Fermentation Lab Part II and III: Assessing Fermentation in S.

Cerevisiae
The main goal of your fermentation experiments is to investigate the effects of environmental conditions on
fermentation in yeast. This week, youll gain experience performing fermentation assays and gather
preliminary data. Testing experimental techniques and assessing initial results are common steps in scientific
analyses. Methods rarely work perfectly the first time, and evaluating preliminary data helps researchers
develop more effective procedures. Insight can be gained into the inherent variability of data (no assay or
piece of equipment is perfect!), which is important when considering the number of replicates for each sample
and how many times you should repeat your experiment. The magnitude of the independent variables and/or
the controlled variables can be tweaked to better address your hypothesis. Preliminary data are also
frequently used to revise your hypothesis or even generate a completely new one! Youll use the preliminary
data you collect today to develop a hypothesis and design an experiment to test your hypothesis. Next week,
youll perform your experiment and analyze the data. Results from both sets of experiments will be included in
your Fermentation Lab report. IMPORTANT: You need this handout for this week AND next week.

LEARNING OBJECTIVES: PART II and III

Generate a hypothesis and design appropriate experimental procedures to investigate fermentation in yeast.
Gain experience analyzing results, performing statistical analyses, and generating appropriate tables and
graphs to present your date.
Be able to draw conclusions based on your data.
Develop your ability to write and revise a laboratory report that effectively communicates the results of your
analyses in a scientific journal format.

INTRODUCTION
Yeast & Alcoholic Fermentation
Yeast are unicellular eukaryotes and one of the few types of organisms/cells capable of carrying out
alcoholic fermentation. In the absence of oxygen (anaerobic conditions), yeast convert pyruvate produced by
glycolysis into carbon dioxide and ethanol via a two-step process (see reaction below). In the first reaction,
carbon dioxide is released, forming a molecule called acetaldehyde, which is then converted to ethanol. This
process regenerates the oxidized electron carriers (NAD+) necessary to allow glycolysis to continue when there
is not enough oxygen to accept the electrons stripped from glucose by the other processes involved in cellular
respiration. Most species of yeast will only carry out fermentation under anaerobic conditions because much
more ATP can be generated via cellular respiration compared to fermentation ~30 ATP versus 2 ATP. Some
species of yeast, like Saccharomyces cerevisiae (Bakers yeast) will ferment glucose and other sugars in the
presence of oxygen. These species have a much higher tolerance for ethanol, which is toxic to most yeast,
and thus allows them to kill of their competitors. Youll use a common strain of S. cerevisiae for your analyses.

1
Fermentation Experiment: Basic Procedures
We will use the amount of carbon dioxide produced (CO2) as a measure of fermentation. CO2 production
will be estimated by the volume of culture medium (liquid with yeast) displaced during incubation.
Microcentrifuge tubes with holes in the lids will be used as reaction vessels. Yeast in 5% glucose will be
incubated in inverted tubes. As CO2 is formed, some culture medium will be pushed out of the tube. Weighing
tubes before and after incubation will allow us to determine how much culture medium was lost and, therefore,
the volume of CO2 formed. Well assume that the density of the culture medium is 1.0 g/mL, so the weight
difference will directly correspond to the volume of CO2 generated in milliters (mL). To determine the rate of
fermentation, youll measure the amount of CO2 formed at various time points, plot your results as amount of
CO2 versus time, and determine the slope of the line (amount of CO2 per unit time). Below is the experimental
procedure you should follow to collect your preliminary data. Use this procedure as the starting point for
developing the specific experiment youll carry out next week. NOTE: In this procedure, replicates refers to
multiple samples for the same condition. This is essentially the sample size, although that is not the
terminology used for this type of experimental set-up.

1) Obtain 20 microfuge tubes. You will have four replicates (tubes) each for five time points 0 min, 5 min,
10 min, 15 min, and 20 min. Label your zero minute time point tubes 0a, 0b, 0c, 0d. Label your tubes for
the other time points using the same format (e.g. 5a, 5b, 5c, 5d for the 5 min time point).

2) Using a small beaker, obtain ~17 milliliters (mLs) of 5% glucose solution from the stock bottle. To limit
contamination, do NOT put extra back into the stock bottle!

3) With a pipettor, add 0.8 mL 5% glucose solution to each tube.

4) Using a small flask, obtain ~17 mLs of 7% yeast solution from the stock bottle. Do NOT put extra back into
the stock bottle!

5) Fill a 1000 mL beaker with approximately 700-800 mL of 40C water, using hot water from the sink. Check
the temperature with a thermometer and adjust if necessary (it should be close, but doesnt need to be
perfect).

6) Starting with one of your zero minute time point tubes (0a), add 0.8 mL yeast, carefully close the cap (slight
spillage may occur), dry thoroughly, weigh, and record as the pre-weight in Data Table 1. Repeat for the
remaining three tubes.

7) Working quickly, put the four zero minute time point tubes upside-down in your floating rack. Dip the rack
into your beaker of 40C water for one second and then remove the rack. Immediately place tubes right-
side up in your other rack to prevent additional solution in the tubes from leaking out of the holes in the top.

8) Dry the tubes thoroughly and reweigh. Record the post-weights in Data Table 1.

9) Working quickly, add 0.8 mL yeast solution to all of your remaining tubes (16 of them). Carefully close the
lids, dry, and weigh the tubes. Record the pre-weights in Data Table 1 and move on to the next step
immediately.

2
10) Quickly and carefully, put all 16 tubes upside-down in your floating rack. Start a timer and immediately
place the rack in your beaker of water at 40C. Place a glass weight on top to hold them submerged, and
put the beaker in the 40C water bath (the beaker prevents the water from becoming contaminated with the
expelled yeast).

11) Remove tubes at the appropriate times (5, 10, 15, and 20 min). Dry and weigh the tubes immediately after
you remove them. Record all weights in Data Table 1.

12) When you are done, CLEAN your microfuge tubes, tap them on a kimwipe to dry them out a bit, and leave
them in the racks so they can dry completely. Rinse out your beakers and flasks. Make sure that your
bench top is dry.

PROCEDURE: PRELIMINARY ANALYSIS OUTLINE (Part II)

I. Lab Discussion

II. Preliminary Analysis: Collect Data

Follow the experimental procedure described in the introduction. Remember to record all your data!

III. Preliminary Analysis: Analyze Your Data

NOTE: You will analyze your data using Excel. If you are not familiar with Excel, check the Excel User
Guide (provided in lab). You can also ask your lab TA and/or your lab mates.

1) Open Excel, and save the new file on the desktop. Name the file whatever you want so that you know
what it is. Youll need to e-mail the file to yourself (or copy it to a flash drive) so you have it when you
leave lab.

2) Make a table like Data Table 1 and type in the pre-weights and post-weights for your tubes (you dont
need the Time column). Use Excel to calculate the volume of CO2 produced for each sample.
Record the values in your lab handout Data Table 1 (just in case!). Note If you dont know how to get
Excel to do the calculations for you, check the Excel User Guide (provided in lab) and/or ask your lab
TA or labmates.

3) Make a table like Data Table 2 from your handout. Enter the CO2 volumes you just calculated into the
second Excel table AND the one in your lab handout (again, just in case!). This sets up your data in the
appropriate way to generate a graph that will allow you to determine the rate of CO2 production.

4) Select the data in your second table (including row/column titles) and make a Scatter Plot (its under the
Insert menu). Choose the plot type that does NOT have lines connecting the points. Move your graph
to a new sheet so you have a bigger version. Add a title your graph. Label the vertical axis CO2
Production (mLs) and the horizontal axis Time (min).

3
 5) Select the data for replicate a by clicking on one of the points, and add a Linear Trendline. Show the
equation and the R-squared value on the graph. If you dont know how to do this, check the Excel User
Guide! The R-squared value shows you how well your data points fit a straight line. An R-squared
value of 1.0 is a perfect fit. Double click in the text box with the equation and label it Replicate a. You
can click and drag the text box anywhere you want on the graph.

6) Repeat step 4 for the other three replicates, labeling the equations appropriately.

7) The slope of your line represents the rate of CO2 produced. (The slope is the m value in y = mx +b.)
Record the slopes in Data Table 3 of your lab handout. Make a similar table in your Excel spreadsheet
and enter the data.

8) Use Excel to calculate the average (mean) and standard deviation for your dataset (the slopes of all of
your lines).

9) Look at your data and write down any conclusions you can draw from your preliminary analyses.

IV. Develop Hypothesis & Experimental Method

Use the information provided in the Fermentation Lab: Literature Reports handout (provided in lab), what
you learned from the paper you read for lab last week (Hedberg, et. al. 2008), and your observations from
the preliminary analyses you just performed to develop a hypothesis relating to the effect of a specific
environmental parameter on fermentation in S. cerevisiae. Then design an experiment to test your
hypothesis. Things to consider:
You can assess ONE environmental condition.
You can test two different versions of your independent variable. One group (or your lab TA) will
run a control identical to the assay you performed today. Therefore, time is NOT something you
can choose for your independent variable.
You need to make sure that you have access to the materials you need to test your hypothesis.
Check with your lab TA to see if its feasible to obtain the materials you want.
You will have 20 microfuge tubes to use, and you need to use the same number of replicates for
each time point that you used in todays preliminary analyses.
Write your hypothesis and a detailed procedure in the Fermentation Experiment Procedure section of this
lab handout. Identify your dependent, independent, and controlled variables.

BEFORE you leave lab, make sure

your three Preliminary Analysis Data Tables in your handout are complete.
you have your Excel file (e-mailed or on a flash drive).
you have a detailed experimental procedure for next week.
youve checked with your lab TA to make sure that its feasible to obtain all the materials you need for
your experiment.

IMPORTANT: Drafts of your Materials & Methods and Results sections for your preliminary analyses are due
next week! You will need to ADD your analyses from next weeks experiments to BOTH sections for your full
report draft. Check the Fermentation Lab Rubric for specific requirements and the Lab Report Guide for
general guidelines. Use your notes from the Paper Discussion as well!
4
PROCEDURE: FERMENTATION EXPERIMENTS OUTLINE (Part III)

I. Lab Discussion

II. Perform Experiment and Collect Data

Perform the experiment you designed last week. Use Data Tables 4 and 5 to record your data.

III. Analyze Data and Make Figures

1) A template spreadsheet will be provided to facilitate analysis. Open the template file and choose Save
As. Save the file to the desktop using a NEW file name.

2) Using the template, analyze the data for each of your conditions the same way you analyzed your
preliminary analysis data last week.

3) In your handout, make a table similar to Data Table 3 to record your analyzed data, but include
columns for BOTH of your conditions AND the control data (obtained by one group or your lab TA).
This is a back-up copy of your data in case something happens to your spreadsheet file.

4) Use Excel to run a t-test in order to assess whether any differences you observed were statistically
significant. There are notes in the template Excel file describing how to do this, and your lab TA will
talk about t-tests at the beginning of lab.

5) Follow the notes in the Excel file to generate your final bar graph.

6) E-mail your Excel file to yourself or copy it to a flash drive. Youll need these data and graphs for your
lab report!

BEFORE you leave lab, make sure

your Fermentation Experiment Data Tables in your handout are complete.
you have your Excel file (e-mailed or on a flash drive) with ALL parts completed.
youve labeled your graphs appropriately.

IMPORTANT NOTES:
A complete draft of your lab report is due next week. It must be typed and formatted properly. Do NOT
put your name on it! The peer review youll do next week will be anonymous.
Dont forget to ADD your analyses from this weeks experiments to the Materials & Methods and results
sections for your full report draft.
Check the Fermentation Lab Rubric for specific requirements and the Lab Report Guide for general
guidelines. Use your notes from the Paper Discussion as well!

5
Preliminary Analysis Data Tables

Data Table 1: Preliminary Assay (5% glucose)

Time Replicate (Tube) Pre-Weight (g) Post-Weight (g) CO2 Produced (mL)
0a
0b
0 min
0c
0d

5a
5b
5 min
5c
5d

10a
10b
10 min
10c
10d

15a
15b
15 min
15c
15d

20a

20b
20 min
20c

20d

Data Table 2: Amount of CO2 Produced

0 min 5 min 10 min 15 min 20 min
Replicate a

Replicate b

Replicate c

Replicate d

6
Data Table 3: Rate of CO2 Production
Rate of CO2 Production (mLs/min)
Replicate a
Replicate b
Replicate c
Replicate d
AVERAGE
SD

Conclusions:

7
Fermentation Experiment Procedures
Hypothesis:

Experimental Procedure:
- Independent variable:

- Dependent variable:

- Controlled variables:

- Number of replicates:

8
Fermentation Experiment Data

Data Table 4: ____________________

Time Replicate (Tube) Pre-Weight (g) Post-Weight (g) CO2 Produced (mL)
0a
0b
0 min
0c
0d

5a

5b
5 min
5c

5d

10a
10b
10 min
10c
10d

15a
15b
15 min
15c
15d

20a
20b
20 min
20c
20d

9
Data Table 5: ____________________
Time Replicate (Tube) Pre-Weight (g) Post-Weight (g) CO2 Produced (mL)
0a
0b
0 min
0c
0d

5a
5b
5 min
5c
5d

10a
10b
10 min
10c
10d

15a
15b
15 min
15c
15d

20a
20b
20 min
20c
20d

10