Is Yeast Alive? --- Test for Fermentation Metabolism
Copyright 2006 by Dr. Ingrid Waldron
Humans use yeast every day. What is yeast, and what are some common uses of yeast? You can buy yeast to make bread in the grocery store.
This yeast consists of little brown grains. Do you think that these little brown grains of yeast are alive? Why or why not?
To find out whether yeast is alive, we first need to think about what makes something alive. What are some characteristics of living
organisms? To begin to answer the question, "Is yeast alive?” you will test whether the grains of yeast have two characteristics of living things
-- the ability to grow and the ability to use energy (referred to as metabolism).
Discussion of Metabolism
The yeast which is used to make bread is Saccharomyces cerevisiae. Yeast are single-cell eukaryotic microscopic fungi. When they grow using
sugars as a source of energy, yeast produce the gas, carbon dioxide.
This yeast is a facultative anaerobe, which means that when oxygen levels are low or glucose levels are high, sugar is metabolized without
using oxygen, resulting in the production of a small amount of ATP, as well as carbon dioxide and ethanol.
Yeast has been used in baking soda and in fermenting of alcoholic beverages for a thousand years. In bread, the carbon dioxide
produced from sugar causes the dough to rise to make a light, slightly spongy loaf of bread. As the bread bakes, the ethanol
evaporates. Bubbles which contained carbon dioxide provide the fluffy texture of bread. The yeast that we are using in these
experiments is this baker’s yeast.
Brewer’s east is also used to make beer and another type is also used in wine fermentation. There, the production of alcohol is
obviously a major goal. In beer production, the carbon dioxide produced from sugar fermentation is trapped in the solution to make a
fizzy or carbonated drink (carbonated means that it has carbon dioxide under pressure in the solution).
An alert and well-informed student may point out that in typical (aerobic) cellular respiration, although CO2 is generated, an equal number of
molecules of O2 are consumed, so there is no net increase in gas molecules.
C6H12O6 + 6 O2  6 CO2 + 6 H2O + ATP
glucose oxygen carbon water energy
as
dioxide
To respond to this observation, it is important to understand the difference between aerobic respiration and anaerobic fermentation.
The first major step in cellular respiration is glycolysis (see the top of the figure below):
1 glucose  2 pyruvate + 2 ATP
What happens next depends on whether or not oxygen is available to the cells (see the bottom of the figure below). When oxygen is available,
cells can use the Krebs cycle (citric acid cycle) and the electron transport chain to make up to 36 ATPs. This is called aerobic respiration.
2 pyruvate + 6 O2  6 CO2 + up to 36 ATP
When oxygen is not available, yeast cells use a process called fermentation which does not produce additional ATP, but maintains the
conditions needed for continuing glycolysis. Fermentation in yeast cells produces ethanol and CO 2. Obviously, anaerobic respiration yields
much less ATP per glucose molecule than aerobic respiration.
Figure from Johnson and Raven, 2004, Biology, Holt Rinehart and Winston, p. 110
Teaching Points
 The characteristics of life include using energy (i.e. metabolism), ability to grow and develop, reproduction, homeostasis, response to
the environment, evolutionary adaptation, composed of one or more cells, and has genetic material.
 The first experiment indirectly tests for the ability to metabolize, i.e. utilize energy. When sugar is available, the yeast metabolizes the
sugar and produces carbon dioxide, a gas which accumulates in the balloons and causes them to get bigger.
 Replication of each experimental condition is useful to be more confident of your results, since experimental results are often variable
even when you try to maintain the same conditions.
 The second experiment tests for the ability to grow.
 Some things that look dead are actually alive in dormant forms that can survive long periods in difficult environments (e.g. too dry or
lacking in food), until the environment improves and provide the conditions needed for active metabolism and growth.
Is Yeast Alive Teacher Prep Notes
Equipment and Supplies:
If you do not have access to reasonable quality compound microscopes (yeast cells are 5-10 μm in diameter), this lab activity can be done just
as well by simply omitting step 6 in the test for growth. If you have only very limited budget, supplies and equipment, you can do the first half
of the lab activity (the test for metabolism) using yeast, sugar, small water balloons, and the plastic tubes used to hold single cut flowers or very
small bottles which have narrow necks that will fit into the ends of the water balloons. Take care to keep the volume of whatever container you
chose small enough so it and the balloons fill up with carbon dioxide within 25 minutes using a reasonable amount of yeast.
Teacher Preparations:
1. You will need to experiment with your particular size of test tube to determine how much yeast you need for four test tubes. We have
found that approximately 1 g of yeast and 1.5-2 g of sugar per 25 mL test tube provide good results. 1 sugar packet is 4.3 g of sugar.
Alternatively prepare glucose solution and activated yeast solution ahead of time (see materials list below for instructions)
2. At least one day before class, prepare one Petri dish of yeast growth medium per group, as described in the following section.
3. At the beginning of class, have stations ready with what each group will need
4. For experiment 2, have the students use only 10-12 grains of yeast and a small amount of water. If incubating at room temperature allow
3-4 days for growth. If you can incubate at 37º C, then overnight will be sufficient.
Sterile Nutrient Agar Plate Preparation:
It is possible to obtain sterile Nutrient agar medium that, upon micro-waving, can be poured into sterile Petri dishes. More cost efficient, but
requiring an autoclave or pressure cooker, is to prepare the medium from Nutrient Agar Powder
Nutrient Agar + Distilled Water = Yield
Nutrient Agar
Distilled Water
Yield
23 g
1000 ml
50 plates
11.5 g
500 ml
25 plates
9.2 g
400 ml
20 plates
4.6 g
200 ml
10 plates
Pour enough autoclaved agar medium (cooled to approximately 50 oC) into each sterile plastic Petri dish to cover the bottom—about 1/8" to
1/4" deep. If the medium solidifies before you finish pouring, it can be reheated in the microwave.
1. Place agar plates on a countertop to cool and solidify. Agar medium will set like stiff gelatin at room temperature.
2. The agar medium is now ready for storage or use.
Storage: Do Not Freeze! Stack agar plates upside down in the refrigerator. The purpose of placing the plates upside down is to prevent
condensation from dripping down onto the agar surface which could then facilitate movement of organisms between colonies. If plates have
been refrigerated, set them out and allow them to warm to room temperature before using them.
Part A: Scientific Experiment to Tests for Metabolism
We will carry out an indirect test for metabolism. In other words, we will be indirectly testing whether yeast can use energy, which is one of
the characteristics of living organisms. When yeast, humans, and other living organisms use energy, they break down high-energy molecules
like sugar to get the energy they need and give off a gas called carbon dioxide as a by-product of this reaction. We will test whether yeast can
metabolize sugar and produce a gas which we will presume is carbon dioxide. Specifically, we will test whether yeast produces a gas when it
has sugar available as a food vs. when no sugar is available.
Carbon dioxide production can also be measured through an increase in carbonic acid in solution (See “Photosynthesis & Cellular Respiration
Rates in Plants”). When carbon dioxide dissolves in water it reacts to form the weak acid, which lowers the pH of the solution. Therefore, the
use of a pH indicator is another way to measure the occurrence of metabolic reactions.
Organisms have certain pathways where electrons are passed from one molecule to the next in a chain of reactions. Therefore, a more direct
way to measure metabolism is by detecting the occurrence of oxidation-reduction reactions. Metabolic reactions such as those described earlier
always involve energy transfers through redox mechanisms. Whenever such reactions take place, electrons are transferred from one compound
to another. When a molecule accepts an electron it is reduced and when it loses an electron it is oxidized. In order for ATP to be synthesized
from ADP and P, energy must be transferred to ADP to allow for the attachment of the third P. This is a process which occurs continually in
most cells. By adding an oxidizing agent to the solution, one can capture some of the electrons being transferred during these reactions. A
compound named 2,6 dichlorophenol indophenol solution (DPIP), is blue in its normal state. Once reduced by the gaining of electrons, the
compound changes color to clear. Therefore, it can be used as an indicator of oxidation-reduction reactions.
In this experiment, we test yeast metabolism with all three methods above. We will test the effect of sugar in yeast metabolism to see if it is
indeed alive.
Prelab Questions:
1. Do you expect yeast to produce a gas when sugar or other food is available?
2. Do you expect yeast to produce a gas when no sugar or other food is available?
3. Hypothesize as to the effect of sugar on activated yeast solutions.
4. Explain the reasons for your prediction.
5. What is the independent variable in all three experiments?
6. Though it is measured differently, what is the dependent variable in all three experiments?
7. Identify variables which are controlled throughout experiments
8. What is the experimental purpose of tubes or jars with sugar added (A, C, & E)?
9. What is the experimental purpose of tubes or jars with no sugar added (B, D, & F)?
10. Which tubes / jars will be testing yeast production of carbon dioxide by “capturing” carbon dioxide production?
11. Which tubes / jars will be testing yeast producing of carbon dioxide by measuring changes in acidity levels of a solution?
12. Which tubes / jars will be testing yeast metabolism through indication of occurrence of redox reactions?
13. What do pH indicator solutions actually measure? What causes pH indicator solutions to change color? Why can they be used as indicators
of metabolism?
14. What causes DPIP to change color from blue to clear? Why can it be used to measure metabolism?
15. What gas will be trapped in the balloons? How can this be used as measure of metabolism?
16. Why will some of the solutions be “heated”?
17. Since each group is only performing the experiment once, why is step 18 in the procedures so important?
Part A: Measuring Metabolism Through Detection of Oxidation-Reduction Reactions
Note: To save on expensive DPIP, teacher may demonstrate this instead of allowing students to do it in groups
Materials
 2,6 dichlorophenol indophenol solution (Dipip) ( 0.2 gram in 1
liter distilled water)
 5% Activated Yeast Solution (Active Yeast 5g for every 100ml of
distilled water) x200ml
 5% Glucose Solution (5g for every 100ml of distilled water) at
30ºC x 400ml
 Balloon (Small)
 Beaker (250ml, for Yeast solution storage)
 Beaker (600ml, for Glucose & Distilled Water Storage)
 Cabbage pH Indicator Solution (See Acids & Bases Lab for more
instructions)
 Culture tube rack
 Culture Tubes x6
 Distilled water at 30ºC x400ml + (5.8L in prep)
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Electronic Balance
Erlenmeyer Flasks (1 or 2L, for stock solution and water bath) x 3
Graduated Cylinder (500ml)
Hot water bath
Marker
Masking Tape
Parafilm
Syringe (10ml) x3
Syringe (1ml) or pipette
Syringe (60ml) x3
Transparent Jar with Lid x 2
Weigh Boats
Procedure:
1. Prepare two test tubes half filled with pH indicator solution, and place them in the test tube rack.
2. Label the other 4 tubes A, B, C, & D using markers and masking Tape
3. Label 2 transparent jars with lids E & F using markers and masking tape
4. Procure 200ml of Yeast solution into a 250ml Beaker & 400ml of glucose and distilled water into 2 separate 600ml Beakers.
5. Using a 10ml syringe transfer enough activated yeast solution to fill 1/4 of test tubes A-D
6. Using a 60ml syringe, transfer 100ml of 5% activated yeast solution into each of the jars
7. Using a different 60ml syringe, add 200ml of distilled water into jar E
8. Using a different 60ml syringe, add 200ml of 5% glucose solution into jar F
9. Place one test tube filled with pH indicator solution inside each of the jars.
10. Close jars with lid tightly
11. Using a different 10ml syringe, add enough distilled water to fill the solution level in test tubes B & D to ¾ of the tube.
12. Using a different 10ml syringe, add 10ml of warmed 5% glucose solution to fill the solution level in test tubes A & C to ¾ of the tube
13. Place a small water balloon over the mouth of test tubes A & B to capture any carbon dioxide released. Then hold a finger over the end of
each test tube, and shake it vigorously to thoroughly mix the contents.
14. Using a 1ml syringe Add 10 drops of DPIP to each tubes C & D. Cover tube with parafilm and invert 2 or 3 times to mix.
15. Record the starting time
16. Return all unused solutions to stock containers
17. Wait 20 minutes and observe the indicator tubes in jars E & F. Record any changes in color. Also record any changes in the solutions in
the jar during the 20 minutes.
18. During the same 20 minutes, observe what occurs in the test tubes A & C every 2 minutes and record and any changes in the tubes and also
use a tape measure to record the circumference of the balloons
19. Also, observe tubes B & D until the blue color disappears. Record the time of disappearance for each tub
20. Compare results with classmates and when possible calculate averages
Hint: If the yeast grains are capable of metabolism, it will take some time to produce enough carbon dioxide to see changes
Results:
Min
Observation Log
Notes
0
2
4
6
8
10
12
14
16
18
20
Measuring Yeast Metabolism with pH Indicator Solution
Jar E
Observed changes in solution
outside test tubes during 20minute interval
Observed changes in test tube
indicator solution color
Jar F
Min
Yeast Metabolism with Baloons
Group Data
Class Averages
Tube A
Tube B
Tube A
Tube B
2
4
6
8
10
12
14
16
18
20
Yeast Metabolism with DPIP Interception of Redox Electrons
Group Data
Tube C
Tube D
Tube C
Class Average
Tube D
Time of Color Change
Error Log:
Analysis:
Graph your results for the balloon test using a line for each baloon’s circumference. Remember to add title, units, and label axis
Discussion Questions:
Questions:
1. What is the name of the process that is occurring in the experimental jars/tubes?
2. Explain in words, the chemical reaction occurring in the experimental tubes
3. What gas is formed during the process?
4. How could you tell that this gas is being produced even if you did not have ballons or pH indicators?
5. How is this process used in the food-making industry?
6. Did the results reject or failed to reject your hypothesis?
7. Explain any difference between tubes:
a. A & B
b. C & D
c. E & F
8. What do you think would have happened if the solutions were heated more?
9. What errors experience or limitations did you find in this experiment? How would you fix them?
10. While yeast is capable of performing fermentation under anaerobic conditions, it can also perform complete cellular respiration through the
Krebs cycle. Did this experiment actually identify if Yeast was performing fermentation or full aerobic cellular respiration? What could we
measure to verify this? (Think about key differences between yeast’s aerobic respiration and anaerobic fermentation).
11. When you make bread, if you just mix flour, sugar, and water, the dough does not rise, and the bread will be flat and hard. If you include
yeast in the bread dough, then the dough rises and the bread is bigger and fluffier. Can you explain how the yeast helps the bread dough to
rise?
INQUIRY EXTENSION:
 Students design their own variations of this experiment following all steps of the scientific method with focus on manipulating conditions
around the yeast and seeing if it affects the metabolic rate (pH, temperature, light, pollution, etc)
 All experimental designs must include at least 3 levels of the independent variable (control + 2)
 All experimental designs must include repetition (students do it 3 times or do all 3 times all at once, which would require several jars/test
tubes)
 You may use the available foods and chemicals or bring in your own.
 At the conclusion, groups will have to submit full lab reports in accordance with lab report template / rubric
 Before doing the lab, submit the following to the teacher: Problem Statement / Background / Material List /Procedure
Note: Before you write a full background you may want to check you problem statement and a preliminary idea of how to test it with the
teacher. Students often decide to switch experiments after they realize their procedures are not practical. At that point, you may have
wasted your time already writing a full background)
Part B. Procedure to Test for Growth
Instructions
1. Obtain a Petri dish with yeast growth media, and label the bottom with your name, teacher, and class period. (A Petri dish is a flat,
covered dish used by scientists, and the yeast growth media in the Petri dish contains a mixture of substances that yeast requires to grow.)
2. Using a microspoon, spread10-12 grains of yeast across your plate.
3. Using a plastic pipette, add several drops of water on the grains of yeast.
4. Your plates will be incubated at 37° C until the next lab class. How warm or cold is that?
Data:
At the next lab class, inspect your plate. Do you see any signs of yeast reproduction on the plate? Sketch what you see.
Take a sample of the growth and observe it under the microscope (see Cell Observation Lab for instructions on how to prepare slides for
microscope viewing). Draw and describe what you see.
Discussion Questions:
1) Which characteristics of life were examined in this lab?
2) Which characteristics of life were not examined? How could you examine them?
3) Based on your findings, is yeast alive?