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Midterm Lab Manual

Cellular Respiration BIO100A

This manual was adapted from   Online BIO100A – Survey of Laboratory Manual Version 4.0  by Michael Maxwell and Omar Clay. 

Instructions

1. Read A. Background. 

2. Read B. Experiment Overview.

3. Complete Section 1 - Background of the Midterm Lab Report (“Report”).

4. Read the C - Materials and Methods. Gather the materials you will need. Set aside at least 2 hours to complete the experiment.  

5. Read D – Procedure. Follow the instructions to conduct the experiment.

6. As you proceed, record your results in Section 3 – Results in the Report.   

7. Remember to take pictures as you proceed. Make sure that each picture includes a Picture Card, a card with your name, the words “BIo100A, Midterm Lab,” the date, and your signature. Insert each picture in the appropriate location in the Midterm Lab Report (“Report”).

8. Analyze your results and complete Section 4 – Discussion in the Midterm Lab Report.

A. Background

Introduction

In this experiment, you will investigate cellular respiration. By monitoring the volume of CO2 gas produced and the growth of yeast culture (dependent variables), you will investigate the role of sugar, temperature, and another independent variable of your choosing in cellular respiration. You will also learn about yeast, cellular respiration, experimental design and estimating measurement uncertainties. 

Cellular Metabolism

Cellular respiration is a set of biochemical reactions essential to cellular metabolism. In this catabolic process, sugars inside cells are broken down into CO2 and water, and the energy released is captured in adenosine triphosphate (or ATP). ATP can then be further used for cellular work or to build large molecules in anabolic reactions. The production of carbon dioxide (CO2) is the main way that carbon enters the atmosphere. This process is a key part of the carbon cycle. Photosynthesis, the other main part of the carbon cycle, removes CO2 from the atmosphere and uses solar energy to produce sugars.

The first step in cellular respiration occurs in the cell’s cytoplasm and is called glycolysis. In glycolysis, a single glucose (sugar) molecule is broken down into two pyruvate molecules and two ATP molecules. 

1 C6H12O6		2 C3H3O3	+	2 ATP
glucose		pyruvate		Energy

From there, two pathways are possible. First, in the presence of oxygen, aerobic respiration can take place in the cell's mitochondria, where pyruvate and oxygen are used to produce CO2, water, and 36 ATP molecules. The mechanisms by which this occurs are the Krebs cycle and the Electron transport chain.

1 C3H3O3	+	6O2		6CO2	+	6H2O	+	up to 34 ATP
pyruvate		oxygen		carbon dioxide		water gas		energy

Cells can also use fermentation, which occurs in the cytoplasm and does not require oxygen. There are many forms of fermentation. One can occur in the muscle cells of animals when blood cannot supply enough oxygen. The same process provides us with fermented dairy products such as yogurt and cheese through the activity of certain fungi and bacteria. This is known as lactic acid fermentation because it produces lactic acid. Ultimately, lactate can be used to produce some more ATP. Yeast and some bacteria can engage in another form of fermentation that produces CO2 and ethanol (drinking alcohol) and maintains the biochemical conditions necessary for continuing glycolysis in the cytoplasm.

In this lab, you will evaluate the production of CO2 by yeast cells. While this CO2 could come both through aerobic or anaerobic processes, the setup of the experiment will probably be conducive to more aerobic production.

Figure 1. Fermentation and cellular respiration (created with Biorender, biorender.com)

Yeast

Yeast is a single-celled fungus. These cells are typically 3-4 microns in size, but don’t let that fool you into thinking that they are not important to humankind. A particularly important kind of yeast is Saccharomyces cerevisiae. These organisms are used to ferment the sugars of grains (wheat, barley, rice, corn, etc.) and other foods (e.g., grapes) to produce alcoholic drinks (whiskey, beer, wine, etc.) and to make bread rise. Yeast is also used in the production of some cheeses, as well as in the field of bioremediation and ethanol fuel production. Yeast-like fungi are also normal inhabitants in the mouth, vagina, skin, and intestines.

Figure 2. Left: Saccharomyces cerevisiae as seen through a microscope. Right: Active dry yeast is a granulated form in which yeast is commercially sold.

Measurement

The goal of this experiment is to evaluate the production of CO2 under different conditions. For this, you will measure the volume of balloons and the height of the yeast culture. Accurate measurements are essential to guarantee that the results and conclusions are reliable. Modern scientists use the metric system in their measurements. The metric system was developed in France in the late 1700s and is now commonly used in most countries. A main advantage of the metric system is that it is based on multiples of 10. This is much easier than the English system of 12 inches to the foot, 16 ounces to the pound, and 16 cups to the gallon. Americans are often unfamiliar with some of the basic units of the metric system; thus, it can present a difficulty. In this laboratory, you will develop an understanding of the metric system and use it to measure common objects. You will also learn about how to report uncertainty in your measurements. Estimating measurement uncertainty is a critical component of scientific reporting that you will use in later labs.

Metric Units

THIS SECTION IS A REFRESHER ON THE METRIC SYSTEM 

The basic measurements of the metric system are: 

1. Length, expressed in meters (m). 

2. Mass (weight), expressed in grams (g). 

3. Volume (capacity), expressed in liters (l). 

These units have the following English equivalents. One meter is roughly 3 feet (1 yard) or slightly longer than one pace. One gram is very light; one paperclip is about 1 gram, while a nickel is about 5 grams. Standard weights are often expressed in kilograms (1,000 grams). One kilogram is roughly 2 pounds. One liter is roughly 1 quart, so there are about 4 liters in 1 gallon. For each of these units, large or small amounts are expressed by prefixes that reflect multiples of 10, as the following table shows. 

Prefix	Symbol	Power of 10	Length	Mass	Volume
giga-	G	1,000,000,000 =109
mega-	M	1,000,000=106
kilo-	k	1,000=103	Kilometer (km)	Kilogram (kg)	Kiloliter (kl)
hecto-	h	100=102
deka-	da	10=101
		100=1	meter (m)	gram (g)	liter (l)
deci-	d	0.1=10-1
centi-	c	0.01=10-2	centimeter (cm)	centigram (cg)	centilter (cl)
milli-	m	0.001=10-3	millimeter (mm)	milligram (mg)	milliliter (ml)
micro-	μ	0.000001=10-6	Micrometer (μm)	Microgram (μg)	Microliter (μl)

While you probably have a converter in your mobile device or computer, it is a good idea to be familiar with how the metric system units relate to the English system.   

Temperature

The metric unit of temperature is degrees centigrade (º C). The Celsius temperature scale was developed by the Swedish astronomer Anders Celsius in 1742. Nearly every country in the world uses the Celsius system. It can be confusing for traveling Americans when the morning news announces a "nice day" of 24 degrees. This sounds cold, but 24º C is actually 75º F. Temperature relates to the average energy of movement (kinetic energy) of molecules in the environment. 

The Celsius system is based on freezing and boiling points of water:

Celsius (°C) Fahrenheit (°F)

Water boils 100 212

Water freezes 0 32

Absolute zero -273 -395

The exact conversion between the two scales are:

°C = (°F - 32) × 5/9

°F = (9/5 x °C) + 32°

B. Experiment Overview

The goal of this experiment is to investigate the effects of sugar amount and temperature on fermentation.

You will be setting up 6 bottles, each with different ingredients, as shown in the table below.

Bottle	Sugar (tsp)	Yeast amount (tsp)	Water amount (cup)	Temperature
1	1	2	¼	Room temp
2	1	2	¼	Water bath
3	1	2	¼	Water bath
4	1/3	2	¼	Water bath
5	No sugar	2	¼	Water bath
6		2	¼

Bottles 2 and 3 are the positive control. They are there to ensure that the experiment works. There should be a balloon increase and yeast growth in each. They have identical conditions. This will allow you to gauge the precision of your experimental method.

Bottle 5 is the negative control. As there is no sugar, there should be no fermentation, balloon increase, or yeast growth.

In Bottle 4, the variable is the sugar amount.

In Bottle 1, the variable is temperature.

In Bottle 6, you will change a variable of your choice.

C – Materials

· Active dry yeast (rapid rise or instant). You will need a minimum of 5 ¼ oz packages. If dry yeast is not available, you can also use an active sourdough starter, 1 tsp of starter per bottle.  

· Six small bottles- .5L plastic or glass bottles. It is important that they are all the same size and type and that they are clear (no labels). 

· Candy Thermometer (alternatively oven/meat thermometer) Note: must be capable of reading temperatures between 100 -130 F. 

· Six same-sized balloons (5” diameter or bigger) 

· Indelible marker/sharpie 

· Sucrose (sugar) 

· String and Ruler or Tape measure 

· Watch/Clock 

· Measuring Cup and Measuring spoons (for 1/4 cup, 1, ½, and ¼ tsp)

· Kitchen pot or other means to prepare a warm water bath

 

Optional Materials (that you might consider for Bottle 6) 

· Vinegar or other acidic fluids 

· Ammonia or other alkaline fluids 

· pH paper 

· Beef bouillon 

· Fructose or honey

· Sugar substitute

Figure 3. Materials

D – Procedure

1. Collect your materials in a clean, safe place.

2. Decide what alternate experimental conditions you will examine in Bottle 6. You will keep the others constant.

3. Identify the variable you chose in Report Table 1.

4. Note that if you change more than one (amount of sugar, temperature, etc.), you will not be able to analyze your results. For instance, you might choose to investigate whether yeast can grow as well with something other than sugar, such as beef bouillon, NutraSweet, saccharine, or a higher quantity of sugar (e.g., 2 teaspoons). If you do this, be sure to keep the yeast quantity constant at 2 teaspoons and heat the bottle like the others. Other ideas include altering the pH of the water by adding a measured amount of ammonia or vinegar, changing the amount of yeast in the bottle, microwaving the dry yeast before trying to grow them (microwaving might kill the yeast), or seeing if the yeast can grow in the cold by placing  Bottle 6 in the refrigerator instead of the warm water bath.

Figure 4. Left: Labeled, loaded, and ready to grow. Right: CO2 production and yeast growth after approximately 20 minutes of heat. 

5. Make sure that your thermometer and five plastic bottles will all fit inside your kitchen pot (Figure 4). Make sure that your balloons fit tightly on the bottlenecks (Figures 3, 4). You may also want to tie your bottles together with string so that they will not float in the bath water.

6. Fill the pot with water to a height just a bit deeper than the height of the water in the bottles (this way, the bottles won’t float too much when you place them in the bath). Place the thermometer so that you can safely monitor the temperature. Heat the water to 110F (slightly warmer than a hot spa). While the water warms up, prepare the bottles.

7. Make sure that each bottle is clean and dry. Label the bottles 1-6 with a sharpie. Add 2 teaspoons of yeast to each bottle. Add the remaining ingredients.

8. Fill each bottle with 1/4 cup of water. Be sure that each receives the same kind and quantity of water. For instance, if you use tap water, use it with all of the bottles. Write down what kind of water you use. If you are going to run an experiment on pH and have pH paper, measure the pH of the water.

9. Replace the caps on each bottle and swirl the solutions thoroughly to ensure that the yeast cells are well distributed in the solution.

10. Remove the caps and replace them with balloons. Stretch the mouth of each balloon over the mouth of each bottle (Figure 4). Make sure that the balloon/bottle connection is secure; if necessary, use tape or string.

11. Measure the heights of the yeast solutions in the bottles. This is the distance from the bottom of the bottle to the top of the yeast. See Figure 5.

12. Record the heights in the Initial Heights column in Report Table 3.

13. Take a pic of your setup and insert it under Report Picture 1 - Setup

14. Place bottles # 2-5 in the warm water and maintain the temperature between 100 and 120 F for ~20 minutes. Bottle # 6 may or may not be placed in the bath, depending upon your choice of experimental treatment. Bottle #1 should be left sitting at room temperature (it will serve as a temperature treatment). Monitor the experiment carefully. Do not let the bath temperature exceed 120F.

15. Take a pic of your experiment while it’s in progress and insert it under Report Picture 2 – In Progress. Make sure the pic includes the ID card.

16. After 20 minutes, turn off the burner and remove all of the bottles.

17. Take a pic of your bottles after you have removed them. Please LINE THEM UP IN ORDER for the pic. Insert the pic under Report Picture 3 – End. Make sure the pic includes the ID card.

18. Record your immediate observations (balloon sizes, yeast heights, and anything else of note -- for example, “balloon has a hole”) in Report Table 2.

19. Measure the yeast heights in each bottle. Record your measurements in the New Yeast Height column in Report Table 3.

20. Determine the change in yeast height. Record your results in the Change in Yeast Height column in Report Table 3.

21. Determine the circumference (C) and radius (R) of each balloon.

a. To determine C, measure the distance around the balloon at its fattest point. (See middle pic below.)

b. To determine R, measure the distance from the top of the balloon to its base at the bottleneck. (See the right pic below.) Determine R using the formula R = Top to base/3.14.

c. Determine the volume of each balloon using the formula V = 2/19 x C x C x R.

d. Record your balloon measurements and calculations in Report Table 4.

e. Graph your results. Please follow the instructions in the Graphing Video posted in your Brightspace course. Insert your Graph in #6 in the Reports Results Section

Figure 5. Measuring Yeast Height and Balloon Circumference and Radius