiment Human Genetics

materials

MATERIALS FROM: QTy ITEM DESCRIPTION: Student Provides 14 Volunteers

Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq.

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Experiment Human Genetics

Discussion and review Introduction - Personal Inheritance: Science has profited tremendously from the Age of Technology. Each day, new breakthroughs in knowledge occur within the scientific realm. In genetics, a sub- field of biology, organism genetic mapping codes are completed, and gene splicing, cloning, and genetic engineering (GE) are discussed internationally, while governments attempt to regulate to what extent GE will be permitted locally. Dr. Susan Lindquist, a noted scientist and Director of the Whitehead Institute of Biomedical Research, recently said, “Biology is no longer a descriptive science. Rather, it is a science where you really try to understand on a molecular level how life’s processes work—and that’s truly an extraordinary problem.” (Biomaterial World, Fall 2002). Biology now aggressively focuses on the dictation and interpretation of the DNA and RNA codes that design life. Today, terms such as Genomics and the synonymous term “Bioinformatics” light up the screen of biological research. Agricultural companies collaborate to produce bioengineered crops that may hold answers to increasing the production necessary to feed a growing world population, or to boost the nutrition of existing crops. Medical science is accelerating its search for cellular level course corrections for inherited and acquired diseases that affect health. And potential pandemic virus flu warfare is concentrated on research at the cellular level. In this lab, we will focus on the human population as an example of what is currently known about genetic query. But, be aware that the same types of inquiries into knowledge relating to human biology are ongoing for thousands of other species across the world. Eventually, the entire biological classification system will be redesigned based on genetic similarities rather than on phenotypic similarities as it is now. Scientists are already anticipating that life on other planets will be integrated into the classification system that humans have developed, and are discussing how that system will be structured.

All human beings are classified as genus and species/subspecies, Homo sapiens sapiens, the wise Man. Although we exist in different colors, shapes, and sizes. We are all similar enough in our genes that we belong together as a single group within the Linnaean Taxonomic Classification. Our taxonomic “address” is:

Kingdom Phylum Class order Family genus Species Animalia Chordata Mammalia Primates Hominidae Homo sapiens

Our personal phenotype is the result of the expression of thousands of alleles that we received when the sperm and egg from our parents joined. These alleles may not have even begun to operate at the time that we were conceived, but were programmed by other alleles to “turn on” at different times during our life. And so we grow, go through puberty, have children, and age. Ultimately, the process of apoptosis, programmed cell death, also dictated by genetics and helped along by our personal lifestyle choices, will exceed our body’s capability to regenerate itself, and we will be no more. Until then, however, we have much to learn and much living to do. So, let us proceed.

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Each allele contains the genes from one parent, so the paired alleles are not necessarily equivalent. Each allele is a gene for a trait, but it is the combination of the genes, one from each parent, that determines the phenotype of the child. So, if a child’s father has attached earlobes, which is phenotypically recessive (ff), he will pass on a gene for attached earlobes, f, to the union that produced that child. If the mother has unattached earlobes, a phenotypically dominant condition, her genetic makeup for ear lobes can be either FF or Ff. Why? Then came the child. Depending on which gene its mother passed to the union in the egg, it may have inherited attached ear lobes (ff) or unattached earlobes (Ff). If it has attached earlobes, it got the other recessive f gene from the mother, since the father obviously contributed an f recessive gene.

Each of us inherits our physical traits from our parents. Many traits have been identified that are single-gene dependent, but some are sex-linked, like male pattern baldness and male or female body form. Many phenotypes are dependent on expression of multiple alleles (see, for instance, definitions incomplete dominance or epistasis online). In the following exercise, we will investigate a variety of single-gene dependent similarities and differences between ourselves and a population of friends and acquaintances. You will map the findings on a genetic traits chart, and answer questions about the findings.

Then, we will further investigate how genetics can influence our well-being. While viruses, intestinal parasites, bacteria, and so on are part of our existence, humans also are prone to many diseases that result from genetic abnormalities. In today’s biological research, many of the interrelationships between genetics and disease are being established. What might be the result? In the second exercise, you will investigate one genetically facilitated disease in-depth and discover that for yourself.

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Exercise 1: Genetic Traits Chart Search Key Terms: Genetic inheritance, genetics.

Trait Description

Sex Male (XY) or female (XX) – the birth sex of the subject

Ear lobe Unattached ear lobes are “floppy” and lobe attaches to head above the end of the lobe (FF, Ff). Attached ear lobes connect to end of lobe (ff).

FF, Ff ff

To n g u e Rolling

Tongue can be rolled into a U-shape when extended from mouth (RR, Rr). Non-rollers cannot roll tongue into U-shape on extension (rr).

RR, Rr rr

H i t c h - h i k e r ’ s Thumb

Thumbs with end joints that can be bent back at 45° are recessive (ss). Thumbs that cannot bend back significantly are dominant (SS, Ss).

SS, Ss ss

H a i r y Fingers

Hair on any middle finger digit is dominant (MM, Mm). No hair on fingers is recessive (mm).

MM, Mm m m

T h u m b on Top

Upon clasping hands, left thumb on top is dominant (TT, Tt). Right thumb on top is recessive (tt).

TT, Tt tt

P a l m a r Tendons

Bend back hand to expose wrist tendons. Feel wrist to count tendons. Two wrist tendons on both wrists is a dominant (LL, Ll) trait. Three wrist tendons on either wrist is a recessive (ll) trait.

LL, Ll

l l

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Experiment Human Genetics

procedure 1. Formulate a hypothesis about whether a population of friends, family, and/or acquaintances

is similar or different to you in appearance. Obviously, the population that is queried may guide this hypothesis.

2. Choose at least 14 people, including yourself, as subjects. Using the following Trait Description checklist, gather data about each individual, and mark the results on Data Table 1. Note that the first person’s data has been added as an example of how to record the data.

3. After you have queried 14 people, transfer the table results to the genetic traits chart, Figure 1. Begin in the center and work from the center outward. Mark the outer circle at the point that the last allele is found. Note that the first entry is completed for you. In the chart, capital letters equate to a dominant and hybrid gene pair (FF and Ff). The lower case letters equate to a totally recessive gene pair (ff).

Data Table 1: Single-Gene Traits of a Selected Population Subject # > 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Total

XX Female XX

Xy Male

FF,Ff = unattached

ff = attached ff

RR,Rr = roller R

rr = can’t roll

SS,Ss = straight thumb

ss = bent thumb ss

MM,Mm = hair

mm = no hair mm

TT,Tt = left on top T

tt = right on top

LL,Ll = two tendons l

ll = three tendons

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Figure 1: Genetic Traits Chart

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Questions A. What observations can be made about the collected and charted data?

B. Are any two people exactly the same? If so, how closely are they related?

C. Is your hypothesis supported by the findings? What statistical tool might help you to make this decision?

D. In the totals that you recorded in Table 1, did you find that the number of dominant phenotypes were greater than, equal to, or less than the recessive phenotypes?

E. Can you explain what possible genotypes are suggested by this pattern? Does this vary by trait?

F. Would you expect this outcome? Why or why not?

G. How would changing the level of the mapped traits affect the chart? For instance, if the alleles for male or female were on the outer rim of the chart, rather than in the middle, how would the pattern observed differ from Figure 10-1?

H. List several other traits that humans have that are governed by genetics.

I. Would your data vary if you went to another part of the world, for instance, China?

J. Why might collected data show different patterns for different cultural populations?

K. Would you expect that the pattern would be different if collected 100 years ago instead of today? Why or why not?

L. If you had percentages of dominant and recessive genes present for the total number of alleles of a trait for the entire world, would you expect that percentage to change over 100 years? Why or why not?

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Exercise 2: Human Chromosomes and the Human genome Project Search Key Terms: Human genome project, genetic mapping, human genetics, geneticist.

Humans have 23 chromosome pairs in all cells (2n). The only exceptions are the reproductive gamete cells of eggs, polar bodies, and sperm where meiosis has separated the chromosomes (n), in preparation for union during mating. “n” represents the number of single chromosomes that each organism has. Twenty-two human chromosomes are paired analogs (2n = 44); one of each pair was inherited from the mother and one from the father. The last chromosome pair (XX or XY) is composed of the two chromosomes that dictate the sex of the child. This next diagram shows what human chromosomes look like graphically. Since the diagram represents the haploid rendition (n), in real cells one of the chromosomes for sex, labeled X or Y, would not be present. It is here for comparative purposes. The dark bands represent locations in each chromosome that will hold Giemsa dye. Geneticists use the dye as a way to map the chromosome, and locate specific genes on each of the chromosomes.

If these chromosomes were from an actual cell nucleus in the body, they would be double (2n) with the second chromosome of the pair attached at the narrow point of each above chromosome, where the centromere would be located. In the case of the X and Y chromosomes, cells of female humans would have two X chromosomes joined at the narrow centromere location, and cells of male humans would have a long X chromosome and a short Y chromosome attached at that junction. Thus, we can see that the human father always determines the sex of the child produced from a union, since only males have the Y chromosome to pass on during a sexual union. An actual photograph of dyed human chromosomes from a body cell (not a gamete) looks like the lower right image. Note how the bands match the graphic image above.

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Most of what we know about human chromosomes and their genetic content has been discovered over the past twenty years during the exploration to map the human genome. The Human Genome Project was substantially completed in 2000 and published in Nature in February 2001. This massive effort resulted in the identification of nearly every genetic locus on every chromosome in the human body. Why is this important? Because we can now use the genome “map” to conclusively determine if an individual has a genetic abnormality. In the future, cell cloning and chromosomal material replacement may hold promise as a way to correct the more adverse genetic abnormalities.

Some abnormalities in human genetic makeup result in typical physical appearance patterns or metabolic disorders. The ones that are “constitutional” are those that are present at birth, such as Trisomy 21 which is Down syndrome and caused by the presence of three chromosomes #21s in body cells; Klinefelter’s syndrome; DiGeorge syndrome; and XYY. Other abnormalities are “acquired” during life due to the action of viruses and/or cancer on chromosomes. Chronic myelogenous leukemia (CML) is an example where the translocation of parts of chromosomes 9 and 22 occur and then are perpetuated by division within the body.

procedure 1. Select one human genetic disorder from the list bellow. This list is not exhaustive and you may

choose another genetic disease if you wish.

2. Perform online research on this disorder to determine:

a. The origin of the disorder (what chromosome(s) and gene(s) are responsible for the disorder). Images documenting the location of the gene(s) are encouraged

b. The mode of action of the gene(s) to cause the disorder

c. The timeline for the disorder to present itself

d. The presentation issues of the disorder - what it looks like, or what it does to the person

e. The propensity for a specific population or culture of people to be affected, more so than other populations or cultures, and the rationale for this

f. The percentage occurrence in births per 1000 for the specific population and world-wide occurrence

g. What principle organizations are researching or providing support for those affected by the disorder (at least four)

h. What progress each of these organizations has made in addressing the disorder or peripheral issues

i. Whether it is a constitutional or acquired genetic disease

j. Treatment - present and potentially future

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List of Genetically Mediated Diseases in Humans

Abetalipoproteinemia Albinism Allergies Alzheimer disease Amyotrophic lateral sclerosis (ALS) Anemia Autism Batten disease Bloom’s syndrome Breast cancer Bronchiectasis Burkitt lymphoma Canavan disease Celiac disease Cleft palate/Cleft lip Cohen’s syndrome Colon cancer Color blindness Congenital adrenal hyperplasia Crohn’s disease Cystic fibrosis Depression DiGeorge syndrome Down syndrome Duchenne muscular dystrophy Essential pentosuria Fabry disease Factor XI deficiency Familial dysautonomia Familial Mediterranean fever

Fragile X syndrome Galactosemia Gaucher disease Hemochromatosis Hemophilia A Homocystinuria Huntington disease Juvenile-onset diabetes Lupus Malignant melanoma Marfan syndrome Mental retardation Myotonic dystrophy Niemann-Pick A & B diseases Obesity Parkinson’s disease Phenylketonuria Polycystic kidney disease Rheumatoid arthritis Schindler disease Severe combined immunodeficiency Sickle cell disease Small cell lung carcinoma Spina bifida Tay-Sachs disease Testis-determining factor Torsion dystonia Type C disease Ulcerative colitis X-linked mental retardation Zellweger syndrome

3. Write up a report on your findings. Include correctly formatted references to all online pages that you consult to gather your information.

Questions A. What is the purpose of this exercise?

B. What is the prognosis on genetic diseases in the future? Do you believe that microsurgery to “fix” chromosomal aberrations will some day prevent the diseases that are listed in the List of Genetically Mediated Diseases in Humans?

Human Genetics Margaret E. Vorndam, M.S. Version 42-0068-00-01

Lab report Assistant This document is not meant to be a substitute for a formal laboratory report. The Lab Report Assistant is simply a summary of the experiment’s questions, diagrams if needed, and data tables that should be addressed in a formal lab report. The intent is to facilitate students’ writing of lab reports by providing this information in an editable file which can be sent to an instructor.

Observations Data Table 2: Single-Gene Traits of a Selected Population

Subject # > 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Total

XX Female XX

Xy Male

FF,Ff = unattached

ff = attached ff

RR,Rr = roller R

rr = can’t roll

SS,Ss = straight thumb

ss = bent thumb ss

MM,Mm = hair

mm = no hair

TT,Tt = left on top T

tt = right on top

LL,Ll = two tendons l

ll = three tendons

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Experiment Human Genetics

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Experiment Human Genetics

Exercise 1: Patterns in Human Inheritance Traits Questions A. What observations can be made about the collected and charted data?

B. Are any two people exactly the same? If so, how closely are they related?

C. Is your hypothesis supported by the findings? What statistical tool might help you to make this decision?

D. In the totals that you recorded in Table 1, did you find that the number of dominant phenotypes were greater than, equal to, or less than the recessive phenotypes?

E. Can you explain what possible genotypes are suggested by this pattern? Does this vary by trait?

F. Would you expect this outcome? Why or why not?

G. How would changing the level of the mapped traits affect the chart? For instance, if the alleles for male or female were on the outer rim of the chart, rather than in the middle, how would the pattern observed differ from Figure 10-1?

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Experiment Human Genetics

H. List several other traits that humans have that are governed by genetics.

I. Would your data vary if you went to another part of the world, for instance, China?

J. Why might collected data show different patterns for different cultural populations?

K. Would you expect that the pattern would be different if collected 100 years ago instead of today? Why or why not?

L. If you had percentages of dominant and recessive genes present for the total number of alleles of a trait for the entire world, would you expect that percentage to change over 100 years? Why or why not?

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Experiment Human Genetics

Exercise 2: Human Chromosomes and the Human genome Project results Report on your findings. Your report should be limited to a page in length and may incorporate the outline of research points above.

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Experiment Human Genetics

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Experiment Human Genetics

Questions A. What is the purpose of this exercise?

B. What is the prognosis on genetic diseases in the future? Do you believe that microsurgery to “fix” chromosomal aberrations will some day prevent the diseases that are listed in the List of Genetically Mediated Diseases in Humans?

Laboratory summary What have you learned from doing this laboratory?

references

Copyright © 2008 by Margaret E. Vorndam, 1413 County Road 671, Rye, CO 81069

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Experiment Human Genetics

The Properties of Water Margaret E. Vorndam, M.S. Version 42-0129-00-01

Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.

Experiment Summary:

Students will learn about the molecular structure of water and how this structure contributes to water’s unique properties. Students will demonstrate how surface tension allows insects to walk on water and how adhesive properties allow water to move up the stem of a plant. They will compare the densities of solid and liquid water to solid and liquid oil. Students will explore specific heat capacity by measuring the time it takes for both solid oil and ice to absorb heat from the surroundings and undergo a phase change.

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ExpErimEnt

Objectives To explain the unique properties of water:

● Cohesion, surface tension, and tensile strength

● Adhesion and capillary action

● Density

● High specific heat and high heat of vaporization

● Solvent ability

Also:

● To illustrate a situation where the properties of water apply

● To critically review an experiment and explain important variables

● To construct and interpret scientific data graphs

Time Allocation: Six hours

Safety Issues: Glass breakage

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Experiment The Properties of Water

materials

MATERIALS: QTy ITEM DESCRIPTION:

Student Provides 1 Bowl, wide and shallow 1 Dishwashing detergent 1 Food Coloring, blue or green 1 Funnel 1 Olive oil 1 Paper clips, several metal of various sizes 1 Pliers 1 Refrigerator with freezer 1 Strainer 1 Timer 1 Toothpicks 1 Water, distilled 1 Glass jar 1 Small cup or dish 1 Rubber band

From LabPaq 2 Beaker, 50 mL, plastic 1 Cylinder, 50 mL - Graduated, Plastic

1 Dissection-kit with 7-tools - including the following: Bent Probe, Dropping Pipet, Probe, Ruler in pocket, Scalpel with 2 Blades - Note blades are in the pocket, Scissors, Tweezers

1 Pencil, marking 1 Digital scale 1 Test Tube(1), 25 x 150 mm in Bubble Bag 1 Test-tube-rack-12x13-mm 1 Thermometer-in-cardboard-tube

Capillary tubes 1 Capillary tubes (FRAGILE - 3 sizes, taped to cardboard)

Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq.

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Experiment The Properties of Water

Discussion and review Even though the atoms of the water molecule, oxygen and hydrogen, share their electrons in covalent bonds, the water molecule is actually a polar molecule. This is due to the electronegative nature of the oxygen atom relative to the hydrogen atom. This state of the water molecule creates partial negative and partial positive charge regions on the molecule, along with a unique shape, somewhat akin to a U-shaped magnet. This results in a number of very special properties for water, as it exists in the biological world: cohesion, adhesion, surface tension, capillary action, high specific heat, high heat of vaporization1, low density of ice, and water’s solvent ability2. Since water makes up a large portion of all living beings (for instance, humans are ~ 60% water) it is important to know how its properties influence life.

Background

Atoms are small particles that make up the building blocks of life. Atoms range considerably in size but an approximation for the diameter of an oxygen atom is 2.6 angstroms, Å. An angstrom is equal to 10-10 meter, or 0.0000000001 m. Obviously, there need to be millions of atoms present for an object to be visible to our eyes. For instance, a human hair is about 1 million atoms wide.

Atoms are composed of a nucleus3 of positively charged protons (P+), neutrons (N) that have no charge, and negatively charged electrons (e-) that surround the nucleus. The number of protons and electrons is equal in an uncharged atom. Electrons exist around the nucleus in a series of electron energy shells and each shell contains a certain number of electrons to satisfy the orbital filling requirement. Some atoms, such as Neon (Ne), have enough negatively charged electrons to fill the requirements of all shells, and thus, are especially stable. These atoms can exist independently without attachment to other atoms. Atoms are electrically neutral, but can take on an overall positive charge or negative charge, depending on whether they gain or lose electrons to/from other atoms. Atoms may bond with an atom of the same element or of a different element.

Protons (P+) and neutrons (N) make up the nucleus of the atom. Electrons (e-) exist around the nuclear core at discrete energy levels in shells. As the size of the atom increases (more protons, neutrons, and electrons in different elements), there are more energy shells. It is the outermost (or valence) shell that shares its electrons (covalent bonding) with other atoms or transfers or accepts electrons with other atoms (ionic bonding).

1. Heat of vaporization = the amount of heat required to change a gram of liquid into the gaseous state at the boiling point of the liquid. For example, the amount of heat required to create steam from 1 gram of liquid water at 100oC is 0.48 calories per gram which is the specific heat of steam.

2. Solvent ability refers to the ability of a liquid solvent, such as water, to dissolve a solute, such as salt or sugar or oil. Solvent ability is an equilibrium between the solid and dissolved state of the solute, and this equilibrium is dependent on the temperature. The ability of the solute to dissolve in a solvent is also a function of the nature of its bonding. For example, water will readily dissolve polar compounds such as sugar and salt, but does not readily dissolve non-polar compounds such as fats, unless detergent is added to it.

3. Note that the nucleus of an atom is not the same as the nucleus in a cell. An atom’s nucleus is composed of protons and electrons. A cell nucleus contains the genetic material in a biological cell and is composed of millions of molecules.

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Experiment The Properties of Water

The figure above represents a Lithium atom, Li, with an Atomic Number of 3. It has three electrons. The innermost energy shell contains two electrons. The one electron in Lithium’s outermost (valence) shell is available to transfer to another atom. After the electron transfers, Li has a charge of +1.

Molecules are combinations of atoms that are electrically neutral. For instance, oxygen, with the chemical formula, O2, is a molecule of two oxygen atoms that share their electrons. Sugar, glucose, C6H12O6, is also a molecule, but it is composed of the elements carbon, hydrogen, and oxygen.

Water molecules are composed of two hydrogen atoms covalently bonded to one oxygen atom. The molecular formula is H2O, and a single molecule can be represented like this:

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Experiment The Properties of Water

Other compounds that have three atoms can share electrons equally (non-polar covalent bonds). Carbon dioxide, CO2, is an example. It can be represented like this:

Why is water a V-shaped molecule, while other molecules may not be? Nonbonding electrons on the oxygen atom of the water molecule push the hydrogen atoms into the V-shape configuration represented by the H2O figure above. The resulting molecule will have a slightly positive charge on one end (here, in the region of the hydrogen atoms) and a slightly negative charge on the other end (in the region of the oxygen atom)4. This type of bonding is referred to as polar covalent. This unique shape gives water different properties than we observe in the non-polar covalently bonded molecules like CO2. We will explore some of these unique properties of water to see why water is so important to life.

4. For further information on atomic configuration, refer to information about Valences Shell Electron Pair Repulsion Theory (VSEPR).

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Exercise 1: Surface Tension Preparations Required Prior to Performing this Lab Exercise

Clean paper clips of various sizes, tweezers, and wide shallow bowl. Rinse thoroughly and allow to dry.

Search Key Terms: Liquid surface tension, cohesion, tensile strength.

If we look at the surface of a lake, we often can see insects resting on the water. Why don’t they sink? When water is in a liquid form, it shares attractions that are constantly forming and breaking between the water molecules due to the partial charges on the oxygen and hydrogen ends of each molecule. Tensile strength is a measurement of the intensity of the water molecules’ attraction (cohesion) to each other at this surface layer. What allows insects to “walk on water?” In this exercise, we will answer this question.

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Experiment The Properties of Water

procedure 1. Read through the procedures below, and make a hypothesis as to whether the paper clip will

float on water or sink. We know that metal paper clips are denser (heavier) than water, so would we expect a paper clip placed on water to sink?

2. Bend a clean metal paper clip into the shape below. Make sure that it is flat.

3. Weigh the paper clip and record the weight in a table like Table 1.

4. Fill a clean bowl with tap water. Allow the bowl to sit until the water is still before proceeding with the exercise.

5. Pick up the flat, bent paper clip using the cleaned tweezers.

6. Use the tweezers to hold the bent paper clip in a horizontal position, flat in relation to the water surface. Gently lower the paper clip in the horizontal position on to the bowl’s water surface and release it. If the paper clip sinks, try again, until you are able to get the paper clip to float.

7. Weigh several other paper clips, bend them in the shape shown above, and do several trials to attempt to float them as well. Record your results Data Table 1.

Data Table 1: Results of Surface Tension Trials

Trial # Paper Clip Weight, grams Does it Float? Yes/No 1 2 3 4 5 Compacted 6 + Detergent (optional)

8. Use pliers to compact a paper clip that you were able to float previously to as small a shape as possible. Attempt to float it. Record your results in Data Table 1.

9. Optional: Replace one of the paper clips that floated on the water surface again in the bowl. Add a drop of dishwashing detergent off to the side of the water surface, away from the paper clip. Observe what happens.

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Questions A. Why must the paper clips, tweezers, and bowl be clean?

B. If one paper clip did not float, what might be a reason?

C. Was your original hypothesis supported or refuted?

Discussion A. Give two examples where the surface tension of water is important. Why is it important in these examples?

B. Would other liquids have the same surface tension property? Why or why not? How might you test this (for instance, the behavior of a paper clip and olive oil)?

C. Is there a limit to the tensile strength of the water surface? Explain.

D. What experimental variables are important to consider when doing this exercise?

E. What is the purpose of this exercise?

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Exercise 2: Capillary Action Search Key Terms: capillary action, adhesion.

Can water overcome gravity? In this exercise, we will investigate a property of water that is critical to the functioning of all living beings.

We know that our hearts pump blood through capillaries in our bodies to move oxygen, carbon dioxide and other molecules important for life to and from our cells. Plants also have vascular systems that move water and the products of photosynthesis around, but plants do not have hearts. How is it possible for plants to be able to move water? In this exercise, we will investigate another property of water, its ability to adhere to, or be attracted to surfaces.

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procedure 1. Read through the procedures and make a hypothesis about the outcome of this exercise.

What will happen?

2. Use tape to attach three capillary tubes of differing internal diameters to a large upside down test tube, as shown in the illustration. Ensure that the bottoms of the capillary tubes are even, placed just above the lip of the large inverted test tube.

3. Fill a glass jar with water to a height of 2 cm from the table top. Add 5 drops of blue or green food coloring to the water, swirl to mix.

4. Place the upside down test tube with the attached capillary tubes into the glass jar with the dyed water. Allow the test tube with attached capillary tubes to remain in the dye water for ½ hour.

5. At the end of ½ hour, remove the test tube with the capillary tubes, and measure how far up each capillary tube the dyed water has traveled.

6. Record your results in Data Table 2.

Data Table 2: Results of Capillary Action Exercise

Capillary Tube Internal Diameter, mm Height of Liquid, cm

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Graphic Results of Capillary Action Exercise

7. If available, collect these data from at least three other students, and record their data results in Data Table 3. Average the data and construct a scatter graph that provides the results of this exercise as a function of capillary tube internal diameter.

Data Table 3: Results of Capillary Action Exercise

Capillary Tube internal Diameter, mm Height of Liquid, cm

8. Present this graph as Figure 1. Make sure to label the axes of the graph appropriately.

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Experiment The Properties of Water

Questions A. What did you observe at the end time of the exercise?

B. What caused the difference in the results that you obtained?

C. How did your outcome compare to that of the other students?

D. Was there a linear relationship between Internal Diameter and the height of the water? What might explain this?

E. Was your original hypothesis supported or refuted?

Discussion A. Give two examples where the capillary action of water is important. Why is it important?

B. Would other liquids have the same property? Why or why not? How might you test this?

C. Blood in our bodies travels through capillaries of differing internal diameters. This means that our heart can work less hard, since blood contains water as part of the fluid, and the capillary action of water in tubes helps to move the blood along.

1. Why does blood pressure rise as patients experience the progression of atherosclerotic disease?

Does this seem to be counter to what you have learned about capillary action? How do you explain the apparent discrepancy (note: see also c., below)?

2. In plants, vascular system elements called xylem and phloem are lined up like long straws from the root of the plant to the leaves. The xylem of plants moves water and dissolved nutrients up to the leaves. Can you suggest what aids the movement of water up the plant, in addition to the capillary action of water?

3. Why is it harder to suck a beverage through a small diameter straw than through a large diameter straw?

4. What experimental variables are important to consider when doing this exercise? What could explain the difference in outcomes between your data and the data of other students?

5. What is the purpose of this exercise?

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Exercise 3: Density Search Key Terms: water density, specific gravity, specific weight, relative density, heat of vaporization.

We note that when we drink cold liquids containing ice that the ice floats on the liquid. But is this true for all liquids?

Water is present as a liquid at room temperature. If the solid form of water, ice, is added to a glass of water, it floats on top while it melts and cools the liquid. This property is obviously important since if ice sank, there would be no ice cover on lakes in the winter and life for aquatic organisms such as fish would be very different.

Properties of water related to weight and mass are used as standards in physics and chemistry for specific weight (weight per unit volume), a measurement for density (mass per unit volume, such as g/cc) that tells us whether one material will float or sink if placed in another material. The specific weight of liquid water is 1.0 gram per cubic centimeter or g/cc5 at 4oC.

Relative density is a ratio of the density of a substance to the density of a reference material, = ρ substance/ ρ reference. If ρ >1, the substance will sink in the reference material. If ρ <1, the substance will float on in the reference material. Since ρ water=1, materials with ρ>1 will sink in water, and those with ρ<1 will float on water.

Specific gravity, S, is a dimensionless unit that is expressed as a ratio of the density of a substance to the density of water at the same temperature.

Generally these measurements are taken and calculated at the temperature where water is the densest, at 1.0000 g/cc, 4oC. This table shows a comparison of the various forms of water, and the properties that it exhibits based on temperature as it relates to density.

Form of Water

Temperature Density (ρ) of Water, g/cc (= g/ml)oC oF

Ice -5 23 0.9993 Ice 0 32 0.9150 liquid 0 32 0.9999 liquid 4 39 1.0000 liquid 20 68 0.9982 liquid 100 212 0.9584 Gas 100 212 0.0006

Note the points where the physical property values are most dramatically changing, at 4oC where water is densest, at its freezing point where the liquid form becomes solid (ice), and at its boiling point where the liquid form becomes a gas (steam) at 100oC.

When water freezes at 0oC (= 32oF), ice, the solid form of water, results. The specific gravity of ice 5. In specifying specific gravity, specific weight, relative density and specific density, the units are sometimes

omitted.

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Experiment The Properties of Water

is 0.917. Does it float or sink on liquid water? Since ρ ice at 0.915 < ρ liquid at 0.9999 at 0 oC or 1.0000

at 4oC, ice floats on liquid water. How common is this? Generally, we think of solid objects, such as a rock, as being heavier (more dense) than liquids such as water, and would expect them to sink. In the case of water, however, when the molecules of liquid water solidify into ice crystals, they organize into a structure where they are farther apart from each other, and thus, the density decreases. An ice crystal is six-sided, reflecting the attraction of the oxygen and hydrogen atoms into the following pattern.

But is this property of water (the solid form floating on the liquid form) true of other liquids and solids? Let’s find out in one example.

At 0oC, water molecules aggregate into a solid six-sided crystal lattice.

Snowflakes and ice result (Not to scale).

procedure 1. Read through the Procedures, and make a hypothesis about the outcome of this exercise.

What will happen?

2. Prepare the day before: Pour a small amount of olive oil, ~ 5 mL, into a small cup or dish. Place the cup/dish and oil in the freezer compartment of a refrigerator overnight until used below.

3. Fasten a large test tube to a test tube rack with rubber bands to maintain it in an upright position.

4. Fill the large test tube to approximately 3/4 full with olive oil.

5. Remove the frozen oil/dish from the freezer.

6. IMMEDIATELY remove a pea-sized portion of the frozen oil from the dish, and drop it into the large test tube containing the liquid oil. Observe the movement of the solid oil in the liquid

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Experiment The Properties of Water

oil. If necessary, lightly nudge the solid oil with a toothpick to break any surface tension that might be holding the solid oil at the surface of the liquid oil.

7. If you are not absolutely sure that ice floats on water, fill a glass with tap water, and add an ice cube. Record your observations under Results.

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Questions A. What did you observe in this exercise?

B. How does this outcome compare to what you observe about the action of ice (the solid form of water) on liquid water?

C. Was your original hypothesis supported or refuted?

Discussion A. Why is it important that ice floats?

B. Give two examples where the property of water density is important.

C. Do all solids float on their liquid forms? Why or why not?

D. The density of liquid olive oil is about 0.92. What can you say about the likely density of solid oil? Why?

E. Will olive oil float or sink if it is added to liquid water? Why?

F. What experimental variables are important to consider when doing this exercise?

G. Attempt to find another pure substance, like water or oil, in which the solid form floats on the liquid form (Note: do not spend more than 5 minutes on this question!).

H. What did you learn from this exercise?

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Exercise 4: Specific Heat Capacity Search Key Terms: specific heat, heat capacity.

Specific heat capacity is a measure of the amount of energy input required to raise the temperature of 1 gram of material by 1oC. Specific heat is measured in units of calories or joules6 per g-oC. Water is unique in the world of science, because its specific heat capacity is much higher than the specific heat capacity of other materials. In this exercise, we will investigate the property of specific heat capacity.

Specific Heat of Various States of Water and Olive Oil

Specific heat capacity at 25oC in J/g-oC required to change temperature of the state

State Water Freezes at 0oC Boils at 100oC

olive oil Freezes at -6oC Boils at 300oC

Solid 2.090 Paraffin wax = 0.84 liquid 4.1796 1.790

gas 4.039 Unknown, but assumed to be less than for steam

6. A calorie (cal) is defined as the amount of heat input required to raise the temperature of 1 gram of liquid water by 1oC. Again, as with specific gravity, water is the standard to which all other substances are compared. 1 cal = 4.184 Joules (J). A Calorie, also known as a kilocalorie, with a capitalized C and equivalent to 1000 cal, (Cal) is defined as the amount of heat input required to raise the temperature of 1 kilogram of liquid water by 1oC. 1 Cal = 4184 Joules (J)

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procedure 1. Read through the procedures, and make a hypothesis about the outcome of this exercise.

What will happen?

2. Prepare the day before.

Measure 50 mL of olive oil by pouring the oil into a graduated cylinder. Add the oil to a small beaker. Clean the graduated cylinder with soap and water to remove oil residue. Likewise, measure out 50 mL of distilled water using a graduated cylinder. Add the water to a second small beaker. Observe the level of the liquid in each beaker. Make a mark on the outside of each beaker at the height of the liquid inside of the beaker.

Place a toothpick in each beaker contents, leaning the upper portion of the toothpick against the side of the beaker. The toothpick will help to remove the frozen content later. Place both beakers in the freezer compartment of a refrigerator overnight until ready to use.

3. Create the following testing apparatus:

a. Strainer held at correct height with books. One book is placed on top of strainer handle to hold strainer.

b. Funnel inserted into graduated cylinder. Graduated cylinder is used to collect and record the amount of melting liquid.

4. Using a thermometer, indicate the temperature of your testing environment in Data Table 4.

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Data Table 4: Results of Specific Heat Exercise for Water

Ambient (Room) Temperature in Which Exercise was Conducted, oC.

Time, minutes Liquid Collected, mL

All solid ice gone

5. Remove both beakers from the freezer. Compare their appearances. Note the height of the frozen solid oil and ice as compared to the original level of the liquid that you added to the beakers. Return the beaker containing the oil to the freezer.

6. Place the ice from inside the beaker into the strainer. You may need to briefly run the outside of the beaker under warm tap water to loosen the ice from the inside of the beaker. Use the inserted toothpick to help remove the ice from the beaker.

7. Immediately begin to time the exercise with the first time noted as 0 minutes, and mL of liquid in the graduated cylinder as 0.0.

8. At the end of ten minutes, note how much water has collected in the graduated cylinder. Record your results in Data Table 4.

9. Continue to record the mL of water collection with passing time in Data Table 4 until all of the ice has melted. Add rows to the bottom of the table as necessary to include all of your data points. Empty the collected water from the graduated cylinder and shake the cylinder to dry it as much as possible. Set up the testing apparatus as before.

10. Remove the beaker containing the solid oil from the freezer. Place the solid oil into the strainer. You may need to briefly run the outside of the beaker under warm tap water to loosen the solid oil from the inside of the beaker. Use the inserted toothpick to help remove the solid oil from the beaker into the strainer.

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11. Immediately begin to time the exercise with the first time noted as 0 minutes, and mL of liquid in the graduated cylinder as 0.0. You will record your results in Data Table 5 at this time.

Data Table 5: Results of Specific Heat Exercise for Oil

Ambient (Room) Temperature in Which Exercise was Conducted, oC.

Time, minutes Liquid Collected, mL

All solid oil gone

12. At the end of ten minutes, note how much liquid oil has collected in the graduated cylinder. Record your results in Data Table 5.

13. Continue to record the mL of oil collection with passing time in Data Table 5 until all of the solid oil has melted. Add rows to the bottom of the table as necessary to include all of your data points.

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Questions A. Was your original hypothesis supported or refuted?

B. What do you observe concerning the plot line of the oil versus the plot line of the water? What does this mean?

C. How close is your melting rate to the melting rates reported by at least three other students for the water and for the oil?

D. Generally, what might you conclude about the amount of specific heat needed to melt 50 mL of water versus 50 mL of oil?

E. How could you explain the differences in your data observations as to when the water and oil began to melt?

F. What experimental variables are important to consider when doing this exercise? What could explain the difference in outcomes between your data and the data of other students?

Discussion A. How might you design a similar experiment to determine whether the specific heat (amount of heat required to raise 1 gram of a material by 1 ºC) of one substance is more or less than another substance? What variables must you consider in the design of your experiment? Give an example of such an experiment that compares the specific heats for two different substances.

B. An alternative energy-savvy person decides to use 55-gallon drums filled with liquid to warm her house at night in the winter. She has to make a decision about what liquid will be the most efficient at storing heat from sunlight that shines on the drums during the day. Should she use water or oil in the drums? Why?

Laboratory summary What have you learned from doing this laboratory?

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The Properties of Water Margaret E. Vorndam, M.S. Version 42-0129-00-01

Lab report Assistant This document is not meant to be a substitute for a formal laboratory report. The Lab Report Assistant is simply a summary of the experiment’s questions, diagrams if needed, and data tables that should be addressed in a formal lab report. The intent is to facilitate students’ writing of lab reports by providing this information in an editable file which can be sent to an instructor.

Exercise 1: Surface Tension results D. State your hypothesis. What do you think will happen?

Data Table 1: Results of Surface Tension Trials

Trial # Paper Clip Weight, grams Does it Float? Yes/No 1 2 3 4 5 Compacted 6 + Detergent (optional)

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E. Record the results of your trials in the Data Table 1 above.

F. Did the weight of the paper clip affect whether it floated or not?

G. Did the size of the paper clip affect whether it floated or not?

H. Did the shape of the paper clip affect whether it floated or not?

I. Optional: Did the addition of detergent influence the flotation of the paper clip? Explain.

Questions A. Why must the paper clips, tweezers, and bowl be clean?

B. If one paper clip did not float, what might be a reason?

C. Was your original hypothesis supported or refuted?

Discussion A. Give two examples where the surface tension of water is important. Why is it important in these examples?

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B. Would other liquids have the same surface tension property? Why or why not? How might you test this (for instance, the behavior of a paper clip and olive oil)?

C. Is there a limit to the tensile strength of the water surface? Explain.

D. What experimental variables are important to consider when doing this exercise?

E. What is the purpose of this exercise?

Exercise 2: Capillary Action results A. State your hypothesis. What do you think will happen?

B. Record the results of your exercise in the Data Table 2.

Data Table 2: Results of Capillary Action Exercise

Capillary Tube Internal Diameter, mm Height of Liquid, cm

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C. Record the average results of at least three other students’ exercises in a table like Table 3. Why is it important to note how many data sets (N) are used in the average?

N (number of data sets averaged) =

D. Graph your data results from Tables 1 and 2 in a scatter graph, line graph, or bar graph similar to the graph layout in Figure 1. Also present the average results of the other students’ data using a second scatter, line or bar on the same graph so that the results of both your data and other students’ data can be compared. The graph should present two series of data, your results and the average students’ results, with Capillary Tube Internal Diameter on the x-axis, and height of liquid on the y-axis. Note: Do the graph by hand, or you may use a graphing program, and copy the resulting graph to your work area. Place your graph here:

E. What is the advantage of comparing your data to that of the other students’ data? Which set of data would you trust the most? Why?

Data Table 3: Results of Capillary Action Exercise

Capillary Tube internal Diameter, mm

Height of Liquid, cm

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Experiment The Properties of Water

My data from Data Table 1 is recorded in _____ (student to specify color of line) Other students’ average data from Table 2 are recorded in ______ (student to specify color of line)

Questions A. What did you observe at the end time of the exercise?

B. What caused the difference in the results that you obtained?

C. How did your outcome compare to that of the other students?

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Experiment The Properties of Water

D. Was there a linear relationship between Internal Diameter and the height of the water? What might explain this?

E. Was your original hypothesis supported or refuted?

Discussion A. Give two examples where the capillary action of water is important. Why is it important?

B. Would other liquids have the same property? Why or why not? How might you test this?

C. Blood in our bodies travels through capillaries of differing internal diameters. This means that our heart can work less hard, since blood contains water as part of the fluid, and the capillary action of water in tubes helps to move the blood along.

1. Why does blood pressure rise as patients experience the progression of atherosclerotic disease?

Does this seem to be counter to what you have learned about capillary action? How do you explain the apparent discrepancy (note: see also c., below)?

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Experiment The Properties of Water

2. In plants, vascular system elements called xylem and phloem are lined up like long straws from the root of the plant to the leaves. The xylem of plants moves water and dissolved nutrients up to the leaves. Can you suggest what aids the movement of water up the plant, in addition to the capillary action of water?

3. Why is it harder to suck a beverage through a small diameter straw than through a large diameter straw?

4. What experimental variables are important to consider when doing this exercise? What could explain the difference in outcomes between your data and the data of other students?

5. What is the purpose of this exercise?

Exercise 3: Density results A. State your hypothesis. What do you think will happen?

B. Record what actually happened here:

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Experiment The Properties of Water

Questions A. What did you observe in this exercise?

B. How does this outcome compare to what you observe about the action of ice (the solid form of water) on liquid water?

C. Was your original hypothesis supported or refuted?

Discussion A. Why is it important that ice floats?

B. Give two examples where the property of water density is important.

C. Do all solids float on their liquid forms? Why or why not?

D. The density of liquid olive oil is about 0.92. What can you say about the likely density of solid oil? Why?

E. Will olive oil float or sink if it is added to liquid water? Why?

F. What experimental variables are important to consider when doing this exercise?

G. Attempt to find another pure substance, like water or oil, in which the solid form floats on the liquid form (Note: do not spend more than 5 minutes on this question!).

H. What did you learn from this exercise?

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Experiment The Properties of Water

Exercise 4: Specific Heat Capacity results A. State your hypothesis. What do you think will happen?

B. What difference do you observe in the heights of the frozen solid oil and water as compared to the liquid forms prior to freezing? What might explain this result?

C. Record what actually happened in a table similar to Tables 4 and 5.

Data Table 4: Results of Specific Heat Exercise for Water

Ambient (Room) Temperature in Which Exercise was Conducted, oC.

Time, minutes Liquid Collected, mL

All solid ice gone

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Experiment The Properties of Water

Data Table 5: Results of Specific Heat Exercise for Oil

Ambient (Room) Temperature in Which Exercise was Conducted, oC.

Time, minutes Liquid Collected, mL

All solid oil gone

D. Graph your data results from Data Tables 4 and 5. Use a scatter graph or progressive line graph, with time noted on the x-axis, and mL of liquid collected on the y-axis. You should have two scatter plots or lines on your graph (not two separate graphs) —one for water and one for oil. Note: you may use a graphing program, and copy the resulting graph to your work area. Place your graph here:

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Experiment The Properties of Water

E. Calculate the melting rate for each of water and oil. The melting rate is equal to the slope of each of the separate lines of your graph, one for water, and one for oil. The easiest way to calculate the slope for each is to use your graphing program, and ask it to determine the trendline for you, as follows:

F. Share your melting rate for water and the oil with other students, if possible. Make sure to also obtain the ambient (room) temperature from the other students. Why?

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Experiment The Properties of Water

Data Table 6: Comparison of Melting Rates for Specific Heat Exercise

Results Reported By Water, mL/min Oil, mL/min general Ambient Temperature, oC.

Student Student A Student B Student C Student D Average =

Questions A. Was your original hypothesis supported or refuted?

B. What do you observe concerning the plot line of the oil versus the plot line of the water? What does this mean?

C. How close is your melting rate to the melting rates reported by at least three other students for the water and for the oil?

D. Generally, what might you conclude about the amount of specific heat needed to melt 50 mL of water versus 50 mL of oil?

E. How could you explain the differences in your data observations as to when the water and oil began to melt?

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Experiment The Properties of Water

F. What experimental variables are important to consider when doing this exercise? What could explain the difference in outcomes between your data and the data of other students?

Discussion A. How might you design a similar experiment to determine whether the specific heat (amount of heat required to raise 1 gram of a material by 1 ºC) of one substance is more or less than another substance? What variables must you consider in the design of your experiment? Give an example of such an experiment that compares the specific heats for two different substances.

B. An alternative energy-savvy person decides to use 55-gallon drums filled with liquid to warm her house at night in the winter. She has to make a decision about what liquid will be the most efficient at storing heat from sunlight that shines on the drums during the day. Should she use water or oil in the drums? Why?

Laboratory summary What have you learned from doing this laboratory?

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Experiment The Properties of Water

LabPaq by Hands-On Labs

AppEnDix

laboratory Equipment and Techniques

While many of these techniques and equipment are most applicable to specific science disciplines in formal laboratory facilities, knowledge of these items is often required for the study of other science disciplines and when working in a home laboratory.

Dispensing Chemicals: To avoid contamination when pouring liquid chemicals from a reagent bottle with a glass stopper, hold the stopper in your fingers while carefully pouring the liquid into the desired container. When pouring from a screw-cap bottle, set the cap down on its top so that it does not become contaminated or contaminate anything. Be certain to put the correct cap on the bottle after use. Never pour excess chemicals back into a reagent bottle because this may contaminate the reagents. If any liquid spills or drips from the bottle, clean it up immediately.

To obtain samples of a powdered or crystalline solid from a container, it is best to pour the approximate amount of solid into a clean, dry beaker or onto a small piece of clean, creased paper for easy transport. Pour powders and crystals by tilting the container, gently shaking and rotating the solids up to the container lip, and allowing the solids to slowly fall out. If you pour too much solid, do not put any solid back in the container. Also, never put wooden splints, spatulas, or paper into a container of solids to avoid contamination.

Dropping Chemicals: In micro-scale science, you use only small drops of chemicals and it is extremely important that the drops are uniform in size and carefully observed. To ensure uniformity of drop size, use scissors to cut off the tip of the pipet perpendicular to the pipet body; cutting at an angle will distort drop sizes. Turn the pipet upside down so the dispensing chamber behind the dropper is full of liquid. Then hold the dropper in front of your eyes so you can carefully observe and count the number of drops dispensed as you slowly squeeze the pipet. The pipet should be held in a vertical position at eye level to ensure drops are uniform in size and the correct drops are dispensed.

Heating Chemicals: Heat solid and liquid chemicals with great care to prevent explosions and accidents.

● Liquids in Beakers: To heat liquids in beakers or flasks, ensure that these containers are well supported above the heat source. Generally, the beaker or flask is placed on wire gauze supported by an iron support attached to a stand. The heat source is placed under the beaker or flask.

● Liquids in Test Tubes: When heating liquids in test tubes, always use a test tube rack or holder. Evenly heat the test tube contents by carefully moving the test tube back and forth in the flame. Heat the test tube near the top of the liquid first; heating the test tube from the bottom may cause the liquid to boil and eject from the tube.

Heating Sources for Micro-Scale Techniques: For micro-scale science experimentation, the most commonly used heat sources are alcohol burners, candles, and burner fuel. Alcohol burners can be a problem because their flame is almost invisible, and they cannot be refilled while hot. Candles, while effective for heating small quantities of materials, tend to leave a sooty, carbon residue on the heated container that obstructs observations. A chafing dish (burner fuel) is actually the

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best of these alternatives because it has a visible flame, is easily extinguished, and does not leave excessive flame residue. Regardless of the type of burner used, never leave an ignited heat source unattended.

Mass Measurement Equipment: Note that weighing scales are often called balances since weights are calculated using balance beams. Triple and quadruple beam balances are the most common measuring equipment found in laboratories. However, with today’s precision technology, digital top-loading balances are becoming increasingly popular.

● Digital Top Loading Balance: This scale is initially zeroed by pressing the zero button. If you are using weighing paper or a small beaker, first tare the paper or beaker by placing it on the scale and pressing the tare button. This will produce a zero reading, and the weight of the paper or beaker will be excluded from the weighing process.

● Hanging Spring Scales: Measurements are taken by suspending the item from a scale, often within a container. Spring scales are not easily tared, so the container weight should be separately calculated and subtracted from the combined weight of the item and the container.

Volume Measurement Equipment: To obtain accurate measurements from any glass volume measurement container, such as a beaker or graduated cylinder, you must identify and correctly read a curved surface known as the meniscus. The meniscus of water and water-based solutions concaves downward and is read at the very bottom of its curve. When using plastic containers, a meniscus will not form.

Pipet: Pipets are small tube-type containers with openings at one end if made of plastic or at both ends if made of glass. They come in a range of volumes and are generally used to transfer specific amounts of liquids from one container to another.

Berel Pipet: These soft and flexible pipets are made of polyethylene plastic and are extensively used in LabPaqs. They have long, narrow tips and are used to deliver chemicals and to collect products. Berel pipets come in different sizes, and their tips can have different diameters and lengths. You can modify them to serve diverse purposes such as chemical scoops, gas generators, or reaction vessels.

Volumetric Flask: Volumetric flasks are pear-shaped flasks with long necks used for the preparation of solutions whose concentrations need to be very accurate. Flasks come in a variety of sizes ranging from a few milliliters to several liters, and their volume levels are precisely marked. When the liquid level inside a volumetric flask is such that the meniscus lines up with the calibration mark on the neck, the volume of the liquid is exactly as stated. Unlike volumetric flasks, the markings on beakers, Erlenmeyer flasks, and most other laboratory containers are very good approximates but are not intended to be exact and precise volume measurements.

Well Plates: These microplates are plastic trays containing numerous shallow wells arranged in lettered rows and numbered columns. Similar to test tubes and beakers, you can use the wells to observe reactions, to temporarily store chemicals during experiments, and to hold pipets. The most commonly used plates are 24-well and 96-well.

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Distilled Water and Deionized Water: Tap water frequently contains ions that may interfere with the substances you are studying. To avoid such interference, use distilled or deionized water any time water is needed for dilution of concentration or the preparation of experimental solutions. Wash used glassware with soap, rinse with tap water, and rinse again with distilled water.

Potential Laboratory Hazards Recognizing and respecting potential hazards is the first step toward preventing accidents. Please appreciate the grave dangers the following laboratory hazards represent. Work to avoid these dangers and consider how to respond properly in the event of an accident.

Acid Splatter: When water is added to concentrated acid, the solution becomes very hot and may splatter acid. Splattering is less likely to occur if you add acid slowly to the water. Remember this AAA rule: Always Add Acid to water, never add water to acid.

Chemical Spills: Flesh burns may result if acids, bases, or other caustic chemicals are spilled and come into contact with skin. Flush the exposed skin with a gentle flow of water for several minutes at a sink or safety shower. Neutralize acid spills with sodium bicarbonate – simple baking soda. If eye contact is involved, use the eyewash station or its substitute. Use the spill containment kit until the spill is neutralized. To better protect the body from chemical spills, wear long-sleeved shirts, full-length pants, and enclosed shoes when in the laboratory.

Fires: The open flame of any heating source, combined with inattention, may result in a loose sleeve, loose hair, or some unnoticed item catching fire. Except for water, most solvents, including toluene, alcohols, acetones, ethers, and acetates, are highly flammable and should never be used near an open flame. As a general rule, never leave an open flame or reaction unattended. In case of fire, use a fire extinguisher, fire blanket, and/or safety shower.

Fume Inhalation: To avoid inhaling dangerous fumes, partially fill your lungs with air and, while standing slightly back from the fumes, use your hand to waft the odors gently toward your nose. Lightly sniff the fumes in a controlled fashion. Never inhale fumes directly! Treat inhalation problems with fresh air, and consult a physician if the problem appears serious.

Heated Test Tube Splatter: Splattering and eruptions can occur when solutions are heated in a test tube. You should never point a heated test tube towards anyone. To minimize this danger, direct the flame toward the top rather than the bottom of the test tube. Gently agitate the tube over the flame to heat the contents evenly.

Horseplay: A laboratory full of potentially dangerous chemicals and equipment is a place for serious work, not for horseplay! Fooling around in the laboratory is an invitation for an accident.

Shattered Glassware: Graduated cylinders, volumetric flasks, and certain other pieces of glassware are not designed to be heated. If heated, glassware is likely to shatter and cause injuries. Always ensure you are using heatproof glass before applying it to a heat source. Take special caution when working with any type of laboratory glassware.

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Use, Disposal, and Cleaning instructions for Common materials

These procedures are not repeated for each experiment, because it is assumed students will always refer to them before beginning any experiment. Properly cleaning the laboratory after experimentation is a safety measure!

instrument Use

● Small quantities of chemicals are usually packaged in thin stem pipets. The drop size dispensed from small dropper bottles is different from that of the pipets. Most experiments require pipet-sized drops. It may be necessary to squeeze a few drops of chemical from a dropper bottle into a well plate, and then use a clean, empty pipet to suck up and drop the chemical.

● Once dispensed, do not return chemicals to their dropper bottles as this could cause contamination. To avoid over-dispensing, squeeze out only a few drops of chemicals into a well plate at a time. Squeeze out more as needed.

● To use burner fuel, unscrew the cap, light the wick, and place the can under a burner stand. Extinguish the fuel by gently placing the cap over the flame to deprive it of oxygen. Leave the cap sitting loosely on top of the wick when you are not using the fuel in order to avoid unnecessary evaporation and ensure an ample supply of fuel for all experiments. Allow the fuel to cool completely before tightly screwing on the cap for storage. If you screw the cap on while the fuel is still hot, you may create a vacuum that will make it very difficult to reopen the fuel can in the future.

● To reseal a pipet, heat the tip of a metal knife and press the pipet tip onto the hot metal while twirling the bulb. Never simply hold a flame to the tip of the stem!

● To minimize contamination, avoid touching the surfaces of clean items that might later come in contact with test chemicals.

Storage and Disposal

● Items in LabPaq auxiliary bags are generally used multiple times or for several different experiments. Always clean and return unused auxiliary items to the bag after completing an experiment.

● Blot up used and leftover chemicals with paper towels and place in a garbage bin or flush down a drain using copious amounts of water. The quantities of chemicals used in LabPaqs are very small and should not negatively impact the environment or adversely affect private septic systems or public sewer systems.

● Discard non-chemical experimental items with household garbage but first wrap them in newspaper. Place these items in a securely covered trash container that cannot be accessed by children and animals.

● LabPaqs containing dissection specimens will usually contain specific information regarding

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their handling. After completion of any dissecting work, wrap dissection specimens in news or waste paper, seal in a plastic bag, and place in a closed trash bin for normal garbage disposal.

Cleaning Instructions

● To clean a thin-stemmed plastic pipet, squeeze the bulb to draw up and then expel tap water from the bulb several times. Repeat this process with distilled water. Dry the pipet by repeatedly squeezing the bulb while tapping the tip on a clean paper towel. Then use gravity to help dry the pipet by forcefully swinging the pipet into a downward arch while squeezing the bulb. Lay the pipet on a clean paper towel or place it in a test tube stand and allow it to air dry.

● Use a mild liquid dishwashing detergent mixed with warm water to loosen solids or oils that adhere to experimental glassware, plastics, and equipment and to clean laboratory equipment and the laboratory area after an experiment. Use tap water to rinse washed items well and remove all traces of detergent.

● Use a soft cloth or a test tube brush to loosen and clean residue from the surfaces of experimental glassware, plastics, and equipment.

● Use a final rinse of distilled water to clean tap water mineral residue from newly washed items, especially beakers, cylinders, test tubes, and pipets.

● Dry test tubes by placing them upside-down in the test tube rack. Air dry other items by placing them on paper towels, aluminum foil, or a clean dishtowel.

Important Notice Regarding Chemical Disposal: Due to the minute quantities and diluted and/or neutralized chemicals used in LabPaqs, the disposal methods previously described are well within acceptable levels of disposal guidelines defined for the vast majority of local solid and wastewater regulations.

Since regulations occasionally vary in some communities, you are advised to check with your local area waste authorities to confirm these disposal techniques are in compliance with local regulations and/or if you should seek assistance with disposal.

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Material Safety Data Sheets An important skill in the safe use of chemicals is the ability to read a Material Safety Data Sheet (MSDS). An MSDS is designed to provide chemical, physical, health, and safety information on chemical reagents and supplies. It provides information about how to handle, store, transport, use, and dispose of chemicals in a safe manner.

An MSDS also provides workers and emergency personnel with the proper procedures for handling and working with chemical substances. An MSDS provides basic information about physical data, toxicity, health effects, first-aid procedures, chemical reactivity, safe storage, safe disposal, required protective equipment, and spill cleanup procedures.

It is important to know how to read and understand an MSDS. An MSDS is generally organized into the following sections:

Section 1: Product Identification

Chemical name and trade names

Section 2: Hazardous Ingredients

Components and percentages

Section 3: Physical Data

Boiling point, density, solubility in water, appearance, color, etc.

Section 4: Fire and Explosion Data

Flash point, extinguisher media, special fire fighting procedures, and unusual fire and explosion hazards

Section 5: Health Hazard Data

Exposure limits, effects of overexposure, emergency and first-aid procedures

Section 6: Reactivity Data

Stability, conditions to avoid, incompatible materials, etc.

Section 7: Spill or Leak Procedures

Steps to take to control and clean up spills and leaks and waste disposal methods

Section 8: Control Measures

Respiratory protection, ventilation, protection for eyes or skin, or other needed protective equipment

Section 9: Special Precautions

How to handle and store, steps to take in a spill, disposal methods, and other precautions

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View MSDS information at www.hazard.com/msds/index.php. You can also find a link to MSDS information at www.HOLscience.com. If there is ever a problem or question about the proper handling of any chemical, seek information from one of these sources.

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How to Write Lab Notes and Lab Reports Important Note: Each instructor has a specific way they want

students to keep lab notes and summarize their lab activities. Your instructor will give you instructions regarding the formats you should

use. The following information will familiarize you with how lab Notes and lab Reports are usually prepared in the real world of science and

give you a better understanding of the scientific method. it will also be useful if your instructor requires you to write formal lab reports as is

often required in college courses.

Generally, two basic records are compiled during and from scientific experimentation. The first record is your lab Notes which you will record as you perform your experiments. Entries in your lab notebook will be the basis for your second record, the lab Report. The Lab Report formally summarizes the activities and findings of your experiment and is normally submitted to your instructor for grading.

lab Notes

Scientists keep track of their experimental procedures and results as they work by recording Lab Notes in a journal-type notebook. In laboratories these notebooks are often read by colleagues, such as directors and other scientists working on a project. In some cases, scientific notebooks have become evidence in court cases. Consequently, Lab Notes must be intelligible to others and include sufficient information so that the work performed can be replicated and there can be no doubt about the honesty and reliability of the data and the researcher.

Notebooks appropriate for data recording are bound and have numbered pages that cannot be removed. Entries include all of your observations, actions, calculations, and conclusions related to each experiment. Never write data on pieces of scratch paper to transfer later, but always enter the data directly into the notebook. When you record erroneous data, neatly draw a light, diagonal line through the error, and write a brief explanation as to why you voided the data. Also record information you learn from an error. Mistakes can often be more useful than successes, and knowledge gained from them is valuable to future experimentation.

As in campus-based science laboratories, independent study students are usually expected to keep a complete scientific notebook of their work which may or may not be periodically reviewed by the instructor. Paperbound 5x7 notebooks of graph paper work well as lab notebooks. Since it is not practical to send notebooks back and forth to instructors for each experiment, students usually prepare formal Lab Reports and submit them along with their regular assignments to the instructor.

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Lab Notes of experimental observations can be kept in many ways. Regardless of the procedure followed, the key question for deciding what kind of notes to keep is: Do I have a clear enough record that if I pick up my lab notebook or read my Lab Report in a few months, I can still explain to myself or others exactly what I did?

Lab Notes generally include these components:

● Title: Match this title to the title stated in the lab manual.

● Purpose: Write a brief statement about what the experiment is designed to determine or demonstrate.

● Procedure: Briefly summarize what you did to perform this experiment and what equipment you used. Do not simply copy the procedure statement from the lab manual.

● Data Tables: Always prepare tables before experimenting, so they will be ready to receive data as it is accumulated. Tables are an excellent way to organize your observational data, and where applicable, the Procedure section advises a table format for data recording.

● Observations: Record what you observed, smelled, heard, or otherwise measured? Generally, observations are most easily recorded in table form.

● Questions: Thoughtfully answer the questions asked throughout and at the end of experiments. The questions are designed to help you think critically about the experiment you just performed.

● Conclusions: What did you learn from the experiment? Base your conclusions on your observations during the experiment. Write your conclusions in your best, formal English, using complete sentences, full paragraphs, and correct spelling.

Some general rules for keeping a lab notebook are:

1. Leave the first two to four pages blank so you can add a Table of Contents later. Entries in the Table of Contents should include the experiment number and title.

2. Neatly write your records without being fussy.

3. Do not compile a complete Lab Report in your lab notebook. Instead, record what you did, how you did it, and what your results were. Your records need to be substantial enough that any knowledgeable person familiar with the subject of your experiment can read the entries, understand exactly what you did, and repeat your experiment if necessary.

4. Organize all numerical readings and measurements in appropriate data tables.

5. Always identify the units (for example: centimeters, kilograms, or seconds) for each set of data you record.

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6. Always identify the equipment you are using so you can refer to it later if you need to recheck your work.

7. Capture the important steps and observations of your experiments using digital photos in which you are pictured. Photos within your Lab Report document both what you observed and that you actually performed the experiment.

8. Record more rather than less data. Even details that may seem to have little bearing on your experiment (for example, time and temperature variances when the data were taken) may turn out to have great bearing on your future results analysis.

9. Make a note if you suspect that a particular data set may not be reliable.

10. Never erase data. If you think an entry in your notes is in error, draw a single line through it and note the correction, but do not erase or scratch it out completely. You may later find that the information is significant after all.

Errors: Although experimental results may be in considerable error, there is never a wrong result in an experiment. Whatever happens in nature, including the laboratory, cannot be wrong. If you made your observations and measurements carefully, your results will be correct. Errors may have nothing to do with your investigation, or they may be mixed up with so many other unexpected events that your report is not useful. Even errors and mistakes have merit and often lead to our greatest learning experiences. Errors provide important results to consider; thus, you must think carefully about the interpretation of all your results, including your errors.

Experiment Completion: The cardinal rule in a laboratory is to fully carry out all phases of your experiments instead of “dry-labbing” or taking shortcuts. The Greek scientist, Archytas, summed this up very well in 380 BCE:

In subjects of which one has no knowledge, one must obtain knowledge either by learning from someone else or by discovering it

for oneself. That which is learned, therefore comes from another and by outside help; that which is discovered, comes by one’s own efforts independently. To discover without seeking is difficult and rare, but if

one seeks, it is frequent and easy. If, however, one does not know how to seek, discovery is impossible.

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lab Reports

This lab manual covers the overall format that formal lab reports generally follow. Remember, the lab report should be self-contained so anyone, including someone without a science background or lab manual, can read it, understand what was done, and understand what was learned. Data and calculation tables have been provided for many of the experiments in this lab manual, and you are encouraged to use them. Computer spreadsheet programs such as Microsoft® Excel® and websites like nces.ed.gov/nceskids/Graphing/Classic/line.asp can also greatly facilitate the preparation of data tables and graphs. Visit www.ncsu.edu/labwriter/ for additional information on preparing lab reports.

Lab reports are expected to be word processed and look organized and professional. They should be free of grammar, syntax, and spelling errors and be a respectable presentation of your work. Avoid writing in the first person as much as possible.

Lab reports should generally contain and clearly distinguish the sections discussed in detail below. The presentation and organization skills you will develop by producing science lab reports is beneficial to all potential career fields.

Lab report Format

Title Page

This is the first page of the Lab Report and consists of:

a. Experiment number and/or title

b. Your name

c. Names of lab partner(s)

d. Date and time experiment was performed

e. Location if work was performed in the field

f. Course name and section number

Section 1: Abstract, Experiment, and Observation

Abstract: Even though the abstract appears at the beginning of the lab report, you will write it last. An abstract is a very concise description of the experiment’s objectives, results, and conclusions and should be no longer than a paragraph.

Experiment and Observation: In chronological order, carefully and concisely describe what was done, what was observed, and what, if any, problems were encountered. Describe what field

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and laboratory techniques and equipment you used to collect and analyze the data on which the conclusions are based. Insert photos and graphic illustrations in this section; graphics should be in .jpg, .tif, or .gif format to minimize electronic file size.

Show all your work for any calculations performed. Title every graph and clearly label the axes. Data point connections should be “best-fit curves,” which are smooth, straight or curved lines that best represent the data, instead of dot-to-dot data point connections.

Include all data tables, photos, graphs, lists, sketches, etc., in an organized fashion. Include relevant symbols and units with data. Generally one or two sentences explaining how data was obtained is appropriate for each data table.

Note any anomalies observed or difficulties encountered in collecting data as these may affect the final results. Include information about any errors you observed and what you learned from them. Be deliberate in recording your experimental procedures in detail. Your comments may also include any preliminary ideas you have for explaining the data or trends you see emerging.

Section 2: Analysis – Calculations, Graphs, and Error Analysis

Generally, the questions at the end of each experiment will act as a guide when preparing your results and conclusions. The analysis is written in paragraph form and no more than one or two pages long. As you write, consider the following:

a. What is the connection between the experimental measurements taken and the final results and conclusions? How do your results relate to the real world?

b. What were the results of observations and calculations?

c. What trends were noticed?

d. What is the theory or model behind the experiment?

e. Do the experimental results substantiate or refute the theory? Why? Be sure to refer specifically to the results you obtained.

f. Were the results consistent with your original predictions of outcomes or were you forced to revise your thinking?

g. Did errors occur(for example, environmental changes or unplanned friction)? If so, how did these errors affect the experiment?

h. Did any errors occur due to the equipment used (for example, skewed estimates due to a lack of sufficient measurement gradients on a beaker)?

i. What recommendations might improve the procedures and results?

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Error Analysis: In a single paragraph, comment on the accuracy and precision of the apparatuses used, include a discussion of the experimental errors, and include an estimate of the errors in your final result. Remember, errors are not mistakes. Errors arise because the apparatus and/or the environment inevitably fail to match the ideal circumstances assumed when deriving a theory or equation. The two principal sources of error are:

● Physical phenomena: Elements in the environment may be similar to the phenomena being measured and may affect the measured quantity. Examples include stray magnetic or electric fields or unaccounted for friction.

● Limitations of the observer, analysis, and/or instruments: Examples include parallax error when reading a meter tape, the coarse scale of a graph, and the sensitivity of the instruments.

Human errors and mistakes that are not acceptable scientific errors include: calculator misuse (for example, pushing the wrong button or misreading the display); misuse of equipment; faulty equipment; incorrectly assembled circuits or apparatuses.

Section 3: Discussion, Results, and Conclusions

Discussion: Carefully organize your discussion to include consideration of the experiment’s results, interpretation of the results, and uncertainty in the results. This section is written in paragraph form and is generally no more than one to two pages in length. Occasionally, it will be more appropriate to organize various aspects of the discussion differently. While not all of the following questions will apply to every experiment, consider them when writing your lab report.

Results:

a. What is the connection among your observations, measurements, and final results?

b. What were the independent or dependent variables in the experiment?

c. What were the results of your calculations?

d. What trends were noticeable?

e. How did the independent variables affect the dependent variables? For example, did an increase in a given independent variable result in an increase or decrease in the associated dependent variable?

Interpretation of Results:

a. What is the theory or model behind the experiment you performed?

b. Do your experimental results substantiate or agree with the theory? Why or why not? Be sure to refer specifically to your experimental results.

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c. Were these results consistent with your original beliefs or were you forced to reevaluate your prior conceptions?

Uncertainty in results:

a. How much did your results deviate from expected values?

b. Are the deviations due to error or uncertainty in the experimental method? Can you think of ways to decrease the amount of uncertainty?

c. Are the deviations due to idealizations inherent in the theory? What factors has the theory neglected to consider?

d. In either case, consider whether your results display systematic or random deviations.

Lab Notes and Lab Reports undoubtedly sound complex and overwhelming at first, but don’t worry. They will make more sense to you when you begin performing the experiments and writing reports. After writing your first few Lab Reports, the reports will become second nature to you. Refer to the sample Lab Report in this manual.

laboratory Drawings

Laboratory work often requires you to illustrate findings in representational drawings. The very act of drawing an observation enhances and deepens your understanding of what you have seen and your findings. Clear, well organized drawings are an excellent way to convey observations and are often more easily understood than long textual descriptions. The adage “a picture is worth a thousand words” really is true when referring to Lab Notes.

Give yourself ample drawing space and leave a white margin around the actual illustration so it is clearly visible. Also leave a broad margin along one side of your drawing to insert object labels. Use a ruler to draw straight lines for the labels and connecting lines to the corresponding objects. The following images provide examples of how laboratory drawings might look when they are included in a formal Lab Report.

Students often believe they can’t draw; however, with a little practice, anyone can illustrate laboratory observations. A trick many artists use is to form a mental grid over the scene and draw within the grid. For example, quickly make a free hand drawing of a diagram below. Now, mentally divide the diagram into quarters and try drawing the diagram again. In all likelihood, the second, grid-based drawing yielded a better result.

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Final Cleanup Instructions Congratulations on completing your science course’s lab assignments! We hope you had a great science learning experience and that what you have learned in this course will serve you well in the future. Studying science at a distance and performing laboratory experiments independently are certainly not easy tasks, so you should be very proud of your accomplishments.

Since LabPaqs often contain potentially dangerous items, it is important that you perform a final cleanup to properly dispose of any leftover chemicals, specimens, and unused materials. Please take a few minutes to protect others from possible harm and yourself from future liability by complying with these final cleanup instructions.

While you may wish to sell your used LabPaq, this is not advisable and would be unfair to a potential purchaser. It is unlikely that a new student trying to utilize a used LabPaq would have adequate quantities or sufficiently fresh chemicals and supplies to properly perform all the experiments and to have an effective learning experience. Further, it is doubtful that adequate safety information would be passed on to a new student in the same way it was presented to you. This is a significant concern and one of the reasons why a new user would not be covered by LabPaq’s insurance. Instead, you would be responsible for any problems experienced by a new user.

Chemical Disposal

● Due to the minute quantities, low concentrations, and diluted and/or neutralized chemicals used in LabPaqs, it is generally sufficient to blot up any remaining chemicals with paper towels and dispose of them in a trash bin or flush remaining chemicals down a drain with copious amounts of water. Empty dispensing pipets and bottles can be placed in a normal trash bin.

● These disposal methods are well within acceptable levels of the waste disposal guidelines defined for the vast majority of state and community solid and wastewater regulations. However, since regulations can vary in some communities, if you have any doubts or concerns, you should check with your area authorities to confirm compliance with local regulations and/or if assistance with disposal is desired.

Specimen and Supply Disposal

● To prepare any used dissection specimens for normal garbage disposal, wrap them in news or waste paper and seal them in a plastic bag before placing them in a securely covered trash container that will prevent children and animals from accessing the contents.

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● Non chemical supplies can also be discarded with household garbage, but should first be wrapped in news or waste paper. Place such items in a securely covered trash container that will prevent children and animals from accessing the contents.

lab Equipment

● Many students choose to keep the durable science equipment included with their LabPaq as most of these items may have future utility or be used for future science exploration. However, take care to store any dangerous items, especially dissection knives and breakable glass, out of the reach of children.

● Please do not return items to LabPaq as we are unable to resell items or issue any refunds.

Best wishes for a happy and successful future!

The LabPaq Team

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Using Statistics This short introduction to statistics is designed to assist you in performing a few simple statistical analyses on some of your experiments. This brief introduction to statistics only scratches the surface. If you want to learn more about statistics, consider reviewing a statistics textbook, downloading one of the many statistics tutorials on the Internet, or taking a statistics course. The final section will show you how to use Microsoft® Excel® to calculate most statistics automatically.

Statistics is a branch of applied mathematics. It specifically deals with the collection and interpretation of quantitative data and the use of probability theory to estimate population parameters.

Statistics can be categorized into two sub-groups: descriptive statistics and inferential statistics. Descriptive statistics describe large amounts of data in an abbreviated form. They describe important characteristics of the data, including the mean, median, range, variance, standard deviation, etc. Inferential statistics use data obtained from a small group of elements called the sample to make estimates and test hypotheses about the characteristics of a larger group of elements called the population.

Descriptive Statistics

There are a number of measures of central tendency used to describe the center of a distribution and the scatter of observations around the center.

● Mean: The mean is the arithmetic average of all observations in a distribution. The mean is equal to the sum of all observations divided by the sample size.

● Mode: The mode is the most common value or class in the distribution.

● Median: If all of the observations are arranged in rank order from smallest to largest, the median is the value bound by 50% of the observations on each side. If the number of observations in the distribution is odd, the median is simply the middle value in the ranked observations. If the number of observations is even, then the median is the mean of the two most central observations.

The measurement of the scatter of observations around the center of a distribution is extremely important. What if you had two means from two populations and the means of both were 50, but the values going into sample #1 were 1, 50, 100 and the values going into sample #2 were 49, 50, 51. It would appear that these are two very different distributions, but with the same mean.

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The scatter of observations around the center of a distribution can be depicted in the following ways:

● Range: The range of a distribution is the difference between the largest and the smallest value and is typically expressed as range = 1 – 22, meaning the lowest value was 1 and the highest was 22.

● Variance and standard deviation: The variance is a measure of the average squared deviation from the mean. Variance differs from the range in that the variance takes into account the distribution of all data points; the range simply describes the single lowest and highest extremes. To calculate variance, take the deviation (or differences) of each value xi (that is, the ith value of x) from the mean, (that is, Xi - ). Square these differences and divide by the number of values minus one (that is, n-1).

The standard deviation (s) is the square root of the variance. The advantage of the standard deviation is that if the data conform to a normal distribution, 95% of the values will fall within two standard deviations (actually 1.96s) on either side of the mean.

If you were contrasting the weight of two populations of acorns, it might be nice to see a statement like “Acorns from Plot A (mean = 4.58 g, S.D. = .59, range = 3.99 – 4.91) were heavier than the acorns from Plot B (mean = 3.64, S.D. = .71, range = 2.99 – 4.29).”

At this point you could also give the results of a statistical test comparing these two samples. See the upcoming section on Hypothesis Testing.

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Inferential Statistics

There are two types of statistical inferences: hypothesis testing and estimation of population parameters. Hypothesis testing refers to a general class of procedures for weighing the strength of statistical evidence or for determining whether the evidence supporting one hypothesis over another is sufficiently strong. Hypothesis testing is one of the most important tools statistical applications bring to real-life problems. Most often, decisions are required concerning populations on the basis of sample information. Statistical tests are used in arriving at these decisions.

There are five ingredients to any statistical test:

1. Null Hypothesis 2. Alternate Hypothesis 3. Test Statistic 4. Rejection/Critical Region 5. Conclusion

Following is a simple example of hypothesis testing and the application of a null hypothesis and an alternative hypothesis.

One may wish to test whether or not a coin is fair (that is, whether there is an equal chance of it coming up heads or tails when tossed). The null hypothesis is that the coin is fair; the alternative hypothesis is that the coin is biased. If a series of coin tosses produce a result that is only 4% likely given a fair coin, one would reject the null hypothesis, assuming 95% confidence is required. By contrast, if the experiment produces a result that is 30% likely given a fair coin, one would fail to reject the null hypothesis that the coin is fair. It is not permissible to accept the alternative hypothesis. Only acceptance or failure to reject the null hypothesis is allowed in hypothesis testing. If a test fails to reject the null hypothesis, it is said to lack sufficient power to accept the alternative hypothesis.

The null hypothesis states that there is no effect or difference between procedures and is denoted by H0. The objective of hypothesis testing is to either accept or reject the null hypothesis

The alternative hypothesis states that there is a statistically significant difference in the outcome of an experimental procedure. Typically, the null hypothesis is stated first, followed by the alternative hypothesis (Ha). This alternative can be stated simply as there is a true difference. Only one of the two statistical hypotheses can be true.

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Consider a simple example in which one wishes to compare the size of male and female fish. The null hypothesis might be that the males and females are the same size (that is, the samples obtained were drawn from the same underlying population). The alternative hypothesis is that males and females are different sizes. The null hypothesis is tested with an appropriate statistic. If one rejects the null hypothesis, one is left with the alternative that there is a difference (that is, males and females are of different sizes). This sounds pretty simple, but statistical tests provide a formal means to tell us if the evidence is sufficiently compelling to reject the null and decide that something is going on and might be worthy of further investigation.

Hypotheses can be directional (for example, males are smaller than females) or non-directional (for example, males and females are of different sizes), and this determines whether to use what is called a one-tailed or a two-tailed test.

● Example: Two-Tailed Hypothesis

Ho = There is no difference in size between male and female fish. Ha = There is a difference in size between male and female fish.

● Example: One-Tailed Hypothesis

Ho = Male fish are not smaller than female fish. Ha = Male fish are smaller than female fish.

In the example for a one-tailed test, failure to reject the null hypothesis might mean that there was no difference in size of male and female fish or that female fish were bigger than male fish.

Decision Making and the Level of Significance

After stating the hypothesis, one must select and carry out an appropriate statistical test. Each test is based upon a different test statistic [given the symbols, t (for a t-test), F (in an analysis of variance), r (for a correlation analysis), χ2 (for a chi-square test), etc.]. By plugging the values from a sample into a formula for the statistical test, one ends up with an observed value for the test statistic. One must then compare the observed value of the test statistic with a theoretical distribution of values that one would obtain if the null hypothesis was true. This distribution of expected values is generated from the assumptions that underlie the test and, in the case of parametric tests, from some of the data that was collected, such as the variance among multiple observations within a group. These distributions are typically summarized in tables that are published in statistics books or are readily available on the Internet (search: Statistical Tables).

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With these tables, one can ask how likely is it that we would have obtained the observed results. The table provides the entire distribution and determines how much of the distribution lies beyond the observed value of the test statistic. This yields the P-value, or the probability of obtaining one’s observed results or something more extreme under the assumption that the null hypothesis is correct. For example, a P-value of 0.13 means that if the null hypothesis were true, 13% of all possible samples would lead to results as extreme as those found (that is, with the same or more extreme differences between the two groups).

The smaller the P-value, the less likely it is that the null hypothesis is true. But how small should the P-value be before one rejects the null hypothesis? This cut-off is given the symbol α (alpha) which by convention is typically set at 0.05. In other words, 5% of the times when the null hypothesis is correct, one will conclude that the null hypothesis is wrong. This is called a Type I error. The probability of a Type I error is equal to α.

To interpret the results, one compares the P-value to α. If P<α (that is, if P<0.05) then one rejects the null hypothesis. If P> α or P= α, one tentatively accepts the null, recognizing that it might be wrong, but there is insufficient evidence to reject it. If able to calculate a P-value exactly from a distribution or from a statistics software program, it's useful to report the exact value as P=0.05 rather than P<0.05 or P>0.05. In the second and more common case, the entire distribution is not published, so one cannot exactly determine the P-value. Instead, the tables provide particular values of the test statistic associated with different P-values or levels of significance or α. If one’s observed test statistic is greater than this critical value of the test statistic, one can reject the null hypothesis because P < α.

The T-Test One of the most common comparative statistical tests is the t-test. Also called the student’s t-test, it is used when there are just two sets of normally distributed data to compare. Normally distributed data means that the data distribution looks like a bell-shaped curve. There are several types of t-tests, each designed mathematically for a specific application. Here we will look at the t-test used to compare two independent samples which one would use in an experiment where the average height of plants in the two squares sampled are compared.

In this exercise, you will ask if the difference between the mean heights of the plants in the two plots are statistically significant. Your null hypothesis is that the species of plants present and the conditions in which they have grown have made no difference in their height and that the mean heights of the two plots are essentially the same, allowing for some minor variance:

Ho: µ1 = µ2

The alternative hypothesis is that differences in the plant species and growing conditions have made a difference and that the mean heights are not the same:

Ha: µ1 ≠ µ2

If the mean heights are not the same, then the question is whether the mean height of one sample is significantly larger or smaller than the other. As you have two means, you will use a

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two-tailed test. Don’t worry if this sounds confusing. Following are step-by-step examples of how this analysis is performed.

Sample Problem 1: Weights of acorns collected from two different plots

1. Calculate the mean (average) of the weights in grams. Add all data point values for each plot and divide by the number of data points.

Plot A Plot B 2.33 2.02 2.52 1.90 2.23 2.13 2.70 2.50 2.00 2.30 2.42 2.21 2.54 2.21 2.60 1.80 2.44 2.64 2.53 2.14

● Plot A: (2.33 + 2.51 +2.12 +2.7 +2 +2.42 +2.54 +2.6 +2.44 +2.53)/10 = 2.419

● Plot B: (2.02 +1.9 +2.13 +2.5 +2.3 +2.5 +2.3 +2.21 +2.21 +1.8 +2.64+2.14)/10 = 2.185

2. Calculate the variance (s²) of each plot:

a. Square each data value and enter it in a data table: 2.33² = 5.4289, etc.

b. Add all the data values in the last row: 2.33+2.51+2.12 +……= 24.19.

c. Add all the squared data values: 5.4289 + 6.3001 + ------- = 58.94 (rounded). Plot A Plot B x x2 x x2 2.33 5.4289 2.02 4.0804 2.51 6.3001 1.9 3.61 2.12 4.4944 2.13 4.5369 2.7 7.29 2.5 6.25 2.0 4.0 2.3 5.29 2.42 5.8564 2.21 4.8841 2.54 6.4516 2.21 4.8841 2.6 6.76 1.8 3.24 2.44 5.9536 2.64 6.9696 2.53 6.4009 2.14 4.5796 24.19 58.9359 21.85 48.3247

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3. Enter the values calculated in step 2 into the equation:

Plot A: s2 = {58.94 – [(24.19*24.19)/10]/9} = 0.047 Plot B: s2 = {48.32 – [(21.85*21.85)/10]/9} = 0.065

4. Calculate the t-value.

a. Calculate the numerator of the equations above: . Subtract the mean of plot B from the mean of plot A: 2.419 - 2.185 = 0.234.

b. Multiply the difference of the means (0.234) by the √ n (for the sample in the example n=10, and the √10 is 3.162) which = 0.74.

c. Calculate the denominator: Take the square root of the sum of the two variances calculated earlier: √(0.047 + 0.065) = 0.334.

d. Divide the numerator by the denominator: 0.74/0.334 = 2.24 = t.

e. The calculated t-value is 2.24.

5. Now look at the table of critical values for t and compare the values in the table to your calculated t.

In order to use the critical values table, you need alpha (α) and degrees of freedom (df). The total number of data points is n, in your case 20 acorns. For a t-test involving two independent means, df = n – 2. In your case, n = 20 so df = 20 – 2 = 18.

Alpha refers to the degree of confidence. The degree needed to accept the null hypothesis is normally 5% or 0.05. Since you are using a two-tailed test, your alpha has to be split between the two tails, giving an alpha of 0.025 for each tail.

Go to the table and look under 0.025 and 18 df. You can find such tables on the Internet with a search of t-test critical values.

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From the table you see that the value under α = 0.025 and df = 18 is approximately 2.1

Interpretation of the results: Since the calculated t-value of 2.24 is greater than the critical value of 2.1, accept the null hypothesis that the two means are statistically equal. It indicates that the difference between the means is insignificant at the 95% level (100% minus alpha). In other words, the means of the two samples differ by less than can be accounted for by minor variations and the size of the sample.

Sample Problem 2: Weights of acorns collected from two different plots with different number of data points in each plot.

1. Calculate the mean (average) of the weights in grams. Add all data point values for each plot and divide by the number of data points.

Sample 1 Sample 2 2.33 2.02 2.51 1.9 2.12 2.13 2.7 2.5 2.0 2.3 2.42 2.21 2.54 2.21 2.6 1.8 2.44 2.64 2.53 2.14 2.5 2.55

Plot A: (2.33 + 2.51 +2.12 +2.7 +2 +2.42 +2.54 +2.6 +2.44 +2.53+2.5+2.55)/12 = 2.437

Plot B: (2.02 +1.9 +2.13 +2.5 +2.3 +2.5 +2.3 +2.21 +2.21 +1.8 +2.64+2.14)/10 = 2.185

2. Calculate the variance (s²) of each plot:

a. Square each data value and enter it in a data table, Ex: 2.33² = 5.4289, etc.

b. Add all the data values: 2.33+2.51+2.12 +……= 29.24

c. Add all the squared data values: 5.4289 + 6.3001 + ------- = 71.69 (rounded).

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Plot A Plot B X x2 x x2 2.33 5.4289 2.02 4.0804 2.51 6.3001 1.9 3.61 2.12 4.4944 2.13 4.5369 2.7 7.29 2.5 6.25 2.0 4.0 2.3 5.29 2.42 5.8564 2.21 4.8841 2.54 6.4516 2.21 4.8841 2.6 6.76 1.8 3.24 2.44 5.9536 2.64 6.9696 2.53 6.4009 2.14 4.5796 2.5 6.25 2.55 6.5025 29.24 71.6884 21.85 48.3247

3. Enter the values calculated in step 2 into the equation:

Plot A: s2 = {71.69 – [(29.24*29.24)/12]/10} = 0.04 Plot B: s2 = {48.32 – [(21.85*21.85)/10]/9} = 0.065

4. An additional step is needed to calculate pooled variance since you have an unequal number of data points in each plot.

sp 2 = (n1-1)s1

2 + (n2-1)s2 2

n1 + n2 – 2

So, what does this mean?

• n1-1 = number of data points in plot A minus 1 • s1

2 = variance for plot A • n2-1 = number of data points in plot B minus 1 • s2

2 = variance for plot B • n1 + n2 – 2 = number of data points in plot A + plot B minus 2 (which also = df)

sp 2 = (11)0.04 + (9)0.065 = 1.025 = 0.051

12 + 10 – 2 20

5. Now, having adjusted the variance for different sample sizes, you can calculate the t-value using a slightly different equation.

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t(pooled) =

= 2.437 – 2.185 = 0.2545

= 0.051/12 + 0.051/10 = .00935

√.00935 = 0.0967

Final step: 0.2545/0.0967 = 2.63 calculated t = 2.63

6. Now find the critical t-value in the table. Using alpha = 0.05/2 = 0.025 and df = 22 – 2 = 20, ee find that tcritical = 2.086.

Interpretation of the results: Since the calculated t-value of 2.63 is greater than the critical value of 2.086 you reject the null hypothesis that the two means are equal. It indicates that the difference between the means is significant at the 95% level (100% minus alpha).

The Chi-Square Test The chi-square (χ2) test is one of the most useful non-parametric statistical tests for the biologist. It is used with count data or frequencies organized in a matrix defined by two or more variables.

The chi-square test is based on the differences between the observed results and the expected values (those results that would be obtained if the null hypothesis were true). The formula for χ2 is as follows:

where o is the observed frequency and e is the frequency expected under the null hypothesis of no difference between groups.

Example: Suppose a fishery’s biologist samples adult fish from two lake populations: 100 from Lake 1 and 150 from Lake 2. The biologist records whether or not the lakes are infested with a nematode parasite that encysts in their muscles. The biologist wants to know whether the presence of the parasite is independent of the lake from which they were taken.

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a. Arrange the data in a data table.

b. Calculate the sums for each table row and column.

Data Table 1 – Observed values Site # fish w/parasites # fish w/out parasites Total Lake A 15 85 100 Lake B 50 100 150 Totals 65 185 250

c. Compute the table of expected values. For example, the expected value for the number of fish with parasites in Lake 1 = (100x65)/250=26 [that is, (the row total x the column total)/total].

Data Table 2 – Expected values Site # fish w/parasites # fish w/out parasites Total Lake A 26 74 100 Lake B 39 111 150 Totals 65 185 250

Notice the row and column totals are the same in the tables of expected and observed values.

d. Compare the observed and expected frequencies using the χ2 statistic. χ2= (15-26)2/26 + (85-74)2/74 + (50-39)2/39 + (100-111)2/111 = 10.5

e. Determine the degrees of freedom for the test = (2 rows-1) x (2 columns-1) = 1 df.

f. Compare the calculated χ2 value (10.5) with the value for 1 degree of freedom from a stats table. Since your calculated value is greater than 3.84 (from the table), you can reject the null hypothesis that the presence of parasites in fish is independent of the lake.

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Table 2 – Chi-square

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Preparing a Water Bath Hot or cold water baths may be used in experiment-run chemical reactions. In some cases, scientists use warm water baths to incubate biological samples at a specific temperature, generally as a way to cause a reaction. In other cases, the heat of a water bath aids in driving a chemical reaction to an endpoint. Alternatively, scientists may employ cold water baths for other reactions.

Typically, scientists work in an environment of 70°F. However, because ambient temperature is not necessarily the same as the temperature in which a particular biological reaction occurs, scientists simulate the actual temperature using a water bath which approximates the temperature of the reaction. Water has a high heat capacity and will maintain a particular temperature longer than many other liquids, making it suitable for simulating reaction temperatures.

In a typical laboratory, scientists have access to a water bath with a built-in thermostat and integrated heating coils. In a home laboratory, commonly available household items can be used to build a water bath. When preparing water baths to heat substances in test tubes, a stove and thermometer are required. When cooling a sample, a refrigerator or an ice bath is used instead.

Hot Water Bath

MATERiAlS lABEl oR BoX/BAg QTy iTEM DESCRiPTioN

Student provides 1 Heatproof cooking spoon, rod, or stick 1 Hot pads, potholders, or oven mitts 1 Rubber bands 1 Saucepan 1 Stove, hot plate, or crock pot 1 Tap water 1 Wash cloth labPaq provides 1 Celsius thermometer 1 Glass stirring rod 1 Safety glasses 1 Test tube rack 1 Test tubes

Time Allocation: Allow 30 minutes to set up and heat the water bath.

Safety Issues: Specifically review the safety procedures for heating glass.

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prOCEDUrE

1. Use rubber bands to attach the Celsius thermometer to a wooden spoon or similar heat-proof instrument. Secure the thermometer at the top and bottom of the spoon, so it is secure and cannot slip. The bottom of the thermometer should be located ¼ inch or more above the bottom of the spoon, so it does not contact the bottom of the pan.

CAUtiOn: Glass thermometers are fragile. Do not subject the thermometer to unsupported pressure.

Figure 1: Thermometer attached to spoon.

2. Select a saucepan that can hold the test tube rack and can hold enough water to cover about ¾ of the height of the test tubes when they are in the test tube rack.

3. Fill the saucepan with tap water to the anticipated level of solute liquid in the test tubes. For example, if the total liquid content in the test tubes will be 4 cm, fill the pan with water to a depth of approximately 4 cm. If the test tubes will contain varying amounts of liquid, fill the pan to a depth matching the highest anticipated solute liquid level.

note: If long-term heating is required, add water to raise the depth to a level slightly above the test tube liquid level to compensate for water evaporation.

4. Place the pan on the stove and place the Celsius thermometer attached to the spoon in the water bath.

note: It is important that the thermometer does not touch the bottom of the pan. If necessary, secure the spoon to the handle or the top rim of the pan with a piece of bent, flame-proof wire to prevent it from submerging in the water bath.

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Figure 2: Water bath setup.

5. Turn on the stove and bring the water to the temperature indicated in the experiment.

note: The temperature of a simmering water bath is just at boiling (100°C at STP). The thermometer is not needed to maintain a simmering temperature, as long as the bath maintains a very slow boil. At higher elevations, the boiling point is depressed by a few degrees, but the temperature will be close enough to 100°C to conduct the experiment.

6. When the water bath reaches the correct temperature, lower the stove’s heat to maintain the temperature during the experiment. Use the Celsius thermometer in the water bath to monitor the water temperature while heating the test tubes.

note: It is sometimes difficult to maintain a water bath’s temperature with an electric stove. Two ways to control this problem are: 1) use a larger pan of water, because water maintains a more even temperature in large volumes or 2) slide the pan off the burner after the water reaches the correct temperature and return the pan to the burner if the temperature drops more than a few degrees. A range of ± 5°C is an acceptable variation in a water bath.

7. Submerge a folded wash cloth in the pan to cushion the glass test tubes from vibrations caused by boiling or heating water. This addition may create a slight decrease in the water temperature, but if the stove heat is set to maintain temperature, the water bath will return to the correct temperature. Do not adjust the stove heat.

8. Place the test tubes in the test tube rack.

9. Use hot pads to carefully place the test tube rack into the water bath, making certain that water cannot overflow into the test tubes. If the bath water appears too deep, remove some of the water before adding the test tube rack.

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10. Leave the test tube rack in the water bath for the experiment’s specified time period.

11. At the conclusion of the time period, use hot pads to carefully remove the test tube rack and test tubes from the water bath.

12. Continue the experiment.

13. If any reagents spill into the water bath during the experiment, thoroughly wash the pan with liquid detergent and rinse it completely after the experiment is over.

Cooling Water Bath

MATERiAlS lABEl oR BoX/BAg QTy iTEM DESCRiPTioN

Student provides 1 Cup or small jar 1 Tap water 1 Refridgerator 1 Ice labPaq provides 1 Test tubes

Time Allocation: Allow 15 minutes to set up and cool the water bath.

procedure

1. A temperature of 0oC can be achieved at the melting point of ice. Therefore, place ice in a coffee cup or small jar.

2. Add tap water.

3. Place the cup or jar in the refrigerator.

4. Allow the tap water and ice to stand for 10 minutes before proceeding with the experiment.

5. Test tubes can be placed directly into the ice water for cooling. Ensure that the water level does not overflow into the test tube. If needed, simply pour out some of the ice water to avoid this possibility.

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