Biological Sciences Department

BIO 161
Introduction to Cell and Molecular Biology Lab

Week 4: Respiration and Fermentation

Read the material below and complete the Pre-Lab in your Lab Report Booklet.

Objectives

1) Explain aerobic respiration in both plants and animals

2) Explain the link between carbon dioxide production and aerobic respiration

3) Explain and identify aerobic respiration and fermentation in microorganisms

4) Understand the redox states of intermediate molecules in aerobic respiration and

fermentation

5) Identify similarities and differences between aerobic respiration and fermentation by

connecting the experimental results to the appropriate cellular process

Introduction
Living organisms use chemical energy to perform their cellular functions. Many organisms convert the
chemical energy stored in food into adenosine triphosphate (ATP), a type of chemical energy used to
power a large number of cellular activities and enzymatic reactions. If oxygen is present, ATP can be
generated from food molecules using aerobic cellular respiration. When oxygen is used for
respiration, carbon dioxide is produced as a by-product.

In the following activities, you will investigate aerobic respiration occurring in different organisms.
Keep the following equation in mind as you prepare for and work through these lab activities:
Part I: Carbon Dioxide as Evidence of Respiration
Since CO2 is a by-product of cellular respiration, it can be used as an indicator that cellular respiration
has taken or is taking place by different organisms. In this activity you will predict which organisms
have undergone cellular respiration by predicting which ones will produce CO2 as a by-product. You will
compare your predictions to actual samples suspended over red cabbage extract, a pH indicator that
changes color in the presence of carbon dioxide. When CO2 is present, the solution becomes acidic
(purple) due to the formation of carbonic acid (H2CO3) in water (see equation, below). If the acid is
neutralized or CO2 is removed from the solution, the extract becomes basic and returns to its original
blue color.
Procedure:
1) Predict which samples (listed below) will produce CO2 and change the tube of cabbage extract from
blue to purple?

2) Discuss your predictions in groups. Try to come to a consensus if there is disagreement!

3) Your instructor will show you tubes of cabbage extract sealed with the samples listed. Record your
observations in your Lab Report Booklet.

Table 1: Does it Respire?

tube What's inside?

1 No sample...just air.

2 seedling (young growing plant)

3 dry seeds (not germinating)

4 fresh leaf in the light

5 live cricket

6 a rock...

7 fresh leaf in the dark

8 dead cricket (dead for several days)

Part II: Cellular Respiration: Rest vs Exercise

Humans breathe to facilitate aerobic cellular respiration for the purpose of ATP production. Oxygen is
breathed in to create an aerobic environment and CO2 is expelled from the lungs as waste from the
process of respiration. In this activity, you will investigate the effect of rest versus exercise on
respiration.

Procedure:
1) Obtain the bag apparatus to capture air.
 2) Prepare 4 tubes with 20 ml cabbage extract: 1) no air, 2) room air, 3) air at rest, and 4) air after
exercise.

3) Using the aquarium pump on your lab bench, fill the apparatus bag with room air. Make sure the
in clamp is open and the out clamp is closed!

4) SLOWLY bubble the room air from the bag into Tube #2. This time, make sure the out clamp is
open and the in clamp is closed.

5) Insert a straw into the in end of the apparatus tubing. You will use this to breathe into the bag. You
dont want to put the actual tubing IN your mouth, right?!

6) Have one group member blow into the apparatus and fill the bag while sitting quietly (at rest). Slowly
bubble into the Tube #3.

7) The SAME person in step 6 should perform 2 minutes of aerobic exercise (we recommend vigorously
running up and down the stairs at either end of building 53), then breath IMMEDIATELY into the bag.
Another group member should accompany the "breather" to have the bag apparatus ready as soon as
the exercise is done.

8) Bubble the exercise air into Tube #4.

TEST YOURSELF!
What is the purpose for Tube 1 (no air)? (click for answer)

Negative control. Use this tube for a comparison against all your other tubes receiving some form of
air.

TEST YOURSELF!
Why should you use the same person for both steps 6 and 7? (click for answer)

A good experiment should eliminate extraneous variables. Since every person is different, using
different people can cause differences in the changes between rest and exercise to be falsely
represented.

9) Add and count drops of ammonia to Tubes 2-4 until they reach the original cabbage extract color.
NOTE: add drops SLOWLY and swirl the tubes with each addition. Ammonia is basic and so removes
protons from the cabbage extract, increasing the pH of the solution.

10) Record the number of ammonia drops required for each tube in your Lab Report Booklet.

Part IV: Respiration in Yeast

Be familiar with this figure before coming to lab and before beginning Activities 3 & 5.
Part III & V: Respiration in Yeast
Activities 3 and 5 in your Lab Report Booklet are a part of the same procedure. The results of one are
related to the other!

In Activity 5, you will investigate respiration in yeast cells under both aerobic and anaerobic conditions.
You will qualitatively compare the production of CO2 using limewater solution. Limewater (Ca(OH)2) is
called such because of the saturated calcium (think limestone!). If CO2 is bubbled into the solution it
will begin to appear turbid (or 'cloudy') as a white precipitate forms. The precipitate is calcium
carbonate (CaCO3). The tubes in the image below contain limewater. Exposure to CO2 results in CaCO3
formation. The tube on the right would be accurately described as having 'high turbidity'.
 In Activity 3, you will analyze the SAME yeast from your experiment in Activity 5 to compare before and
after Activity 5 whether the yeast cells are dormant or metabolically active. This can be done using the
stain methylene blue. Methylene blue is blue in its original oxidized state. If NADH (an electron donor
generated in glycolysis) is present, it donates an electron to methylene blue, which turns clear when
reduced.

TEST YOURSELF!
Can you distinguish between aerobic and anaerobic respiration using methylene blue? Why or why
not? (click for answer)

No. Methylene blue dye changes color when it receives an electron from NADH. NADH is generated in
glycolysis which is necessary for BOTH aerobic and anaerobic respiration. You can only distinguish
between dormant and active- not the TYPE of activity (respiration or fermentaion). (Youll need to
complete Activity 5 to figure out which TYPE of activity your yeast are doingif any!)

Procedure:
NOTE: The portions of the procedure written in blue correspond to Activity 3 results.

1) Measure out 200 ml of room temperature tap water in a flask.

2) Obtain a vial of dried yeast and pour ALL of the yeast into your flask.

3) Lightly swirl the flask to suspend yeast.

4) Prepare a tube with 20 ml limewater. The limewater bottle will probably have while solids in the
bottom. Do not shake the bottle...you don't want those solids in your sample.

5) On a slide, place a small drop of methylene blue. Do not add a full drop from the dropper. A half drop
is PLENTY!

6) Use a transfer pipet to add a drop of your hydrated yeast suspension to the methylene blue dye.
Youll want the drop of yeast to be roughly the same size as the drop of methylene blue you added.

7) Use the pipet to swirl the yeast and dye together.

8) Place a cover slip on the slide and view at 40x magnification.

9) For about 100 cells (2-3 fields of view) determine if the cells are generating NADH and record your
count in your Lab Report Booklet.
10) Your yeast flask will be assigned one of four conditions: A1, A2, N1, or N2. See the table below for
the details of your flask condition.

Table 1:Conditions for Yeast Flasks. Your instructor will assign your group one set of
conditions. If you are viewing this on a phone, click the "+" to expand each or rotate to
landscape mode.

Conditions

Sample Warming Aeration (stirring) Sugar (20 ml sucrose)

A1 Yes Yes No

A2 Yes Yes Yes

N1 Yes No No

N2 Yes No Yes

***This is a great place to STOP and THINK about your predictions for EACH condition: which flasks do
you expect to perform aerobic respiration? Anaerobic? Why?

12) Place your flask on a hot plate and turn heat on the lowest setting. NOTE: the lowest setting is too
hot for the yeast. Turn the heat on and off every couple of minutes to keep the yeast at the appropriate
temperature.

13) For aeration (flasks A1 and A2) add a stir bar and use a stir/hot plate.

14) For sugar (A2 and N2) add 20 ml sucrose to the flask.

15) Place the rubber stopper connected to tubing on your flask. Place the free end of the tubing in your
limewater sample. Record the starting time.
16) Record the turbidity (cloudiness) of the limewater in your Lab Report Booklet every 5 minutes for 15
minutes.

17) Remove the stopper and smell your flask. Record the results.

18) Make sure you record the results from steps #16 and #17 for all the other flasks, too!

19) On a clean slide, place another small drop of methylene blue.

20) Remove a drop of the yeast suspension (now 15 minutes old) and add it to the methylene blue on
your new slide.

21) Repeat step #7-9 to complete your cell counts.

Review Questions
(These questions may be assigned by your lab instructor. You are not responsible to complete the
review questions if they are not assigned.)

1) Write a follow-up hypothesis for this weeks lab. Imagine that you had access to any biological
material you wanted (deep sea animals, desert plants, Olympic athletes, etc.). Think of a testable
hypothesis you could address using the techniques and equipment from this weeks lab. Be creative
but make sure that your hypothesis follows the format described in the Lab Report Guide.

2) A rock covered with green spots is placed on a screen in a test tube above some cabbage extract;
after several hours, the solution remains blue. What can you conclude from the experiment?

3) Your group observes that dead animals along the roadside often increase in size several days after
they have been killed. In the spirit of scientific investigation, you collect the gas from inside one of
these dead animals and bubble it through limewater. The limewater turns cloudy.

a) What does the cloudy result indicate?

b) If the animal is dead, how are these results possible? Explain your answer in detail.

c) Describe a positive control and a negative control your group should perform along with the
procedure described above. (Gas from the road kill is the test...what controls should you do to help you
interpret the limewater results?)

4) Which produces the most energy in the form of ATP? Explain your choice in as much detail as
possible.

a) Aerobic respiration

b) Lactic acid fermentation

c) Alcohol fermentation

d) All of the above produce the same amount of ATP energy but through different chemical
reactions

e) None of the above produce ATP

5) Carbon dioxide is passed into a solution of cabbage extract indicator until the solution turns purple.
A sprig of elodea is then placed in this purple solution. After a few hours in the sunlight, the purple
solution turns blue again. Suggest an explanation for the color changes observed in the cabbage
extract.

For Questions 5 through 8, use the following three answers. An answer may be used once, more than
once, or not at all. For each question, explain your answer.

a) Process occurs only under aerobic conditions

b) Process occurs only under anaerobic conditions

c) Process occurs under both aerobic and anaerobic conditions

6) Manufacturing of beer or wine: ____

7) ATP production: ____

8) Accumulation of lactic acid in a runners muscles causes her to drop out of a race: ____