Write a Report for Comparing cell contents of plants from different environments

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Rutgers University – Newark

College of Arts & Sciences Department of Biological Sciences

General Biology II (21:120:102)

Lab Learning Objectives & Lab Learning Activities

CONTENT

General Information .................................................................................................... 3 1

Laboratory Safety Rules ............................................................................................... 4 2

Laboratory Syllabus ..................................................................................................... 5 3

Microscope Use ........................................................................................................... 7 4

4.1 The Compound Microscope ................................................................................. 7

4.2 The Dissecting Microscope ................................................................................... 9

Lab Report Format ..................................................................................................... 11 5

Lab 1 – Viewing and Measuring Cells ........................................................................ 12 6

6.1 Exercise 1 - Features of the compound microscope .......................................... 12

6.2 Exercise 2 – Procedure for viewing specimens .................................................. 14

6.3 Exercise 3 – The image under a compound microscope.................................... 15

6.4 Exercise 4 – Mirror images ................................................................................. 16

6.5 Exercise 5 – Features of the dissecting microscope .......................................... 17

6.6 Exercise 6 – Use of the dissecting microscope................................................... 18

6.7 Exercise 7 – Calculating total magnification of objects ..................................... 19

6.8 Exercise 8 – Determining the diameter of the field of view. ............................. 20

6.9 Exercise 9 – Using the diameter of the field of view to measure cells .............. 21

6.10 Exercise 10 – Preparing a wet mount of a naturally pigmented (red onion) cell.23

6.11 Exercise 11 – Staining a wet mount of an unpigmented (human cheek) cell. ... 24

6.12 Exercise 12 – Estimating the size of a unicellular organism............................... 26

Lab 2 – Enzymes and Cell Function ............................................................................ 28 7

7.1 Exercise 1 – Browning of fruit ............................................................................ 31

7.2 Exercise 2 – Effect of temperature on enzyme-catalyzed reactions ................. 36

7.3 Exercise 3 – Effects of pH on enzyme-catalyzed reactions ................................ 39

Lab 3 – Cell Membranes and Water Balance ............................................................ 44 8

8.1 Exercise 1 – Movement of water into a "model cell" ........................................ 47

8.2 Exercise 2 – Using the plasmolysis threshold to estimate the concentration of solutes and water inside living cells .............................................................................. 52

8.3 Exercise 3 – Comparing solute concentrations (isotonic points) inside plant cells from different environments ........................................................................................ 56

Lab 4 – Photosynthesis .............................................................................................. 61 9

9.1 Exercise 1 – Separation of leaf pigments by paper chromatography ................ 62

9.2 Exercise 2 – Internal features of a leaf ............................................................... 64

9.3 Exercise 3 – Stomata .......................................................................................... 66

9.4 Exercise 4 – The effect of carbon dioxide and pH on the rate of photosynthesis68

9.5 Exercise 5 – The effect of the color of light on the rate of photosynthesis ....... 71

Lab 5 – DNA Fingerprinting ........................................................................................ 74 10

10.1 Exercise 1 – extraction DNA from cells .............................................................. 75

10.2 Exercise 2 – Practice using pipettes to load wells .............................................. 77

10.3 Exercise 3 – DNA fingerprints of unknown DNA samples .................................. 78

General Information 1

 Course Coordinator: Rola Bekdash ([email protected]); Boyden Hall 313; Extension 1267)

 Laboratory Supervisor: Sahil Wadhwa ([email protected]); Biology Learning Center, Boyden Hall 217; Extension 5108)

 Laboratory Supervisor Specialist: Albert Le Goff ([email protected]); Boyden Hall 111; Extension 1220)

Teaching Assistants

 TBD Location of the General Biology Laboratory: Boyden Hall 223A & 223B

Weekly Laboratory Sessions

Please attend the Lab session that you did register for. The duration of each lab session is 3 hours.

Section 1 Monday 1 – 3:50 pm

Section 2 Monday 1 – 3:50 pm

Section 3 Tuesday 8:30 – 11:20 am

Section 4 Tuesday 8:30 – 11:20 am

Section 5 Tuesday 2:30 – 5:20 pm

Section 6 Tuesday 2:30 – 5:20 pm

Section 7 Wednesday 8:30 – 11:20 am

Section 8 Wednesday 8:30 – 11:20 am

Section 9 Wednesday 11:30 am – 2:20 pm

Section 10 Wednesday 11:30 am – 2:20 pm

Section 11 Thursday 8:30 – 11:20 am

Section 12 Thursday 8:30 – 11:20 am

Section 13 Thursday 1 – 3:50 pm

Section 14 Thursday 1 – 3:50 pm

mailto:[email protected]

Laboratory Safety Rules 2

1. Wear your white lab coat before starting your lab session and bring your dissection kit with you.

2. Do not start any procedure until your Teaching Assistant (TA) has described and discussed the exercise and the potential hazards associated with it.

3. Wear protective eye wear for all dissection exercises as well as exercises using hazardous

materials. All students are required to get their own safety goggles to lab EACH day whether or not they are needed for the procedure.

4. Wear disposable gloves when you are doing dissection, working with preserved specimens, handling blood smaples or working with chemicals.

5. Keep long hair tied back if you are working with dissection specimens or other hazardous

material.

6. Do not eat or drink or bring any food or beverages into the lab. 7. Notify your TA and the lab supervisor of any accidents, including minor cuts, punctures, or

spill of chemicals. 8. If you have any medical condition (e.g. allergies, medication, and pregnancy) that may

increase your sensitivity to certain chemicals or procedures, you should notify your TA and consult your physician.

9. Dispose all sharps such as needles, razor blades, syringes, slides, Pasteur pipettes and capillary tubes in the “sharp containers”.

10. Dispose all live specimens and used gloves in the the container that has an autoclave bag.

11. Clean your work area with disinfectant at the end of each lab session and return all chairs to their designated places.

Laboratory Syllabus 3

WEEK *LABORATORY TOPIC/QUIZZES *REVIEW & LAB PREPS

Week 1 1/22 – 1/24

No Labs this week

Week 2 1/28 – 1/31

Lab # 1 – Cells

Week 3 2/4 - 2/7

Review Lab # 1 Quiz 1 on Lab #1

Prep Lab # 2

Week 4 2/11 - 2/14

Lab # 2 – Enzymes

Week 5 2/18 - 2/21

Review Lab # 2 Quiz 2 on Lab #2 Finish Lab#2 Report

Prep Lab # 3

Week 6 2/25 - 2/28

Lab # 3 – Membranes

Week 7 3/4 - 3/7

Review Lab # 3 Quiz 3 on Lab #3 Finish Lab#3 Report

Prep Lab # 4 & Review Lab Exam I

Week 8 3/11 - 3/14

Lab Exam I (Labs # 1, 2 & 3)

Week 9 3/18 - 3/21

Spring Recess – No Labs this week

Week 10 3/25 - 3/28

Lab # 4 – Photosynthesis

Week 11 4/1 – 4/4

Review Lab # 4 Quiz 4 on Lab #4

Prep Lab # 5

Week 12 4/8 - 4/11

Lab # 5 – DNA Technology

Week 13 4/15 – 4/18

Review Lab # 5 Quiz 5 on Lab #5

Review Lab Exam II

Week 14 4/22 – 4/25

Lab Exam II (Labs # 4 & 5)

(*) PLEASE READ THE LABORATORY TOPIC BEFORE COMING TO CLASS

YOU ARE REQUIRED TO BRING A DISSECTION KIT YOU ARE REQUIRED TO ATTEND AND PARTICIPATE IN ALL THESE LAB SESSIONS. IF YOU MISS MORE THAN TWO LAB SESSIONS, YOU WILL BE ASKED TO WITHDRAW FROM THE COURSE. YOU CANNOT MAKE-UP FOR ANY MISSED LAB SESSION. IT IS YOUR RESPONSIBILITY TO KNOW WHAT YOU MISSED AND COVER IT. MAKE-UP FOR LAB QUIZZES MAY BE DONE WITH THE APPROVAL OF YOUR TA/PTL WITH THE SUBMISSION OF VALID DOCUMENTATIONS.

*NO MAKE-UPS FOR LAB EXAMS. A MISSED LAB EXAM WILL GET A ZERO GRADE.

Grading distribution of the Laboratory Lab grade represents 25% of the total course grade.

Lab Exam I 10 %

Lab Exam II 10 %

Lab Review/pre Sessions/Quizzes (5 sessions) Quiz 1 (1%) Quiz 2 (0.5%) Lab2 Report (0.5%) Quiz 3 (0.5%) Lab3 Report (0.5%) Quiz 4 (1%) Quiz 5 (1%) Total 5 %

In addition to the five lab sessions, you are all required to attend and participate in the five Lab Review/Prep sessions. These sessions are worth 5% of the total course grade. These sessions are designed to help you prepare for your next week lab and to review the results/findings of the lab that you finished the week before. You will have quizzes. You are also required to submit with your partner two lab reports based on data obtained from Labs 2 & 3. So, come fully prepared to do your experimental work, record your data and finish the 3 hr lab session in a timely manner. Problems Any health-related problems that may arise from unexpected accidents (chemical spills, minor cuts ...) during lab time, please inform your TA, the lab supervisor and/or the lab supervisor specialist.

Microscope Use 4

4.1 The Compound Microscope

Figure 1: Major features of a compound microscope: occular lens, 2-3 objective lenses, coarse and fine adjusatment knobs, and iris diaphragm.

Using a compound microscope 1. Place the prepared slide on the microscope stage so that mounted specimen is centered over the hole in the stage. Use the stage clips to secure the slide in place. 2. Always begin with the lowest power objective. Rotate the nosepiece to bring the shortest objective (usually 4x) into position. 3. Turn the coarse adjustment knob to move the objective downward toward the slide until the point of the objective lens is just above (but not touching) the slide. 4. Turn on the microscope light and look into the eyepiece, holding your eye about 1/2 inch from the eyepiece. While looking into the eyepiece, use the coarse adjustment knob to raise the body tube slowly until the specimen comes into focus. Sharpen the image with the fine adjustment knob. If you see nothing, you may need to recenter the specimen over the hole in the statge. 5. Note that the contrast between light and dark can be adjusted using the substage, iris diaphragm. If the light is too bright, the image may be“washed out” (invisible). 6. While watching through the ocular lens, move the slide to the left or right. Which direction did the image of the specimen move? Now move the slide away from you or towards you. What direction did the image move now? 7. For greater magnification, rotate the nosepiece to bring a higher power (longer) objective into position. Whenever a higher power objective is in place, use only the fine focus adjustment to refocus! 8. Turn off the sub-stage light whenever you are not looking through the microscope! The bulbs are expensive and burn out quickly. 9. When you have finished using the compound microscope:

a. Turn off the light source. b. Rotate the nosepiece to the low power objective. c. Unplug the microscope and coil the wire loosely over the body tube. d. Cover the microscope.

4.2 The Dissecting Microscope

The dissecting microscope differs from the compound scope in many ways: a. Total magnification is much lower (maximum either 30 or 45 power). b. Objective lens is equipped with a "zoom" adjustment. c. Two ocular lenses give a stereoscopic (3-D) view of the specimen. d. Only one focus adjustment knob. e. Longer "working distance" (between specimen and objective lens). Figure 2. Major features of the dissecting scope: stage (for specimen), ocular lenses, objective (zoom) lens, coarse focusing adjustment, light sources (both above and below specimen).

 Goggle images

Using a dissecting microscope 1. Choose the proper illumination (lighting). Some specimens are better viewed using reflected light (with the light source above the specimen), others with transmitted light (with the light source under the specimen). 2. The total magnification of the microscope can be calculated by multiplying the power of the ocular lens (engraved on the side of the eye piece) by the power of the objective (zoom) lens (read from the dial on the zoom control knob). 3. The orientation of the image is intuitive – when you move the specimen, the image moves in the same direction – unlike the compound microscope.

Lab Report Format 5

You will write two lab reports based on data from labs 2 & 3. A scientific report usually consists of the following:

1. Title 2. Introduction 3. Materials and Methods 4. Results 5. Discussion & Conclusion

Title The title should be less than ten words, is straightforward and reflects the factual content of the experiment. Introduction The introduction defines the subject of the report. It must outlines the scientific objective(s) for the experiments performed and give the reader sufficient background to understand the rest of the report. A good introduction will answer several questions, including the following:

 Why was this study performed?

 What is the specific purpose of the study? The specific hypotheses and experimental design pertinent to investigating the topic should be described. Materials and Methods Materials and methods used in the experiments should be reported in this section. Provide enough detail for the reader to understand the experiment without overwhelming him or her. Generally, this section attempts to answer the following questions:

 What materials were used? How were they used?

Results The results section should summarize the data from the experiments without discussing their implications. The data should be organized into tables, figures, graphs, photographs, and so on. All figures and tables should have descriptive titles. Figures and tables should be self- explanatory; that is, the reader should be able to understand them without referring to the text. All columns and rows in tables and axes in figures should be labeled. Discussion & Conclusion This section should not just be a restatement of the results but should emphasize interpretation of the data, relating them to existing theory and knowledge. Suggestions for the improvement of techniques or experimental design may also be included here. In writing this section, you should explain the logic that allows you to accept or reject your original hypotheses. Provide a conclusion based on the results that you got. By Warren D. Dolphin, Iowa State University (Modified by Dr. Bekdash)

Lab 1 – Viewing and Measuring Cells 6

All living things on earth are basically similar: all are composed of one or more cells. The chemical reactions that support life occur inside cells. Cells don't arise spontaneously; they arise only from other cells. Each cell contains all the organism's hereditary information. This information is passed from parent to offspring through cells. How large is a cell? Are all cells about the same size? Why are cells so small? What limits cell size? You’ll soon know answers to all these questions! Cells are usually way too small to be seen with the naked eye. The human body is made up of trillions of cells. One of the largest human cells, the human ovum (egg cell) is only about the size of the period at the end of this sentence. Since most cells are much smaller than that, most of what we know about cells has been gained with the aid of microscopes. Lab learning objectives You will have mastered the content of this minicourse when you are able to: 1. Given a compound light microscope, locate, name and describe the functions of the light source,

ocular lens, objective lenses, iris diaphragm, specimen stage, and coarse and fine focus adjustment knobs.

2. Demonstrate proper use of the compound light microscope. Given a prepared slide, properly mount

the slide, adjust the light source, and focus on the specimen first at low power and then at high power.

3. Compare and contrast the compound microscope and dissecting microscope with regard to: range

of magnification, how the specimen is illuminated (transmitted vs. reflected light), focusing mechanism, depth of focus, appearance of image (2- vs. 3-dimensional), and spatial relationships (up, down, right, left) between specimen and image.

4. Demonstrate the proper techniques for preparing wet mounts of living cells.

5. Use the diameter of the field of view of your compound light microscope to measure the overall size

of various single-celled protozoa that use different modes of locomotion

PART I: THE COMPOUND MICROSCOPE

6.1 Exercise 1 - Features of the compound microscope

The microscope you will use first is called a "compound" microscope because it uses a combination of two lenses to magnify the image. The lens closest to your eye is called the "ocular" lens (oculus is Latin for eye). The lens closest to the object is the "objective" lens.

l. Use Figure 1 to identify the parts of your compound microscope referenced in bold in Exercise 2: the eyepiece (ocular lens), nosepiece with 3 objective lenses, substage iris diaphragm (or disc aperture diaphragm), coarse and fine focus knobs, and light source. Figure 1: A Compound Microscope

Turn off the microscope light whenever you are not looking through the microscope! The light bulbs for the compound scope are expensive ($20 each) and burn out quickly.

6.2 Exercise 2 – Procedure for viewing specimens

1. Remove the dust cover and place the microscope in a convenient position away from the edge of

the table. 2. Wipe the tips of the objective with lens paper. Do not use facial tissue or a handkerchief, because

these contain fibers that can scratch the lenses. 3. Obtain a prepared slide of the letter "e," clean the slide with lens paper, and place the slide on the

microscope stage so that the letter is centered over the hole in the stage. Use the stage clips to secure the slide in place.

4. Always begin with the lowest power objective. Rotate the nosepiece to bring the shortest

objective (labeled 4x) into position. 5. While watching from the side, turn the coarse adjustment knob to move the objective downward

toward the slide until the point of the objective lens is just above (but not touching) the cover slip of the slide. Watch carefully so that the objective does not touch or crack the cover slip. This is important when viewing commercially prepared slides and even more important when viewing the thick “wet mounts” you’ll be making yourself.

6. Turn on the microscope light and look into the eyepiece. Your eye should not be too close to the

ocular lens. Instead, the eye should be about 1/2 inch from the eyepiece (see Figure 1). Keep both eyes open. (Although awkward at first, this practice minimizes eye strain and lets you view and sketch the subject at the same time.)

7. While looking into the eyepiece, use the coarse adjustment knob to raise the body tube slowly until

the specimen comes into focus. If you see nothing, make sure the specimen is centered over the hole in the stage, then repeat steps 5 & 6.

8. Sharpen the focus with the fine adjustment knob. Note that the contrast between light and dark

can be adjusted using the substage iris diaphragm. 9. For greater magnification, rotate the nosepiece to bring a higher power (longer, 10x power)

objective into position.

10. Whenever a higher power objective is in place, use only the fine focus adjustment to refocus! Otherwise you risk cracking the cover slip with the objective lens, especially when viewing the slides you have wet mounted yourself. You should not need to turn the fine adjustment knob very far, because the objective lenses are designed to be "parfocal," to minimize the amount of refocusing needed after changing magnifications.

PART II: RELATIONSHIP OF SPECIMEN AND IMAGE It is frequently necessary to move the specimen around on the stage to locate the feature you are interested in. The relationship between the specimen and its image may not be what you expect!

6.3 Exercise 3 – The image under a compound microscope

1. Hold the letter "e" slide right side up (that is, the way it normally appears on a printed page) and

place it on the stage of your microscope. Following the procedures outlined in Exercise 2 (steps 4- 8), bring the "e" into focus under the low power ("scanning") objective.

2. You put the specimen (the "e") right-side-up on the microscope stage. Is the image you see of the

"e" also right side up? Is the opening to the left or right. Is the base of the e up or down? Draw the image of the e as it appears under the microscope:

e as seen with the naked eye e as seen through compound microscope

1. What happens to the image you see as you move the slide of the specimen to the left or right?

Does the image move in the same or the opposite direction? What happens when you slide the specimen toward you and away from you? Does the image move in the same or the opposite direction? Record your observations in Table 1.

Table 1: Relationship between the actual orientation of the specimen and the apparent orientation of the image. (Enter "same" or "opposite" for each category.)

Specimen (as it is on the microscope slide when seen with the naked eye)

Image in compound microscope

Image in mirror (Place letter e on the table in front of vertical mirror)

Image in dissecting microscope

When bottom of "e" is toward you

opposite opposite same

When open side of "e" is on right

same

If you move "e" sideways (left or right)

If you move "e" toward and away from you (up & down)

6.4 Exercise 4 – Mirror images

1. Is the microscope image the same as a "mirror image" of the specimen? To find out, get a small

mirror from the Central Study Area and use it to study the relationship between the specimen ("e") and its mirror image. Hold the mirror vertically on the desk and facing you. Place the "e" flat on the table top, between you and the mirror so that when you look at the "e" directly, its orientation is the way it should be when you are reading. Now look at the image in the mirror. In what way is the image in the mirror similar or different from the “e” printed on the slide? Specifically: (a) If the bottom of the printed is toward from you, is the bottom side of the reflected “e” also away from you? (b) If the open side of the printed “e” is to the right, is the open side of the reflected image also to the right? (c) When you move the “e” to the right, does its image move right or left? (d) When you move the “e” toward you, does its image move away from or toward you? Enter your observations (same or opposite?) for the left/right and toward/away categories in Table 1.

2. Referring to Table 1, compare the compound microscope’s image with the mirror image. Is the

image of the "e" in the microscope the same as a mirror image?

Compound microscope image vs. Mirror image of the “e”

a. In what ways are the microscope image and mirror image similar? b. In what ways are they different?

PART III: THE DISSECTING MICROSCOPE

6.5 Exercise 5 – Features of the dissecting microscope

1. Locate the dissecting microscope in your carrel or in the Central Study Area. Identify the major

features of the dissecting scope: stage (for specimen), eyepieces, objective lens, zoom knob, focusing knob, and light sources above and below specimen (Figure 2).

2. The dissecting microscope differs from the compound scope in many ways: a. Total magnification is much lower (maximum either 30 or 45 power). b. Objective lens is equipped with a "zoom" adjustment. c. Two ocular lenses give a stereoscopic (3-D) view of the specimen. d. Only one focus adjustment knob. e. Longer "working distance" (between specimen and objective lens). Figure 2: The Dissecting Microscope

3. Why do some microscopes have two eyepieces? Dissecting microscopes always have two eyepieces, which are connected to two separate objective lenses. The two separate optical systems work in parallel to give a stereo, 3-D image. Although some compound microscopes also have two eyepieces, both eyepieces are connected to the same objective lense and so never can produce a 3-D image. In a compound microscope the second eyepiece simply serves to reduce eyestrain during prolonged use.

6.6 Exercise 6 – Use of the dissecting microscope.

1. Illumination (lighting) of the specimen can be from above or below. Since the e is printed on

translucent paper, it should be clearly visible under both lighting conditions. So examine an opaque object instead -- like the leg of an insect. Is the specimen more easily seen using reflected light (with the light source above the specimen) or transmitted light (with the light source under the specimen)?

2. Total magnification of the dissecting microscope can be calculated by multiplying the power of the

eyepiece engraved on the side of the eyepiece by the power of the objective (zoom) lens read from the dial on the zoom control knob. First set the zoom lens so that the "e" appears as small as possible and calculate total magnification. Then set the zoom so the “e” appears as large as possible and calculate the highest magnification.

3. How does the orientation of the image you see compare with the actual letter "e" as you move the

slide around. Enter your observations in the right column of the Table 1.

Power setting Power of eyepiece (ocular lens) x

Power of zoom objective lens =

Total magnification

Minimum zoom

Maximum zoom

4. Summarize the appearance of the letter e in the four cases you observed: Naked eye Compound scope Mirror Dissecting scope

PART IV: ESTIMATING THE SIZES OF CELLS When looking at objects through a compound microscope, it is difficult to get a feel for just how big (or small) they really are. We need some kind of "ruler". The actual size of an object seen under the microscope can be estimated by first measuring the diameter ("width") of the viewing field (the circle of light seen through the eye piece). You can then estimate the length of the specimen as a fraction of diameter of the field of view. For example, if you estimate the diameter of the field of view to be, say, 6 millimeters (mm) and you see that the specimen is about half as long as the field is wide, then the specimen is about 3 mm long. To estimate the diameter of the field of view, complete exercises 8 and 9 below. When performing these calculations, it helps to keep in mind that the higher the power, the smaller the diameter of the field of view. Always!

6.7 Exercise 7 – Calculating total magnification of objects

1. In the table below, record the magnification of the ocular lens of your compound microscope. It is

engraved on the side of the eyepiece barrel and is usually l0X. When nothing is indicated, the power is 10X.

a. Power of ocular lens ______

2. Similarly, record below the magnification of each of your objective lenses.

a. Power of objective lenses ______ ______ ______ 3. To calculate total magnification, simply multiply the magnification of the ocular lens by the

magnification of the objective lens. Compute the total magnification for each objective lens on your microscope:

Power setting Magnification of objective lens x

Magnification of ocular lens =

Total magnification

Low

Medium

High

6.8 Exercise 8 – Determining the diameter of the field of view.

1. First determine the diameter of the field of view (circle of light) for the microscope under low power. Place a clear plastic, metric ruler on the microscope stage across the center of the field of view and focus on the ruler with the lowest power objective.

2. Move the ruler so that one of the millimeter lines falls exactly at the edge of the circle of light. Then count the number of millimeter-long spaces needed to get to the opposite side. The diameter is approximately _____ millimeters. (For our purposes you can round off to the nearest whole number of millimeters).

3. The unit commonly used for measuring microscopic specimens is the micrometer (µm), a unit equal to 1/1000 of a millimeter. There are 1000 micrometers (µm) in one millimeter (mm). To convert diameter in mm to diameter in µm, multiply by 1000.

The diameter of your field of view is _____ mm, which is ________ µm.

4. At higher magnifications, the field of view (lighted circle) covers a much smaller portion of the specimen, and the image of the plastic ruler becomes so large that it can no longer be used to measure the field of view. (Try it!). However, the diameter of the field of view under higher magnifications can be calculated from the diameter of the visual field that you measured under low power. This is because as magnification increases, the diameter of the field of view decreases proportionally. Example: The easiest way to think about this is with an example. Suppose you used the ruler and measured the diameter under low power (40x) to be 6 mm. The diameter under medium power (100 power) would be simply 40/100ths of 6 mm. The diameter under high power (450 x) would be simply 40/450ths of 6 mm.

Now calculate the actual diameters for your microscope at medium and high power and enter them in Table 2. You can just use the example above and plug in the values for diameter and power you determined for your own microscope. Or you may find it useful to use the boxed equation below… but then again, maybe not!

Unknown diameter = Diameter measured x The power of the low magnification under higher power under low power The power of the higher magnification

Table 2: Relationship between magnification and the diameter of field of view.

Power Total magnification

Diameter of field of view (millimeters) 1

Diameter of field of view (micrometers) 1

Low

µm

Medium

µm

High

µm

1 For our purposes, you can round off to nearest 0.1 millimeters (mm) or 100 micrometers (µm).

6.9 Exercise 9 – Using the diameter of the field of view to measure cells

1. The actual size of any microscopic object can now be estimated by comparing the length of the specimen to the known diameter of the field of view. To estimate the length of a cell as it appears under low, medium, or high power: a. Determine the diameter of the field of view at this power (from Table 2).

b. Estimate the number of cells that would fit, end-to-end, along the diameter of the field of view. (See diagram below.)

c. Divide the diameter of the field by this number. 2. Estimating cell diameters: In the circles below are two imaginary cells. The cell on the left is shown under medium (100x) power. Its length is about 1/8th the diameter of the field. From Table 2, what is the calculated diameter of the field at 100X? _____ µm.

So how long is this cell? _____ µm. 100 x 430 x 3. A different species of cell is shown on the right, as it appears under high (430x) power. As you can see, its length is about one half the diameter of the field. From Table 2, what is the diameter of the field of view at high power? _____ µm. So how long is this cell? _____ µm. 4. According to your calculations, the cell on the right is smaller than the cell on the left. But looking at the diagrams, it is the cell on the right that looks larger. How can this be?

PART V: MEASURING LIVING CELLS

6.10 Exercise 10 – Preparing a wet mount of a naturally pigmented (red onion) cell.

To view living cells under a microscope, you first have to "wet" mount them on a glass slide. Prepare wet mounts of onion cells as follows: 1. Place a clean microscope slide on a paper towel. Put a drop of water (less than 1 cm diameter) in the center of the slide. 2. Obtain a wedge of red onion from the Central Study Area. With your fingers or forceps, remove a portion of the thin (cellophane-like) tissue that lines the outer surface of each scale-like leaf. With scissors or a razor blade, cut off a small (0.5 cm square) piece of epidermis and place it in the drop of water on the slide. 3. Place a cover glass over the specimen by holding the cover glass at a 45-degree angle against one edge of the water drop. Let the water spread along the width of the cover glass before slowly lowering the glass down over the material. Try to avoid trapping air bubbles under the cover glass. With your compound microscope, view your wet mount of onion epidermis. Use the circles below to represent the field of view (circle of light) as seen through your microscope. In the left-hand circle, sketch a few of the onion epidermal cells under high power. Record the magnification you are using (100x, 430x). For the onion cells, draw the highest magnification that allows you see at least one whole onion cell in the field of view. Use the circle on the left for this drawing.

Onion epidermal cells ( x) Human epidermal cells (430 x)

6.11 Exercise 11 – Staining a wet mount of an unpigmented (human cheek) cell.

1. Place a second clean microscope slide on a paper towel. Put a drop of water (less than 1 cm

diameter) in the center of the slide.

2. Obtain human epidermal cells from the inside of your own mouth. Shake a clean toothpick out of the dispenser. Gently scrape the inside surface of your cheek with the toothpick. Put the cheek cell scrapings into the water droplet. Spread out the scrapings by gently tapping the toothpick in the water droplet.

3. Place a cover glass over the specimen by holding the cover glass at a 45-degree angle against one

edge of the water drop. Let the water spread along the width of the cover glass before slowly lowering the glass down over the material. Try to avoid trapping air bubbles under the cover glass.

4. Unlike red onion cells, human cheek cells are not naturally pigmented. Cheek cells are almost

completely transparent and need to be stained. Place a small drop of methylene blue dye at one edge of the cover slip and touch a paper towel to the opposite side of the cover slip. The dye will be drawn under the cover slip and across the cheek cells, staining them.

5. After the dye has been in place for one minute, put a drop of clean water near the edge of the cover

slip and again touch a paper towel to the opposite side. This will remove excess dye, but leave the cells stained.

6. View the slide under the microscope. In the right-hand circle above, sketch a few of the cheek cells

under high power (over 400 x)

Table 3: Relative sizes of cells: List the lengths of several kinds of cells, including your three protozoans and any other specimens measured by other students in the lab.

Specimen Cell length (µm) Key features

Onion epidermis

Human epidermis (cheek cells)

A protozoan with cilia (Paramecium)

A protozoan with a flagellum Name:

A protozoan with pseudopodia Name:

Virus (influenza) Limit for light microscopes Bacteria (anthrax) Red blood cell (human) Airborne pollen Limit for naked eye Human ovum Grain of sand

0.02 µm 0.5 µm 1.0 µm 7.0 µm 25 µm 100 µm (= 0.1 mm) 100 µm (= 0.1 mm) 500 µm (= 0.5 mm)

6.12 Exercise 12 – Estimating the size of a unicellular organism

1. Water from ponds contains many interesting unicellular organism. One of the most common is the single-celled Paramecium – a fast moving organism covered with beating, hair-like cilia. Place a drop of Paramecium culture onto a clean microscope slide. For best results, take a sample of the "sludge" from the bottom of the container. 2. To see the fast-moving Paramecium better, add a drop of "Proto-slo" to the sample on you slide and stir using a dissecting needle or toothpick. Proto-slo slows down swimming organisms by "thickening" (increasing the viscosity of) the water. 3. Add a cover slip by holding a cover glass at a 45-degree angle against one edge of the water drop. Permit the culture solution to spread along the width of the cover glass. Then slowly lower the cover glass. Try to avoid trapping air bubbles under the cover glass. 4. Use the low power objective to find a Paramecium. Center the Paramecium in the field of view before switching to higher power. 5. Sketch the Paramecium under medium and high power in the circles below. Estimate its length, using the diameter of the field of view. Enter your estimate in Table 3. Paramecium (medium x) Paramecium (high x)

Sketch two additional protozoans (other than Paramecium). Use your drawings to estimate the size of these protozoans, and enter results in Table 3. Name _______________________ ( x) Name ____________________ ( x) Length ________ µm Length ________ µm Locomotion type ___________________ Locomotion type __________________ Based on the cells you have measured, are all protozoans about the same size? Are any of the species significantly larger -- say, 10-times larger -- than the others? CHECK OUT PASS: 1. Return your carrel to its original condition. An instructor will check your carrel and give you a "check out pass" to turn in at the front desk as you leave. 2. When you have finished using the compound microscope: a. Turn off the light source. b. Rotate the nose piece to the low power objective. c. Unplug the microscope and coil the wire loosely over the body tube. d. Cover the microscope. 3. Rinse off and dry the slides and cover slips you used for wet mounts. Any broken cover slips should be placed in the “broken glass” container near the fish tanks.

Lab 2 – Enzymes and Cell Function 7

YOU NEED TO WORK WITH A PARTNER FOR THIS LAB AND ALL REMAINING LABS THIS SEMESTER. Please plan ahead. Partners share the data they collect, but the reports you write for labs #2 and #3 must be written individually.

Growing, reproducing, digesting, and the many other processes of "life" involve thousands of different biochemical reactions. Without enzymes, almost none of these biochemical reactions would proceed quickly enough to sustain life. Enzymes are catalysts that can help break larger molecules into smaller molecules, or help join two molecules together, all while remaining unchanged themselves. Enzymes are involved in everything from photosynthesis to the fertilization of eggs by sperm, from the digestion of food to the clotting of blood. Enzymes are proteins that catalyze (speed up) vital biochemical reactions by reducing the "activation energy" needed to get the reaction going.

Without enzymes, the temperature inside living cells is too low, and the concentration of reacting molecules is too dilute, to sustain the biochemistry of life. Enzymes are extremely efficient. Minute quantities of an enzyme can accomplish at low temperatures what otherwise would require much higher temperatures and/or harsh chemical reagents. For example, one ounce of the stomach enzyme pepsin can digest two tons of egg white in a few hours. Without pepsin, digesting this much egg white would require 10-20 tons of strong acid working for 24-48 hours at high temperature. Enzymes are so extraordinarily efficient for four reasons: (1) Enzymes can be used over and over again because they are not themselves changed by the reactions they catalyze. In the process of converting one molecule (the substrate) to another (the product), the enzyme binds temporarily with the substrate to form an enzyme-substrate complex. The enzyme returns to its original form as soon as the transformation of the substrate into the product is complete.

Figure of “Lock-and-key model” is from Wikipedia.com

(2) Enzymes are extremely specific. They are very choosy about what substrates they will bind with and what reactions they will catalyze. Most enzymes bind with only one particular kind of molecule (like a "lock and key") and cause only one particular kind of change in that molecule. Some enzymes specialize in synthesis (joining two substrates) while others specialize in splitting the substrate into products. (3) Enzymes are extremely reactive, much more reactive than ordinary chemical catalysts. For example, hydrogen peroxide (H2O2) by itself slowly decomposes into water and oxygen. A small

amount of powdered iron will act as a catalyst and speed up this decomposition several fold. But a single molecule of the enzyme catalase (found in human blood) can, in one minute, split more than five million peroxide molecules! Catalase is one of the fastest acting enzymes known. Other enzymes operate on their substrates at rates ranging from 1000 to 500,000 molecules per minute. (4) Most enzymes function best within a narrow range of temperature and pH (acidity). For example, as temperature rises, the rate of an enzyme-catalyzed reaction will at first increase because the enzyme and substrate molecules move around more quickly and so encounter each other more often. But above a certain temperature most enzymes become denatured (lose their shape) and so lose their catalytic activity. In this lab, you will learn about some of the qualitative characteristics of enzyme catalyzed reactions (Part I) and then quantify the effects of environmental factors (temperature, pH) on the rate of enzyme- catalyzed reactions (Part II).

Lab learning objectives

You will have mastered the content of this minicourse when you are able to: 1. Explain why life on earth could not exist without enzymes. Using specific examples, explain how the

metabolic efficiency of living organisms is improved by the extreme reactivity and specificity of enzyme catalysts (See Introduction.)

2. Use a spectrophotometer and other qualitative and quantitative techniques to measure the activity

of an enzyme under different environmental conditions. 3. Graph and analyze the effects of temperature and acidity (pH) on the rate of an enzyme catalyzed

reaction. 4. Explain, at the molecular level, why enzymes work better at certain temperatures and pH’s. Include

the effects of pH and temperature on molecular motion and shape. PART I: A SIMPLE ENZYME-CATALYZED REACTION When you cut open an apple, banana or pear, you cut through many cell walls and release the complex contents of many cells. Left exposed to the air, the white surface of the fruit will turn brown in a short time. This familiar color change is a good example of an enzyme catalyzed reaction. The brown color comes from the reaction of oxygen in the air with catechol in the fruit to form benzoquinone. Catechol (the substrate) is clear and benzoquinone (the product) is brown. This browning reaction is catalyzed by catecholase, an enzyme that occurs naturally in the fruit.

catecholase Catechol + 1/2 O2 ------------> benzoquinone + H2O

(clear) (brown)

This simple browning reaction can be used to illustrate several important properties of enzymes in general. In Part I, you will make some preliminary qualitative observations on the nature of enzyme catalyzed reactions by recording how environmental factors affect the browning reaction. For example, must the fruit be exposed to air to turn brown? Does temperature affect the rate of browning? Is the browning rate changed by the presence of an acid, like lemon juice? You will be asked to use the observations you make in Part I to formulate qualitative hypotheses about how enzymes work. In Part II you will test your hypotheses quantitatively.

7.1 Exercise 1 – Browning of fruit

In this exercise you will compare the relative amount of browning that occurs in banana slices placed under six different conditions. Methods 1. Work with a partner. 2. Set up the six experimental treatments (described below) before you obtain a piece of banana. The comparison of browning rates will be easier if all the slices begin browning at the same time.

 To observe the effect of contact with air (oxygen):

 Slice 1: leave open to the air at room temperature.

 Slice 2: immediately wrap tightly in saran wrap.

 To observe the effect of temperature:

 Slice 3: place in a beaker of ice.

 Slice 4: place in a petrie dish on a warming plate.

 To observe the effect of acidity (pH):

 Slice 5: keep moist with distilled water in a petri dish.

 Slice 6: keep moist with lemon juice in a petri dish. 3. Acidity is measured in terms of a unit called pH. On the pH scale, any value below 7 is considered acidic, anything above 7 is basic, and 7 is neutral. Acids typically have pH values between 2 and 6. The stronger the acid, the lower the pH value. Use litmus paper to determine the acidity (pH) of lemon juice and water. Lemon juice pH = ______ Distilled water pH = ______ 4. From a fresh banana, cut 6 equal, unpeeled slices. Working quickly, place one slice in each of the conditions described in step 2. 5. Check the sections every 3-5 minutes for the first half hour. In Table 1 record (a) the time you first detect browning, and (b) the extent of subsequent browning (if any).

Between observations, begin Part II.

Results Table 1: Extent of browning under different environmental conditions.

Minutes elapsed to first browning

Extent of further browning

Slice #1 (open to air)

Slice #2 (wrapped)

Slice #3 (cold)

Slice #4 (warm)

Slice #5 (distilled water)

Slice #6 (lemon juice)

Analysis and Discussion l. Look at the browning rates of slice #1 (exposed to air) and slice #2 (wrapped in plastic). a. Which one turned brown faster? Why? (With what is the catechol reacting?)

b. If your observations are not what you expected, consider this. Ripening fruit releases ethylene, a gas that speeds the ripening process. Saran Wrap Classic is made of polyvinyl and Saran Wrap with Cling Plus (formerly HandiWrap) is polyethylene. How might this affect browning rate? c. How could you test whether the wrapping material is increasing browning rate?

2. If you use the browning rate of slice #1 (left out at room temperature) as your standard of reference (called your experimental "control") : a. What effect (if any) did cooling have on the browning rate? b. What effect (if any) did warming have on browning rate? c. What do these observations suggest about the effect of temperature on enzyme-catalyzed reactions? -- Based on these observations, what should happen to the rate of browning as temperature increases? State this in the form of a hypothesis. HYPOTHESIS A: "As temperature increases, the rate of an enzyme catalyzed reaction (browning) should ___________________________." (Choose one: increase, decrease, or remain the same) 3. Comparing the browning rates of slice # 5 (treated with distilled water) and slice #6 (treated with lemon juice). a. What effect did lemon juice have on the rate of browning? b. What do these observations suggest about the effects of acidity on enzyme activity? -- Based on these observations, what should happen to browning rate as pH increases? (Remember, the stronger the acid, the lower the pH.) State this in the form of a hypothesis. HYPOTHESIS B: "As pH increases (meaning the environment becomes less acidic), the rate of an enzyme catalyzed reaction (browning) should _________________________________." (increase, decrease, or remain the same) These last two hypotheses are based on the qualitative observations you made in Part I. They may or may not be correct. In Part II, you will test these last two hypotheses quantitatively.

PART II: EFFECTS OF ENVIRONMENTAL FACTORS ON RATE OF ENZYME-CATALYZED REACTIONS In this section you and a partner will design and carry out experiments to test the two hypotheses you made at the end of Part I concerning the effects of temperature and pH on enzymes. The quantitative techniques you will be using will allow you to measure rates of the browning reaction far more accurately than before. The experiments are intended to give you first hand experience with the methods of scientific inquiry. They also reveal some interesting features of enzyme-catalyzed reactions. The Spectrophotometer The Spec 20 is a device that measures how "dark" (opaque) a liquid is. The Spec 20 shines a beam of light through the liquid in a test tube and measures how much of the light is absorbed as it passes through the test tube. Measurements are expressed in terms of "absorbance" on a scale from 0 to 2.

You will be using the Spec 20 to measure the rate of the browning (benzoquinone formation) under different conditions. As more and more (clear) catechol is converted to (brown) benzoquinone, less and less light will be able to pass through the test tube, and the percentage of light absorbed will increase. We will be using absorbance to measure the amount of benzoquinone produced under different temperatures and acidities (pH's).

Follow this overall, 3-step procedure whenever you need to use the Spec 20. For exercises 2 and 3, you will need to use the Spec 20 to measure rates of reaction. Use these three steps as a reference when you do exercise 2 (page 9) and exercise 3 (page 12). 1. Prepare a reference solution (or "blank")

The starting solution of enzyme and substrate is not perfectly clear and so absorbs some light even before the browning reaction begins. To correct for this fact, you will be using a "blank," a test-tube filled with the starting solution as a reference. You use the amount of light absorbed by the blank as your reference point. The Spec 20 can be adjusted to treat the amount of light absorbed by the blank as "zero". Any additional light absorbed can then be attributed to newly formed benzoquinone. To make a blank:

a. Label a small test tube with the letter "B" for "blank". This test tube will hold the reference

solution for setting the "0% absorbance" level on the Spec 20. b. Use a pipette and pipette pump to measure 1 ml of potato extract and 6 ml of distilled

water into the tube. c. Cover the tube with a small square of parafilm and invert to mix.

2. Calibrate the Spec 20 (i.e., standardize the internal light level): a. Set the wavelength knob on the Spec 20 to 540 nanometers, a wavelength in the “green” portion of the light spectrum. We use 540 nanometers in this experiment because benzoquinone (the product) absorbs this wave length better than any other. b. With the sample compartment empty and the cover closed, set the transmittance to zero by using the Zero Control (left hand) knob or by following the directions posted next to the Spec 20. c. Wipe off the "blank" tube with a Kimwipe (to remove light-absorbing fingerprints) and insert it into the sample compartment and close the cover.

d. Set the absorbance reading to zero by using the Absorbance (right hand) knob, or by following the posted instructions. Calibrated in this way, the Spec 20 will measure only increases in absorbance above the reference level established by the “blank”. 3. To measure the absorbance of your samples in Exercises 2 & 3: a. Remove the blank. (Blanks get old fast. When setting up the testube series you will be using for each experiment, make a fresh blank at the same time.) b. Insert the sample tube into the sample compartment. c. Close the cover of the sample compartment. d. Read "absorbance " off the lower dial. e. Remove sample tube and repeat steps 3b-d with each sample.

In Part II, you and your partner should work together on both the temperature experiment (Exercise 2) and the pH experiment (Exercise 3).

7.2 Exercise 2 – Effect of temperature on enzyme-catalyzed reactions

For Exercise 2, you will be setting up test tubes containing potato extract (a good source of the enzyme catecholase), water, and catechol (the substrate). Even though potato contains catechol, you will be adding extra substrate (catechol from a commercial supplier) to make the reaction go faster. There are several different ways to speed up an enzyme catalyzed reaction. One way is to add more enzyme. Another way is to increase the concentration of the substrate on which the enzyme is working. In this experiment, we will be making the reaction go faster by adding extra substrate (namely catechol). We are not adding extra enzyme. There is plenty of the catecholase enzyme in the potato extract already. Set up all your tubes with everything in them (see below) except the catechol. When everything is ready add the catechol last, so all the reactions start at the same time. 1. Obtain 6 test tubes and a test tube rack. With a wax pencil, mark the tubes near the top with your initials and numbers 1 through 5, plus B (for "blank", the tube that will hold the reference solution used to calibrate the Spec 20.) 2. With a pipette, measure into each of the 5 tubes: 1 ml of potato extract (a rich source of the enzyme catecholase) and 4 ml of water. Avoid picking up any of the particulate matter (cloudy with particles) that has settled to the bottom of the potato extract flask. Any cloudy material will throw off the measurement of browning rate. 3. To make a "blank," put 1 ml potato extract and 6 ml of water into the sixth tube. The extra 2 ml of water makes up for the 2 ml of catechol that you will not be adding to the control blank. Cover all 6 tubes with parafilm, invert to mix, and stand the tubes in rack. 4. Do not add catechol yet. Put one sample tube into each of the 5 different water baths in the Central Study Area. In Table 2 record the actual temperatures of each of the baths. 5. BEFORE ADDING THE CATECHOL to your samples, use a thermometer to make sure the temperature of the solution inside your test tube has actually reached the temperature of its water bath. This should take 3-7 minutes. 6. Plan ahead! Read steps #7-9 completely before proceeding. Try to run the 5 tubes simultaneously, or closely together in the same sequence, so that reaction times in the 5 samples will be comparable. 7. Add 2 ml of catechol solution to each of the 5 sample tubes. You will need to remove the tube from the water bath, remove the parafilm, add the catechol, put the parafilm back on, and invert tube to mix the contents. Return each tube to its bath for 5 minutes. 8. Use the blank (test tube “B") you made earlier to recalibrate the Spec 20. 9. Exactly 5 minutes after adding the catechol, remove each sample tube from its water bath, dry it with a Kimwipe, insert the tube into the sample holder of the Spec 20, and measure absorbance. Quickly repeat for the other 4 tubes, one at a time, in numerical order. Record these values in Table 2.

Table 2: Effect of temperature on extent of browning

Sample Temp (oC) Absorbance after ( ) minutes

Any color changes? (see #11 below)

Blank

10. If you experience measuring delays, you will need to control for the fact that the reaction product continues to form while you are waiting to measure it. After measuring the absorbance in tubes 1, 2, 3, 4, and 5, measure them again in reverse order (5, 4, 3, 2, 1) and use the average absorbance for each temperature. 11. If the reaction is proceeding correctly, you should see (with your naked eye} a darkening of the solution. This rusty brown precipitate is benzoquinone, the desired product of the reaction. If you see “cloudiness,” it means you mistakenly picked up particulate matter (sludge) from the bottom of the flask. 12. Plot your values for absorbance as a function of temperature using the graph paper below. If necessary, you may change the absorbance scale on the y-axis to 0.5 and 1.0. Graph 1: Effect of temperature on browning rate

Temperature of water bath (oC)

Analysis and Discussion 1. At what temperature(s) was the rate of reaction greatest? ____ At what temperature(s) was the rate lowest? ____ Are these observations consistent with the hypothesis about thermal effects you formulated at the end of Part I? ______ What do these observations suggest about the "optimal" range of temperatures for enzyme catalyzed reactions? State your conclusion in the form of a revised hypothesis. HYPOTHESIS A (Revised): The rate of an enzyme catalyzed reactions is greatest at temperatures that are... 2. At the molecular level, what might explain the rate of the enzyme-catalyzed reaction at low temperatures? (Hint: For a reaction to occur, the two reactant molecules must “bump into’ each other. How does temperature effect the motion of molecules?) 3. At the molecular level, what might explain the rate of the enzyme-catalyzed reaction at high temperatures? (Hint: How does temperature effect the molecular structure of enzymes?)

7.3 Exercise 3 – Effects of pH on enzyme-catalyzed reactions

The pH (acidity) of the environment can affect the molecular bonds that maintain the shape of an enzyme’s “active site,” the site on the enzyme molecule responsible for binding to the substrate or substrates. A pH value of 2 is highly acidic, 7 is neutral, and 11 is highly basic. For Exercise 3, set up test tubes containing potato extract (a good source of the enzyme catecholase), catechol (the substrate), and 5 different pH buffer solutions. Set up all 6 test tubes with everything in them (see below) except the catechol. Add the catechol last, so all the reactions will start at about the same time. 1. Obtain 6 test tubes and use a wax pencil to mark all the tubes near the top with your initial and either numbers 1 through 5 or "B" for the calibration blank. 2. Fill the 5 tubes as follows: Sample 1: 1 ml potato extract, 4 ml buffer for pH 3 . Sample 2: 1 ml potato extract, 4 ml buffer for pH 5. Sample 3: 1 ml potato extract, 4 ml buffer for pH 7. Sample 4: 1 ml potato extract, 4 ml buffer for pH 9. Sample 5: 1 ml potato extract, 4 ml buffer for pH 11. Blank: 1 ml potato extract, 4 ml buffer for pH 7, plus 2 ml distilled water. The 2 ml of water is a substitute for the 2 ml of catechol that will be added to the other test tubes but not to the blank tube. You will need only one blank, because all 5 of the pH buffer solutions are clear and have identical absorbency properties. 3. Cover each tube with parafilm and invert to mix. Stand all 6 tubes in the test tube rack. 4. Now add 2 ml of catechol to the 5 sample tubes, put the parafilm back on, and again invert the tube to mix the contents. You do not need to uncover the tube for the reaction to proceed; the solution has plenty of dissolved oxygen. Keep the test tubes at room temperature. DO NOT PUT THE TEST TUBES IN THE WATER BATH! In this experiment we are measuring the effects of pH, not temperature. 5. If you see "cloudiness,” it means either than you mistakenly picked up particulate matter from the bottom of the flask, or that an unwanted precipitate is forming. A cloudy white precipitate often forms if the pH gets too low (pH = 3). A grayish black precipitate sometimes forms if the pH gets too high (pH = 11). When precipitates are formed by reactions that have nothing to do with “browning,” they can distort your data, causing you to overestimate the rate of browning at very low and very high pHs. So record any color changes and include them in your lab report. 6. About a minute before step 6, use your blank to calibrate the Spec 20. 7. Allow the browning reaction to proceed for exactly 5 minutes. Then insert the sample tubes, one at a time in numerical order, into the Spec 20 and record the absorbances in Table 3. If the reaction ran longer than 5 minutes before you took your measurement, record the exact number of minutes and use

this same time interval when you measure the other sample tubes. Record the time in Table 3. Note any color changes in the test tubes. 8. If you experience measuring delays, you will need to control for the fact that the reaction product continues to form while you are waiting to measure it. After measuring the absorbance in tubes 1, 2, 3, 4 and 5, measure them again in reverse order (5, 4, 3, 2, 1) and use the average absorbance for each pH. Results Table 3: Effect of pH on extent of browning

Sample pH Absorbance after ( ) minutes

Any color changes? (See #5 above)

Blank * zero

1 3

2 5

3 7

4 9

5 11

9. Wash all test tubes and return them to the rack. 10. Plot your values for absorbance as a function of pH on Graph 2. Graph 2: Effect of pH on the browning rate

Analysis and Discussion 1. At what pH values was the rate of reaction greatest? ____ At what pH values was the rate lowest? ____ Are these observations consistent with the hypothesis about the effects of acidity you formulated at the end of Part I? ______ What do these observations suggest about the "optimal” range of pH for enzyme catalyzed reactions? State your conclusion in the form of a revised hypothesis. HYPOTHESIS B (Revised): The rate of an enzyme catalyzed reactions is greatest at pH values that are... 2. The pH (acidity) of the environment can affect the hydrogen bonds that maintain the shape of an enzyme’s “active site,” the site on the enzyme molecule responsible for binding to its specific substrate(s). At the molecular level, what might explain the changes you observed in the rate of the enzyme-catalyzed reaction at low and high pH values? 3. Consider the effects of pH on these two enzymes. A pH value of 2 is highly acidic, 7 is neutral, and 11 is highly basic.

From these data, do all enzymes necessarily have the same optimal pH? Which one of these two enzymes do you think is more typical of the enzymes found in the human body? Where in the body would you find an enzyme whose optimal pH is 2?

Questions For Further Thought 1. Predict what would happen to a living organism exposed to temperatures that fall outside the optimal range for its enzymes. 2. List some familiar adaptations animals have to reduce the effects of temperature extremes on their many vital, enzyme-catalyzed reactions. a. b. c. d. 3. In general, the rates of chemical reactions double for every 10 degree (Celsius) increase in temperature. At the molecular level, what changes account for slower reaction rates of enzyme- catalyzed reactions at high temperatures? (Hint: enzymes are three-dimensional protein molecules. What happens to the shape of protein molecules when they are heated? How would this change the vital “active site” of the enzyme?) -- Why are enzyme catalyzed reactions also slower at low temperatures? (Hint: In a liquid or gas, what happens to the motion of molecules as they cool? Why would this affect the rate at which enzyme and substrate molecules collide and react?)

4. The binding of enzymes with their substrates is most efficient under certain (so called "optimal") pH conditions. An enzyme molecule’s 3-dimensional shape is maintained by hydrogen bonds and other chemical bonds sensitive to pH. How might extremes in pH change the efficiency of the enzyme?

5. Human cells and body fluids contain hundreds of natural buffer systems that keep pH at or very near optimal levels. Potato cells and human cells are both living and so have thousands of biochemical reactions in common. Given your results for catecholase, what value of pH is most likely to be found inside human cells?

LAB REPORT At your next Discussion class, hand-in a lab report to your TA. You and your partner can submit identical cover pages and data tables, but your introduction and discussion must be written by you, in your own words. (1) Cover page: including the title of the experiment (in this case, use "Effects of environmental factors on the rate of enzyme catalyzed reactions"), your name, the date, your discussion leader's name, and the number of your discussion section. Also include the names of all your partners and their discussion section leaders. (2) Introduction: State your two hypotheses about the effects of temperature and acidity (pH) on the rate of this enzyme catalyzed reaction. Explain why each hypothesis makes sense to you. State the prediction you generated from these hypotheses and describe (in general terms) how you tested them. You don’t need to detail the methods (because they are already in the lab guide), but you do need to say enough to show you understand the experiment; e.g., which substance is the enzyme, which is the substrate, and why your Spec 20 data can be used to compare the rate of the reaction under different conditions. (3) Results: On a separate page, summarize your data from the tables and graphs on pages 10 and 13. (4) Discussion: In 1- 2 pages, explain why each of your two curves is shaped the way it is. Explain why your curves went up or down at low, intermediate, and/or high temperatures and pH's. Compare your actual curves to the theoretically-expected shapes for these curves. You should include any relevant parts of your answers to the questions raised in the “Analysis and Discussion" and “Questions for Further Thought” sections of the lab guide.

Lab 3 – Cell Membranes and Water Balance 8

PLAN AHEAD! This lab requires working with a partner and writing a lab report.

Osmosis is the vitally important process by which water moves in and out of cells. Overall, the cells of living organisms are composed of about 75-85% water. The concentration of water is even higher in the fluid inside cells; i.e., the fluid containing dissolved substances that are not attached to membranes. Life depends on having the proper concentration of water inside the cell. If there is too little water, the chemical reactions that support life will not be able to proceed and the cell will die. If there is too much water inside, the cell may burst. In this minicourse, we will examine factors affecting the movement of water in and out of cells. The earliest cells are thought to have originated and evolved in the ocean. Is the internal environment (i.e., the intracellular fluid) of cells still a lot like sea water (97% water, 3% dissolved salts)? In this minicourse, you will also compare the concentration of water and "solutes" (dissolved substances or "salts") found inside plant cells from different environments -- fresh water, marine and terrestrial.

Lab learning objectives You will have mastered the content of this minicourse when you are able to: 1. Define diffusion and osmosis in terms of concentration gradients and the movements of molecules. Explain (in terms of concentration gradients and internal pressure) the effects of osmosis on cells placed in hypertonic, hypotonic and isotonic environments. 2. Use an osmometer to measure changes in internal pressure in model "cells" (dialysis bags) placed in different "environments" (solutions) and graph your results. Explain why internal pressure increases over time and then stabilizes. 3. Describe how the effects of osmosis in plant cells are modified by the presence of a cell wall. Contrast turgor and plasmolysis. 4. Use the plasmolysis threshold of cells to compare the isotonic points (internal solute concentrations) of plant cells from fresh water, marine, and terrestrial environments. PART I: OSMOSIS Molecules (including water, oxygen, carbon dioxide) are in continual motion, colliding and bouncing off each other in random directions. Because the movement of each molecule is random, it is impossible to predict the direction any one molecule will be moving at any given time. However, if you look at huge groups of randomly moving molecules (billions of molecules at a time), predictable patterns emerge. Namely, the "net” (or overall total) movement of molecules is from areas where they are in high concentration to areas where they are in low concentration.

One of the principal ways molecules get in and out of cells is diffusion. Diffusion is defined as the net movement of molecules from areas of higher concentration to areas of lower concentration. For example, if you place a crystal of a purple dye in a glass of water, the purple color spreads out as the dye molecules diffuse through the water. Another example of diffusion is inside the air sacs of your lungs, where the net movement of oxygen is out of the air and into your blood. Because oxygen molecules move in random directions, there will always be some molecules moving in the "wrong" direction. But overall, more oxygen molecules move out of the air than out of the blood, simply because there are more oxygen molecules in the air than in the blood. The diffusion of water molecules is so important to living cells that it is given a special name, osmosis. Osmosis is the net movement of water molecules across a semi-permeable membrane. [Semipermeable membranes allow small molecules to pass through them, but not larger molecules like sugar and dye.] When the concentration of water molecules is higher outside than inside the cell, the net movement of water will be into the cell and the cell will swell. When the concentration of water molecules is lower outside, the net movement of water will be out of the cell and the cell will shrivel. The cell membrane of a living cell is similar to a semipermeable membrane in that it permits some substances to cross it but not others. For example, the cell membrane allows small, uncharged molecules (water, oxygen and carbon dioxide) to diffuse freely, but blocks the diffusion of molecules that are large (e.g., sugar, proteins) or are electrically charged (e.g., sodium and chloride ions). In the exercise below, you will make a simple "model" cell from (kidney) dialysis tubing – plastic tubing manufactured with holes just large enough to be permeable to water but not permeable to large molecules like sucrose (table sugar). Now suppose you filled the dialysis bag with water and a little sucrose, and then placed the bag in a beaker of water. Water will begin to move by osmosis into the bag. But why? Think of it this way: the concentration of water inside the bag is lowered by the presence of the sucrose molecules. (In a sense, the dissolved sugar molecules "dilute" the water, lowering the water's concentration.) Because the concentration of water is higher outside than inside, water diffuses "down" its concentration gradient and into the bag. Unlike water, sucrose is unable to flow down its concentration gradient, because sucrose molecules are too large to get through the pores and out of the bag. Because sucrose molecules can't get out, their presence will keep the concentration of water molecules always lower inside the bag compared to the pure water outside the bag. So water will continue to move into the bag (a) until the bag bursts, or (b) until the internal fluid pressure gets high enough to prevent more water from diffusing in. Notice that because of the presence of sucrose inside the bag, the concentration of water inside can never become the same as the concentration of the water outside. With this background, you can now understand the formal definition of osmosis: the passive movement [= diffusion] of water across a semi-permeable membrane in response to differences in pressure and solute concentrations on either side of the membrane. (We define solute as any dissolved substance. Sucrose is the solute in the above example.)

The Osmometer Osmotic pressure is the tendency of water to move into a cell. An osmometer is a device designed to measure osmotic pressure by measuring the amount of internal hydrostatic pressure needed to stop the movement of water into a cell. The osmotic pressure experienced by a cell differs under different environmental conditions. Figure 1. An osmometer. The height of the standing column of liquid in the glass tube provides a way to measure the hydrostatic pressure of the liquid inside the dialysis bag. The higher the column, the greater the internal pressure. The osmotic pressure for different experimental setups is measured as the height of the column after the fluid level has stopped rising. The fluid stops rising when the water pressure inside the bag (the hydrostatic pressure) has increased to the point where it it is equal to the osmotic pressure and stops any more water from moving in.

8.1 Exercise 1 – Movement of water into a "model cell"

In Exercise 1, as water moves by osmosis into a dialysis bag (our "model cell"), the pressure in the bag increases. You will quantify this pressure change by measuring the height of the water in a glass tube inserted into the bag (See Figure 1). A taller column contains more water, so it weighs more and exerts more downward pressure. 1. Work with one or two partners. Write your partners’ names here:

Partner’s name Discussion leader’s name

2. After looking at the osmometer set up in the Central Study Area, obtain the following materials: 20 cm of dialysis tubing 400 ml beaker scissors 10% sucrose solution string (with dye added) glass tube marking pencil ruler supporting clamps 4. Cut a piece of dialysis tubing 20 cm long. Soak the tubing under running water until soft. Tie a tight knot in one end of the dialysis tubing. Open the other end by rubbing it between your fingers. 5. While your partner holds the bag, carefully pour in 8-10 cm worth of 10% sucrose solution containing dye. (The dye is used as a tracer to let you see if any sucrose solution is leaking out of the bag.) Insert the glass tube into the dialysis bag. Tie the upper end of the bag around the glass tube by wetting a piece of string and wrapping it a few times around the tubing before knotting it. Try to minimize the air pocket left inside the bag. 6. Make sure that: (a) the filled portion of the bag is less than 10 cm long (b) the submerged end of glass tube is about 5 cm from the bottom of the bag (c) the air space inside the bag is as small as possible, (d) that top end of the tube is at least 15-18 cm above the fluid level in the bag, (e) there are no leaks. (There should be no green dye outside the tube.) 7. Hold the bag and glass near the upper knot and carefully rinse off the dialysis bag with water. Avoid squeezing the bag. If properly tied, no dye should leak out. 8. Fill the beaker with about 360 ml of room temperature water from the carboy and lower the dialysis bag into the beaker (See Figure 1). Support the upper end of the glass tube with clamps in such a way that (a) the water level in the beaker is about l.5 cm below the upper end of the fluid inside the dialysis bag, and (b) the bag is not touching the bottom of the beaker.

9. Immediately mark the level of the solution in the glass tube by wrapping a second piece of wet string twice around the tube. Move the string to mark the starting level of the fluid in the glass tube. At 5 minute intervals, measure how far (in millimeters) the top of the column of solution in the tube has risen above the starting level. Record your observations in Table 1, then plot these results on Graph 1. 10. You will need to know: (a) When was the rate of rise the greatest? and (b) At what level (in mm) did the fluid in the tube stop rising (if it stopped)?

While monitoring the fluid level rising in the tube, begin Part II.

Table 1: Changes in internal pressure (height of fluid column)

Elapsed time (minutes)

Distance above mark (millimeters)

Change (# of mm) per 5 min. interval

From Graph 1, at what height did the column of fluid in the tube stop rising? _______mm This height can be used as a measure of the “osmotic pressure” inside our model cell in this particular “environment”.

Analysis and Discussion 1. In terms of water molecules and solute concentration gradients, why does the fluid level in the osmometer rise, at least at first? (Hint: Think about the concentration gradient and the net movement of water at the molecular level.) 2. Look at the numbers in Table 2, and at the steepness of the slope of the graph. Why does the rate of rise (the number of mm of rise per 5 min. interval) change as the experiment progresses? (Hint: Again think at the molecular level.) How might the net inward movement of water molecules be affected by the water pressure (or “hydrostatic pressure”) that is building up inside the bag? 3. When the fluid level finally stops rising, what keeps more water from moving in? Warning: The reason is not that the concentration of water is equal on both sides! The concentration of water inside the bag is always going to be lower, because all the sucrose molecules are trapped inside and so are still “diluting” the water. 4. “What If” Questions: What would have happened … … if all the sucrose had been placed in the beaker rather than inside the bag? … if the glass tube had been only 5 cm long?

PART II: WATER CONTENT OF CELLS Although living tissue is about 75-85% water, the effective concentration of solutes inside cells is not 15- 25%. Most of the non-water components of cells are bound into cell structures and are not in solution, so do not influence osmotic pressure. It is the concentration of solutes (substances actually dissolved in the fluid portion of the cell) rather than overall water content that determines whether water will diffuse into or out of a cell. Just what is the actual concentration of solutes (or "salts") in intracellular fluids, the internal fluids of cells? Is it anything like sea water (97% water, 3% salts) in which the first cells originated and evolved? Let’s find out. In Part II you will learn a technique to estimate the concentration of solutes and water in living cells without killing them. You will use this technique to determine and compare cells from different environments -- fresh water, marine, and terrestrial. The direction of osmosis Whether osmosis will move water into a cell or out of it depends on the "environment" surrounding the cell. Unfortunately, environments are not described in terms of their water content (e.g., 97% water). Instead environments are described in terms of their "solute concentration,” meaning the concentration of substances dissolved in the water (e.g., 3% salt). What is most important is the relative concentration of solutes in the environment compared to the solutes inside the cell. For example, when the environment’s solute concentration is higher than inside the cell, its water concentrations will be lower than in inside the cell, so water will move by osmosis out of the cell, dehydrating the cell. There are three possible environmental conditions:

Environment Solute concentration Water concentration Water movement

Hypertonic Higher than in cell Lower than in cell Out of the cell

Hypotonic Lower than in cell Higher than in cell Into the cell

Isotonic Same as in cell Same as in cell No net movement

1. In hypertonic environments, solute concentrations are higher in the environment than they are inside the cell. (Hyper means more, in this case more solute.) That means water moves out of the cell. 2. In hypotonic environments, solute concentrations are lower outside than inside the cell, and water moves into the cell. (Hypo means less, in this case less solute.) 3. In isotonic environments, solute concentrations are the same outside as inside, so there is no net movement of water. Water moves out at the same rate it moves in. (Iso means equal.) Turgor and plasmolysis Everyone knows that cut flowers need to be kept in water. But why? In plant cells, the cell membrane is surrounded by a rigid cell wall. When plant cells are placed in water -- a hypotonic environment -- water diffuses into the cell, pressure builds inside, and the cell’s plasma membrane becomes pressed tightly

against the cell wall. The cell does not burst, but instead becomes turgid. Turgor pressure is the pressure exerted on the cell wall by the fluid contents of the cell, keeping the flowers erect. When a plant cell is placed in a hypertonic environment, water diffuses out of the cell. Having lost its turgor pressure, the cell’s plasma membrane collapses and pulls away from the cell wall. This shrinking of the cell membrane away from the cell wall is called plasmolysis. Figure 2. Turgid (left) and plasmolyzed plant cells.

8.2 Exercise 2 – Using the plasmolysis threshold to estimate the concentration of solutes and water inside living cells

Under your compound microscope, the cell membrane of a turgid cell is invisible because it is pressed up against the inner surface of the cell wall. But in a plasmolyzed cell, the cell membrane is visible because it has pulled back away from the cell wall. We will use this fact to estimate the concentration of water and solutes inside living cells. In Exercise 2 you will be placing cells into a series of "environments"; i.e., test solutions with known solute concentrations. Your objective is to find the “isotonic points” for each cell type-- the test solution whose solute concentration is equal to the solute concentration inside the cell. At the “isotonic point” point cells are close to the balance point between turgid and plasmolyzed. Viewed under a microscope, an isotonic environment is one in which about half the cells appear turgid and half are plasmolyzed.

Table 2: Using plasmolysis and turgor to determine the solute concentration inside plant cells.

When the cell membrane is ...

The environment is said to be ...

The concentration of solute in the environment is...

plasmolyzed (shrunken & visible) in all cells

hypertonic greater than that inside the cell

turgid (invisible) against the cell wall in all cells.

hypotonic less than that inside the cell

turgid in about half the cells, plasmo- lyzed in about half

isotonic equal to that inside the cell

Procedure 1. Continue to work with 1-2 partners. View the videotape on plasmolysis and how plasmolysis is used to determine the isotonic points of cells. In the videotape, the solute used is sucrose (sugar). We will be using sodium chloride (table salt) as the solute. 2. Obtain a wedge of red onion and peel off a few thin sheets of outer epidermal tissue. To see the difference between plasmolyzed and unplasmolyzed cells, place a sample of onion epidermis in small beakers containing10 ml pure water (0% salt solution) and one with 10% salt solution. Wait for 10 minutes. The cells in pure water (0% salt) will be unplasmolyzed, so can be used as a standard for comparison. 3. To make sure you have actually seen the difference between plasmolyzed and unplasmolyzed red onion cells, sketch some actual cells in the circles below. Unplasmolyzed onion cells ( x) Plasmolyzed onion cells ( x) Being able to see the difference between plasmolyzed from unplasmolyzed cells is crucial for your experiment in Part III. If you are having trouble, be sure to ask for help from one of the learning center instructors.

4. While waiting for the samples in step 2, put additional samples of onion tissue into a series of 4-5 beakers containing concentrations of salt between 0 and 10%. Label a series of microscope slides with 0%, 10%, and your other testing solutions. 5. Allow the samples in step 4 to remain in solution for 10 minutes. Then prepare a wet mount of each specimen. One by one, examine each specimen under the microscope. Work quickly so the specimens do not begin to dry out under the microscope. 6. For each specimen, look only at the cells that contain red pigment. Count 50 cells and record in Table 3 how many of the 50 are plasmolyzed and unplasmolyzed. The natural pigmentation in red onion makes it easier to see whether the cell membrane has pulled away from the cell wall. 7. Calculate the percentage of cells plasmolyzed = Number plasmolyzed_ x 100% Number of cells counted and enter the data in Table 3 on the next page. 8. Repeat steps 5 and 6 (above) for several salt solutions. HINT: You are looking for the concentration at which about half (40-60%) of the cells are plasmolyzed, half unplasmolyzed. This indicates a concentration close to the boundary between plasmolyzed and turgid. If you plan ahead, you won’t need to test all of the concentrations! Look at the results of 0%, 5% and 10%, and then decide whethr you need to test 2 - 4%, or 6- 9%. You may then need to run only one or two additional concentrations to "zero in" on the solute concentration isotonic for onion epidermal tissue. 9. For each of the solutions you test, the percentage of cells plasmolyzed = Number plasmolyzed_ x 100% Number of cells counted Enter these results in Table 3. 10. From Table 3, the isotonic point for red onion appears to be about _____ %.

Table 3: The number of plasmolyzed and unplasmolyzed onion cells in different concentrations of salt solution.

Salt concentration

Number of cells plasmolyzed

Number of cells counted

Percent of cells plasmolyzed

10%

Analysis and Discussion 1. Red blood cells will burst if placed in distilled water (0% salt). Why didn't the onion cells burst? (Hint: what do plant cell have that animal cells don’t.) 2. Unfortunately the isotonic point doesn’t always give a good estimate of the actual concentration of solutes inside the cell. How does the concentration of solutes you found for onion cells compare to the concentration of solutes in sea water? If the solute concentrations are similar, why might this make sense? If they are not similar, what cell structures or mechanisms might help explain the difference? The Role of Evolution by Natural Selection Living cells and their membranes are much more complicated than the “model cell” you made out of semi-permeable dialysis tubing. In some plant species, plasmolysis is due primarily to the loss of water from the vacuole, a large storage chamber for relatively dilute fluids whose solute concentration may be lower than that in the cytoplasm proper. In other plant species, the cells may actually be able to resist plasmolysis in high-salt (hypertonic) environments due to properties of the cell membrane that actively pump solute ions in, or otherwise alter permeability of water and solute ions across the membrane. In your lab report, when you interpret your results from Part III, keep in mind that cells have adaptations that evolved for survival in hypotonic (freshwater) and hypertonic (marine/salt water) environments.

PART III: DESIGN YOUR OWN EXPERIMENT Do plants living in a freshwater pond have the same solute concentrations inside their cells as plants living in the salty ocean or on dry land? All plants are thought to have evolved from a common ancestor – single-celled green algae living in the ocean. In Part III you will design an experiment to test one of the following hypotheses:

Hypothesis 1: Since water is so critical for the life’s biochemical process, all plant cells should maintain the same internal solute concentrations as found in their marine ancestors.

Hypothesis 2: Since plant species evolved for survival in different environments, their internal solute concentrations should be different from their marine ancestors. Which hypothesis do you think is more likely to be true? Circle 1 or 2.

8.3 Exercise 3 – Comparing solute concentrations (isotonic points) inside plant cells from different environments

Using the plasmolysis threshold technique from Part II, design and carry out an experiment to determine whether the isotonic points of terrestrial and freshwater plants are the same or different than the isotonic points of marine plants. Procedure 1. Work in groups of 3-4. Write your partners’ names here:

Partner’s name Discussion leader’s name

2. Select three or four organisms from those available in the Central Study Area. In table 4A-D, record the names of the organisms and where they live. a. freshwater algae b. salt water algae c. terrestrial plants d. other plants, including any you bring in yourself

3. Based on the hypothesis you circled above (that is, that the cells will have the same or different internal concentrations), formulate a reasonable prediction about the outcome you expect. Your prediction should have the general form: Prediction: "If my hypothesis (1 or 2) is correct, then the isotonic point should be (higher/lower/the same) in cells from (aquatic, marine and/or terrestrial) environments than in cells from (aquatic, marine and/or terrestrial) environments. Write below the prediction you plan to test: Prediction: If my hypothesis is true, then… 4. Working with your group, test your prediction by following the procedures used in Exercise 2. Enter your results in Table 4A-D. Table 4A: Plasmolysis of the cells of ____________________________________in different concentrations of salt solution. This species lives in ____________________________________________________

Salt concentration

Number of cells plasmolyzed

Number of cells counted

Percent of cells plasmolyzed

Table 4B: Plasmolysis of the cells of ____________________________________in different concentrations of salt solution. This species lives in ____________________________________________________

Salt concentration

Number of cells plasmolyzed

Number of cells counted

Percent of cells plasmolyzed

Table 4C: Plasmolysis of the cells of ____________________________________in different concentrations of salt solution. This species lives in ____________________________________________________

Salt concentration

Number of cells plasmolyzed

Number of cells counted

Percent of cells plasmolyzed

Table 4D: Plasmolysis of the cells of ____________________________________in different concentrations of salt solution. This species lives in ____________________________________________________

Salt concentration

Number of cells plasmolyzed

Number of cells counted

Percent of cells plasmolyzed

Analysis and Discussion For cells to survive in environments whose salinity is much lower or much higher than 3%, the cells may need to pump salt in (or out) to maintain a suitable internal environment. However, molecular pumps require a lot of energy (ATP), so we would expect cells to have evolved various ways to reduce this energy expenditure. 1. Freshwater plants are surrounded by water in which the salt concentration is much lower than that of sea water. Do any of your isotonic point data suggest that freshwater plant cells might have a lower internal salt concentration than marine plant cells? What advantages and/or disadvantages might this have for freshwater plants? 2. Intertidal organisms that live at the edge of the ocean experience wide fluctuations in salinity. For example, when the tide is in, the intertidal area is covered with sea water (3% salt). As the tide goes out, tidal pools are left behind to evaporate in the sun, greatly increasing salinity in the pools. However, after a rain, freshwater running into the tidal pools can greatly reduce salinity. Do any of your data suggest that marine plant cells may have evolved ways to stablilze their internal environments in the face of the wide fluctuations in salinity found in intertidal environments? Explain.

LAB REPORT Hand in to your discussion leader a typed lab report. You and your group members can submit identical cover pages and data tables, but your introduction and discussion must be written by you, in your own words. (1) Cover page: including the title of the experiment (in this case use "Comparing cell contents of plants from different environments" ), your name, the date, your discussion leader's name, and the number of your discussion section. Also include the names of all your partners and their discussion section TAs. (2) Introduction: State your hypothesis about whether the concentration of solutes inside the cells of plants from different environments should be the same or different. Explain briefly why your hypothesis makes sense to you. State the prediction you generated from this hypothesis and describe (in general terms) how you tested it. You don’t need to detail the methods (because they are already in the lab guide), but you do need to define an isotonic point (especially what you consider to be its relationship to the cell’s internal solute concentration) and explain how you used isotonic points to test your prediction. (3) Results: On a separate page, summarize your data from tables 3 (onion cells) and 4 (three other kinds of cells) into one table, clearly labeled. (4) Discussion: In about 2 pages, explain what an isotonic point is and compare the isotonic points of your specimens with each other and with sea water. Does there appear to be a relationship between isotonic points and environment in which the plants are found? If not, then what cellular mechanisms (salt pumps, impermeable cell membranes, or others?) might these plant cells be using to maintain a stable internal environment despite widely differing external environments? Include some of the analysis and discussion questions raised on the previous page.

Lab 4 – Photosynthesis 9

Almost all life on earth depends directly or indirectly on photosynthesis: the ability of certain organisms (notably green plants) to capture and store the energy of sunlight. Photosynthesis in green plants involves a complex series of reactions, but the overall process uses carbon dioxide and water to make glucose (a simple sugar) and other carbohydrates, with oxygen as a by-product. sunlight

6 CO2 + 12 H20 ----------------> C6H12O6 + 6 O2 + 6 H2O

The sun’s energy is stored in the bonds that hold the glucose molecule together. Photosynthesis occurs inside the green, chlorophyll-rich cell organelles called chloroplasts. Inside the chloroplasts are stacks of "thylakoids" (flattened sacs), aligned to keep the light-absorbing chlorophyll exposed to sunlight. The energy stored in the glucose can later be released by a process called respiration. Respiration involves another complex series of steps, but the overall reaction is roughly the reverse of photosynthesis:

C6H12O6 + 6 O2 -----------------> 6 CO2 + 6 H2O + Energy

During respiration, glucose is broken down into carbon dioxide and water. The energy stored in the glucose is released gradually, a little at each step, and stored in small ATP molecules. The ATP's then carry the energy to wherever it is needed inside the cell. Respiration occurs inside mitochondria, which are cell organelles packed with internal membranes in which the enzymes of respiration are embedded. This minicourse focuses on photosynthesis the effects of environmental factors on the rate of this enzyme-catalyzed reaction.

Lab learning objectives

You will have mastered the content of this minicourse when you are able to: 1. Explain how paper chromatography is used to separate the light-absorbing pigments in a leaf. Prepare and interpret a chromatogram. 2. Given a leaf cross section, identify the major cell layers and explain in what way(s) each layer aids photosynthesis by the chloroplasts. 3. Analyze and discuss the results of experiments concerning the effects of the environment and osmosis on guard cells and stomata. 4. From your own experiments and data from others, describe how the rate of photosynthesis is affected by the following factors: the availability of carbon dioxide, light intensity, light quality, temperature and/or pH.

5. Given what you know about the molecular properties of enzymes, explain why the rate of photosynthesis should be affected by each of the environmental factors listed above.

PLAN AHEAD! You will need to work with a partner for lab #4. A lab report is NOT required this lab.

PART I: LIGHT-ABSORBING PIGMENTS Sunlight is "white" light composed of all the wavelengths (colors) of the visible spectrum: red-orange- yellow-green-blue-indigo-violet (or "Roy G. Biv."). Photosynthesis begins with the absorption of sunlight by pigments in the chloroplasts. These pigments absorb some colors of light better than others. For example, a green leaf contains the pigment chlorophyll. The leaf looks green not because chlorophyll absorbs green light, but because it’s molecular bonds absorb all the colors of the spectrum except green. The green light either reflects back to your eye, or passes completely through the leaf and out the other side.. Since chlorophyll can’t absorb any green light, does that mean all the energy in green light goes to waste? Might the leaf contain other pigments that absorb green light? To find out, do Exercise 1.

9.1 Exercise 1 – Separation of leaf pigments by paper chromatography

Chromatography is a technique used to separate substances in a mixture. It can be used to determine what pigments are present in a leaf. Paper chromatography can separate different pigments only if those pigments (a) have different solubilities, and/or (b) differ in the degree to which they adhere to the paper. Paper chromatography involves placing a drop of leaf extract near the bottom of a strip of paper. The bottom edge of the strip is then dipped into a solvent. As the leading edge of solvent moves up the paper and across the spot of leaf extract, the pigments dissolve and are carried along with the solvent. The more soluble a pigment, the farther it will travel up the strip. Once separated, the pigments can be identified and studied. Methods 1. You will need the following materials: chromatography paper coin (25-cent piece) wax pencil ruler chromatography jar and cork scissors plant leaves (geranium, spinach, etc.) test tube rack solvent (mixture of petroleum ether & acetone) 2. Cut a strip of chromatography paper about l inch longer than the length of the chromatography jar (a flat bottomed test tube). Trim the paper so that it slides easily into the jar. Remove the paper and place your initials on the jar with the wax pencil.

3. Add about l cm of solvent (petroleum ether and acetone) to the empty jar and immediately cork the jar to reduce fumes. 4. Transfer some of the pigment from a spinach leaf to a piece of chromatography paper. To do this (a) place a strip of chromatography paper flat on the desktop, (b) place a leaf over one end of the paper strip, and (c) roll the edge of a coin (a 25-cent piece) back and forth across the leaf so that the pigment squeezes out and makes a thin line across the paper about 2-3 cm from the end of the strip. When you stand the paper upright inside the chromatography jar, the line should be horizontal.

5. Carefully insert the paper into the test tube, so that the paper is touching the solvent but the drop of leaf extract is not submerged in the solvent. Cork the test tube (the end of the paper will stick out past the cork). Observe the separation of pigments at 5-10 minute intervals for 30-40 minutes. Results 1. On the next page, under "Species 1", sketch your chromatogram (paper strip) as it appears after 30- 40 minutes. Indicate the color of each of the spots for your species. Look for the presence of chlorophyll a (bluish green), chlorophyll b (pale green), and auxiliary pigments like xanthophyll (yellow) and carotene (orange, at the solvent edge). 2. Under "Species 2", sketch and label a chromatogram obtained by someone who used a different species of plant. Chromatograms of leaf pigments from two kinds of plants Name of Species 1: Name of Species 2:

Analysis and Discussion 1. How many different pigments (spots) were present -- in your leaf? -- in the leaf of the other species? 2. What causes the differences in color? 3. The two spots closest to the source point are usually "chlorophyll a" (bluish green) and "chlorophyll b" (a pale green). Any other spots indicate the presence of "auxiliary pigments". -- Are the colors of chlorophyll and the auxiliary pigments similar or different? -- Why would having more than one color of pigment be advantageous? 4. Compare the pigments found in your two species of plant. -- Are the colors of the chlorophylls similar in the two species? -- Are the colors of the auxiliary pigments similar in the two species? Why fall colors? We didn’t see the auxiliary pigments in the intact leaf because they are masked by the presence of chlorophyll, which is much more abundant. However, the auxillary pigments do become visible in autumn, just before the leaves fall off the trees, because the chlorophyll breaks down first.

PART II: STRUCTURAL ORGANIZATION OF LEAVES

9.2 Exercise 2 – Internal features of a leaf

All the structures of the leaf can be understood in terms of their role in maximizing the efficiency with which the chloroplasts carry out photosynthesis in different habitats. As you study the structures below, ask yourself, “How does this structure increase the supply of sunlight, carbon dioxide, or water to the chloroplasts?" Examine a prepared slide of a cross section of a leaf under low and high power. Using the numbers in Table 1, locate the structures shown in Figure 1.

Table 1: Tissue layers of a leaf

Tissue layer (Chloroplast-helping) function

(1) Upper epidermis Protective; secretes waxy cuticle (6) that keeps water in, but (being transparent) lets sunlight through

(2) Palisade mesophyll layer Tightly packed parenchyma cells containing most of the leaf's chloroplasts (7)

(3) Spongy mesophyll layer Loosely packed parenchyma cells, which also have chloroplasts; 30-50% is air space (8) that allows atmospheric carbon dioxide to diffuse into mesophyll

(4) Vascular bundles (veins) Moves water and minerals into the mesophyll layer and carries away the products of photosynthesis

(5) Lower epidermis Has stomata (9), openings controlled by guard cells (10), through which carbon dioxide and oxygen pass (and water evaporates)

Figure 1: Cross section of a leaf

9.3 Exercise 3 – Stomata

Stomata are tiny openings in the leaf epidermis which allow gases (oxygen and carbon dioxide) to diffuse in and out. But the leaf loses water for as long as the stomata remain open. To reduce the amount of water lost by transpiration, each stoma is surrounded by two guard cells which regulate the size of the opening. The primary function of stomata and surrounding guard cells is to provide adequate carbon dioxide for photosynthesis while minimizing water loss. During the day when the plant is actively photosynthesizing, the stomata are open to allow the entry of carbon dioxide. At night, the stomata are closed to reduce water loss. Each guard cell is kidney-bean shaped with bands of microtubules running radially around it (See Figure 2). When water enters the guard cells, the microfibrils prevent the cells from expanding their circumference, so they expand their length. The two guard cells are anchored to each other at their upper and lower ends, so lengthening of the bean-shaped guard cells causes their middles to move apart, opening the stoma. The reverse happens when the guard cells lose water; the guard cells lose turgor pressure and the space between them closes. Figure 2. Guard cells and stomata, closed and open

1. Obtain a leaf from the Central Study Area. Use forceps to remove a small piece of the lower epidermis. (It helps to bend the leaf upward so that the lower surface splits open along the fold.) Make a wet mount of the specimen by floating it on a drop of water and covering it with a cover slip. 2. Examine the epidermal tissue under medium power (100x). Then switch to high power (430x) and focus on one of the stomata with its two guard cells. Use the measurement you made of the diameter of your microscope field at 430x in Minicourse 1 to estimate the diameter of the stoma and guard cells. _____ µm 3. In the space below, sketch one of the stomata with its two guard cells. (Don’t just copy the diagram in Figure 2. Draw what you actually see.)

PART III: FACTORS AFFECTING THE RATE OF PHOTOSYNTHESIS In this section you and you partner(s) will do experiments to test the influence of two environmental factors on the rate of photosynthesis. Possible factors affecting photosynthetic rate Your experience with enzyme-catalyzed reactions from minicourse 2 suggest a number of factors that might influence the rate of photosynthesis. (a) pH. Does pH affect photosynthetic rate the same way it affected the browning reaction studied in Minicourse 2? Is there an optimal pH for photosynthesis? (b) carbon dioxide (substrate) concentration. Does photosynthetic rate increase when carbon dioxide concentrations are increased (e.g., by adding sodium bicarbonate)? Does the rate approach some maximum and level off at high CO2 concentrations?

(c) color of light. Are some colors of light better than others? For example, is the rate of photosynthesis faster in blue, red, or green light? Measuring photosynthetic rate The overall equation for photosynthesis suggests several possible variable that might be measured to estimate the rate of photosynthesis. sunlight

6 CO2 + 12 H20 ----------------> C6H12O6 + 6 O2 + 6 H2O

We could measure (a) the rate of consumption of carbon dioxide gas, (b) the rate of formation of glucose, or (c) the rate of release of oxygen gas. For this minicourse, you will measure the rate of oxygen formation using the "floating leaf disk" technique.

In Part III you wil need to work in a group of THREE.

Write the names of your two partners and their discussion section leaders here:

Names of Your Two Partners

9.4 Exercise 4 – The effect of carbon dioxide and pH on the rate of photosynthesis

1. Use a cork borer to cut 100 or more leaf disks from a spinach leaf (spinach is rich in chloroplasts). Cut to either side of the largest leaf veins, not through them. Discard any disks that are bruised or torn. 2. The next step depends on which experiment you are doing: carbon dioxide or pH.

(a) If you are doing the carbon dioxide experiment, set up four 250 ml "side-arm" flasks, each containing 80 ml of one of the four premixed sodium bicarbonate solutions being tested. Use a separate flask for each concentration being tested. Label the flasks with a wax pencil. (b) If you are doing the pH experiment, set up four 250 ml "side-arm" flasks, each containing 80 ml of one of the four premixed pH/sodium bicarbonate solutions being tested. Use a separate flask for each pH value being tested. Label the flasks with a wax pencil.

3. Use forceps to carefully place 25 disks into each flask. Hold the disks by the edge so you don’t crush them! If the disks have not been crushed in the cutting and transferprocess, at this point they should all be floating. 4. Carry the flask(s) over to one of the sink faucets fitted with an “aspirator”. An aspirator generates suction that pulls air out of the flask and out of the leaf disk’s intercellular spaces. Connect the aspirator’s hose to the side arm of the flask. Put a one-hole stopper in the flask and hold your thumb over the hole. 5. Turn on the cold water full force. Watch carefully for 5-10 seconds as air bubbles escape from the edges of the leaf disks, and then release your thumb. DO NOT "BOIL" If you apply the vacuum for too long, the fluid will become turbulent and look like it is “boiling.” This boiling is not caused by a high fluid temperatures, but by low air pressures inside the flask. “Boiling" (too much suction) will permanently damage the intercellular spaces; the discs will never be able to reinflate the spaces and refloat. So take your thumb off the stopper hole as soon as any fluid turbulence starts. 6. After lifting your thumb off the stopper to release the vacuum, gently swirl the liguid around the flask and all the disks should sink. (Note that none of the disks will sink until you take your thumb off the hole!) Wait 10 seconds. You may need to repeat steps 5 & 6 again until fewer than 5-10 disks are left floating. 7. Pour the contents of the flask into a culture dish (a 4” glass bowl). Discard any disks that are still floating. Cover the the sunken disks or keep them away from bright light until you are ready to start timing the experiment! 8. Obtain two (2) partitioned plastic boxes with clear tops. Label the four chambers with the bicarbonate concentration or pH value using a wax pencil. Write on the sides (not the tops) of the boxes, so the labels don’t block the light. Pour fluid from the four labeled flasks into the four corresponding chambers. Use forceps to transfer 20 sunken leaf disks from each of the 4 labeled flasks into the 4 corresponding chambers. Again, hold the disks by the edge so you don’t crush them! Set the

boxes under the lights. Place a large, glass bowl of room temperature water on top of the boxes to act as a heat shield. (See diagram below.) Turn on the light and keep track of the time.

9. Quantify differences between the four experimental treatments by recording the number of disks floating in each petri dish at 5 minute intervals for 45 minutes. To make a count: (a) turn off the light, and slip the petri dish out from under the beaker of water, (b) uncover the dish, gently swirl or jiggle the dish to free any stuck disks, (c) give the disks time to stop moving, then count the number of floating disks, (A disk standing on edge can be counted as 1/2.) (d) wipe off any water that has condensed on the underside of the cover. (e) replace cover, put the dish back under beaker, and turn on the light. 10. Use Table 2 (on the top of the next page) to record the number of re-floating disks. Label the columns with the sodium bicarbonate concentration or pH values you used.

Table 2: The number of leaf disks floating after successive 5 minute intervals under four different conditions of ______________________ . (carbon dioxide or pH)

Time (min.)

10-

15-

20-

25-

30-

35-

40-

45-

Analysis and Discussion 1. What effect does pH -- or bicarbonate (CO2 ) concentration -- have on the rate of photosynthesis? Are the results what you expected? Reconsider the questions asked at the beginning of Part III: (a) pH. Does pH affect photosynthetic rate the same way it affected the browning reaction studied in Minicourse 2? Is there an optimal pH for photosynthesis? (b) Carbon dioxide (sodium bicarbonate) concentration. Does photosynthetic rate increase when carbon dioxide concentrations are increased? Does the rate of reaction keep increasing, or does it level off ?

9.5 Exercise 5 – The effect of the color of light on the rate of photosynthesis

Green leaves look green not because they absorb green light, but because they reflect green light to our eyes. A pigment’s absorption spectrum (Figure 3) quantifies graphically how much of each color (wavelength) the pigment absorbs. Chlorophyll a and chlorophyll b strongly absorb blue light (with wavelengths 400-500 nanometers) and red light (600-700 nm), but absorb very little green light (500- 600 nm). Figure 3: The absorption spectra of chlorophyll a and chlorophyll b

Before starting Exercise 5, make a prediction about how the color of the light might affect the rate of photosynthesis. Be prepared to explain your reasoning to your TA. Your prediction:

Quantifying the effect of light color on the rate of photosynthesis: 1. To do the color experiment, use a cork borer to cut 100 leaf disks from a spinach leaf. Discard any disks that are bruised or torn. 2. You will need only one side-arm flask containing 80 ml of 0.1% sodium bicarbonate. Place 100 freshly cut leaf disks into the sodium bicarbonate solution and aspirate the disks following the prodecure outlined in steps 3-7 of Exercise 4 (see page 9). Asfter the disks sink, pour them into a 4” glass bowl and cover to protect them from bright light. 3. Obtain two (2) partitioned plastic boxes – one with a green/red top, the other with a blue/white top. Fill the 4 chambers with 0.1% sodium bicarbonate solution to a depth of about 1 centimeter. Now transfer 20 sunken leaf disks from the glass bowl into each of the 4 colored chambers. Use forceps to hold the disks by the edge so you don’t crush them! Set the boxes under the lights. Place a large, glass bowl of room temperature water on top of the boxes to act as a heat shield. Turn on the light and record the time. 4. Use Table 3 to record the number of disks re-floating in each chamber at 5 minute intervals for 45 minutes. Follow the counting steps listed in Exercise 3, step 9. Table 3: The number of leaf disks floating after successive 5 minute intervals under four different colors of light filters.

Time (min.) Red Green Blue Clear

10-

15-

20-

25-

30-

35-

40-

45-

Analysis and Discussion 1. Based on your results, what influence does the color of light have on the rate of photosynthesis? 2. Are your results consistent with the predictions you made on page ? -- If so, what can you conclude? If not, were there any problem with your methods, or does your hypothesis need to be revised? 3. Leaf chomotography (Part I) revealed that green leaves contain auxiliary pigments -- the red, yellow and orange pigments that become visible every autumn. These pigments can absorb light energy and pass it over to chlorophyll. Why are auxillary pigments important?

Lab 5 – DNA Fingerprinting 10

Living organisms are remarkably well adapted to their environments: every detail of their morphology, physiology and behavior seems tailored for surviving and reproducing in their particular environment. How did this close correspondence between organism and environment come about? Studies of taxonomy, compatative anatomy and the fossil record indicate that plant and animal species are not constant, but instead evolve, or change through time. Usually the changes make organisms better suited for surviving and/or reproducing in their ever-changing environments. Often, however, the changes in the environment occur too quickly and species go extinct. In fact, 99% of the species which have ever lived on this planet are now extinct. In 1859 Darwin proposed that evolution -- the change through time evident in the fossil record -- was due to natural selection. He called it natural because the direction of the change is determined by the environment (nature), not by a human or supernatural agent. He called it selection because the process is similar to the artificial selection used by humans for breeding more desirable animals and plants; some are allowed to reproduce, others aren’t. Darwin's theory of evolution by natural secetion – along with its corollary, “descent from a common ancestor” -- provides a major unifying theme in biology; it lets us see meaningful patterns in the otherwise disjointed facts of biology. Lab learning objectives You will have mastered the content of this minicourse when you are able to: Explain RFLPs and the procedure for making DNA fingerprints. Extract DNA from onion cells. Use gel electrophoresis to separate DNA samples into RFLP bands. Interpret the resulting DNA fingerprint patterns. PART I: DNA FINGERPRINTING DNA fingerprinting has many applications. It is now routinely used to test samples of blood, hair, saliva and semen left at crime scenes. The results are compared to those of a suspect to establish the individual's guilt. DNA fingerprinting can also be used to establish familial relationships in paternity cases, to track hereditary diseases, and to identify the best matches for organ transplants. This procedure can even be used to ascertain the level of inbreeding in endangered animals and to determine how newly discovered species are related to other organisms on Earth. DNA Fingerprinting is a method of identifying individuals based on the unique RFLP’s (restriction fragment length polymorphisms) of their DNA. When DNA is extracted from white blood cells (or other body tissue) and treated with a restriction enzyme, the enzyme cuts the DNA wherever it finds the sequence of 4-8 bases specific to that restriction enzyme.1 This produces a set of DNA fragments of different lengths. (Polymorphism means “having many forms” – in this case many different lengths.) Different people have very different DNA fingerprints because they have very different distances between the base sequences cut by the enzyme.

Why are the distances between the cuts so different in different people? In 1986, Alec Jeffreys, a geneticist in England, was studying introns – stretches of DNA located in between the genes. Introns are sequences of "nonsense" or "junk" DNA that do not code for a specific protein. Jeffreys found that some introns are made up of short DNA base pair sequences that repeat over and over again. The number of repetitions varies greatly from person to person; for example, in one person a particular base pair sequence may repeat ten times, while in another person the same base sequence may repeat twenty-five times. Intron lengths vary so much from person to person that after a restriction enzyme has cut the DNA, the resulting set of intron fragments is unique for every individual (except in the case of identical twins). Jeffreys used gel electrophoresis to separate the DNA fragments according to their length. The technique involves placing a sample of the restriction fragments into a “well” in the gel and turning on an electric field. Since all the fragments are negatively charged, they all start to migrate toward the positive end of the gel, but the shorter fragments move faster and so move farther across the gel. The fragments eventually spread themselves into a series of bands (Figure 1), each band composed of fragments of similar length. (Jeffreys had to use radioactive probes and X-ray film to visualize the bands, but you will be using ultraviolent “black” light.) Because every person has a distinctly unique RFLP banding pattern, Jeffreys called this technique "DNA fingerprinting." 1 Restriction enzymes are found in bacteria and probably evolved as a defense against invading viruses. More than 600 restriction enzymes are commercially available. Figure 1: The RFLP banding patterns of a child, the mother, and the alleged father are shown here. Is the alleged father really the biological father of the child? Hint: Half of a person's genome comes from each parent.

10.1 Exercise 1 – extraction DNA from cells

Summary of extraction method: Onion cells are difficult to lyse (rupture) because plant cells are surrounded by a cell wall (a tough barrier made of cellulose). (1) Breaking up the onion in a blender helps to release the cellular contents. (2) The chopped material is then mixed with lysing solution, which contains (a) a detergent to break open nuclear membranes, (b) sodium chloride to clump DNA molecules so they will precipitate out when alcohol is added, and (c) a citrate ion to prevent any DNAase in the cells from digesting the DNA.

http://en.wikipedia.org/wiki/Bacteria

http://en.wikipedia.org/wiki/Virus

(3) The heat treatment of 65° C is just hot enough to denature the DNAase (a protein) without degrading the DNA (a nucleic acid). (4) The plant material debris is then filtered off and cold alcohol added to the filtrate. DNA is soluble in water but insoluble in alcohol, so the DNA in the filtrate precipitates out along the interface where the filtrate makes contact with the alcohol. When a spooling rod is inserted into the interface and rotated, the precipitated DNA fibers wrap around the rod. Extraction procedure (Work in pairs!) In the Central Study Area transfer 7 ml. of the liquefied onion to a 20-ml test tube. Pouring onion sludge into the small opening of the tube is tricky, so use a small beaker to fill the test tube about one- third full. Add 7 ml cell lysis solution to the tube. When this is added, the 20-mL tube should be two-thirds full. Hold your thumb over the top and invert the tube once or twice to mix the contents. Do not shake it. Place the tube in a water bath at 65° C for 15 minutes. (While waiting, practice pipetting by doing Exercise 2.) Remove the tube from the water bath. Place a funnel into the mouth of a clean, larger (35-ml) test tube. Use a small piece of cheesecloth to act as a filter in the funnel. Slowly pour the onion solution into the funnel, letting it drip for a while. If necessary, squeeze the liquid through, but do not allow solids to fall into the test tube. Obtain 10 ml. of the chilled DNA precipitation solution (95% ethanol). Hold the tube containing the lysate (the solution of onion DNA) at a 45° angle and slowly allow the precipitation solution to slide down the surface of the tube to form a nice layer on top of the lysate. Observe the DNA as a cloud of fine, viscous threads where the 2 liquids meet. This is called the interface. Insert a glass rod into the test tube until its tip is just below the interface. Turn and twist the rod, gently passing it back and forth between the 2 layers of liquid. The DNA should wrap around the rod like yarn on a spool. List several adjectives to describe the DNA you have extracted (show it to your TA): DO NOT USE the extracted onion DNA for the DNA fingerprints in Exercese 3. For that exercise you will be given fresh samples of “crime scene” and “suspect” DNA.

10.2 Exercise 2 – Practice using pipettes to load wells

Loading DNA samples into the tiny wells of a thin gel is a delicate operation and requires practice. If a well is punctured by the pipette, the sample will drain out and be lost. In the Central Study Area obtain a petri dish filled with agar and containing a series of wells – the practice pipetting station. You will also need a small sample tube of loading dye. At your carrel you will find a micropipetter and a box of pipette tips. Put a new tip on your micropipetter and open the tube of loading dye. Press down on the plunger on the top of the pipetter, let it rise slowly as you draw up the dye. Once the practice loading dye has been drawn into the micropipette tip, carefully introduce the tip to the end of a well pointing towards the middle of the well itself. Position the tip so that it just enters the opening of the well. Be careful not to push the tip into the bottom of the well. Slowly expel the practice loading dye. When expelled slowly, the sample will sink into the well even if the tip is not actually in the well but above it. You and your group should take turns loading the wells in the petridish, so that you will be ready to load the gel for the DNA analysis. Show your practice plate to a lab instructor. You must show your practice wells to your TA to get the DNA for Exercise 3.

10.3 Exercise 3 – DNA fingerprints of unknown DNA samples

You must work with a partner on this exercise. Step 1: Loading the gel. Obtain a sealed E-Gel from a lab instructor. Plug in the power base at your carrel. Open the package and insert the E-gel into the power base. The E-gel is inserted right edge first. Press firmly at the top and bottom until you hear it snap into place. A red light will steadily illuminate when the E-Gel cassette is properly inserted. If the red light does not come on, ask for help from a lab instructor.

Step 2: Uncovering the wells. The wells in the E-Gel are held open by a plastic comb. Remove the comb by carefully pulling it straight up. Dispose of the plastic comb in the refuse boxes provided in the Central Study Area. Do not reinsert the comb into the wells.

Step 3: Loading the wells. Obtain one “crime scene” and three “suspect” samples of DNA from a lab instructor. Using the micropipetting technique you practices in Exercise 2, dispense 20 ul of DNA from Suspect #1 into Well #2. Remember to hold the tip of the micropipette just inside the well. Take care not to puncture the bottom of the well.

Using a fresh pipette tip for each sample, continue loading the remaining samples into wells # 4, 6 and 8, following the sequence below.

DNA sample from Sample vial Load into well number

Suspect #1 Clear 2

Suspect #2 Yellow 4

Suspect #3 Green 6

Suspect #4 Orange 8

Crime scene (Unknown)

Color __________ From Box # _____

Step 4: Running the gel. After loading the E-Gel, push the button again - briefly - to start the 35-minute electrophoresis run. The light will change to a steady green light. Step 5: Turning off the unit. Current through the E-Gel will automatically cease at the end of 35 minutes. The E-Gel Power Base will signal the end of the run with a flashing red light and beeping. Press the button to turn the unit off. The beeping will continue for 5 minutes if it is not turned off. Step 6: Viewing the gel. Remove the E-Gel from the Power Base. Take it to the Central Study Area to view it under an ultraviolet (UV) light.

Questions 1. Assume your gel results represent RFLP banding patterns of DNA fingerprints. Which DNA samples appear to have been derived from the same individual? Why? 2. What would the banding pattern of a DNA fingerprint look like if the investigator forgot to add restriction enzyme to the DNA samples before loading them into the gel? Show your TA (a) your test tube with extracted DNA from Exercise 1, and (b) your finished E-gel under UV light.