CELL RESPIRATION
Introduction to cell respiration laboratory
This laboratory consists of three sets of experiments that illustrate
the oxidation of glucose for the production of ATP.
1.)
The photosynthesis vs. respiration experiment illustrates
the importance of respiration to plants.
2.)
Fermentation products will be tested for to demonstrate
the importance of yeast in the fermentation process.
3.)
An enzymatic reaction of the Krebs cycle will be studied
to show how enzyme activity may be manipulated by changing
substrates.
CELL RESPIRATION
Introduction to comparison of photosynthesis and respiration
It is well known that in the presence of light plants perform
photosynthesis. It is less well understood that at the same time plants are
also performing cell respiration.
To demonstrate this we will determine whether CO2 is consumed
or produced as Elodea is placed in either a light or dark environment.
The change in CO2 will be detected by the pH indicator phenol red.
Think about the chemical equations for respiration and
photosynthesis.
Which one releases CO2 causing an increase and which one uses
up CO2 which will cause a decrease?
CELL RESPIRATION
Introduction to comparison of photosynthesis and respiration (cont.)
Phenol red is yellow under acidic conditions (high H ion
concentration), pink under basic or alkaline conditions (low H ion
concentration) and orange under neutral conditions.
A change in CO2 will cause a directly proportional change in H
ion.
If the CO2 concentration decreases, the H ion concentration will
also decrease and the solution will change to pink.
If the CO2 concentration increases, the H ion concentration will
also increase and the solution will change to yellow.
CELL RESPIRATION
Experimental procedure for comparison of photosynthesis and
respiration
Step 1: Fill four test tubes approximately 2/3 full with tap water
and add 15 drops of phenol red. What tap water should turn pink.
Is Glendora tap water acidic or basic?
Step 2: Adjust the pH of the tap water to neutral, orange by
gently (remember this word gently) blowing into the solution
with a straw. Repeat this procedure with all four tubes. It is
important that all four tubes are the same orange color at the
beginning of the experiment.
CELL RESPIRATION
Experimental procedure for comparison of photosynthesis and
respiration (cont.)
Step 3: Place a piece of Elodea (cut end up) in two of the tubes.
Each set of tubes will contain one with Elodea and a control
without.
CELL RESPIRATION
Experimental procedure for comparison of photosynthesis and
respiration (cont.)
Step 4: Place two tubes, one with Elodea and one without
between the lights on the counter.
CELL RESPIRATION
Introduction to fermentation
Fermentation is a pathway for the oxidation of glucose that
produces ATP in the absence of oxygen. Fermentation is similar to
respiration because it begins with glycolysis. Fermentation differs from
respiration because it does not involve the Krebs cycle or an electron
transport chain.
Fermentation yields waste products other than CO2 and H2O. In
humans, fermentation yields lactic acid and in yeast fermentation yields
ethyl alcohol in addition to CO2.
Since fermentation lacks a Krebs cycle and an electron transport
chain, the only energy products come from glycolysis. The 2 NADH are
used to produce the alcohol or lactic acid. The total amount of ATP
produced from one molecule of glucose is 2.
CELL RESPIRATION
Experimental procedure for fermentation
Tests performed by the Instructor:
Test for carbon dioxide (CO2)production: The instructor will
demonstrate the reaction between CO2 and barium carbonate by using a
straw to breath into a tube of barium carbonate. Note the reaction when
the instructor performs this activity. Students will not perform this
activity because barium carbonate is toxic.
The instructor will then place a tube from a beaker containing
yeast in a sucrose solution into a second tube of barium carbonate to test
for CO2 production by the yeast.
CELL RESPIRATION
Experimental procedure for fermentation (cont.)
Tests performed by the Instructor (cont.):
Test for ethyl alcohol production:
The instructor will
assemble a distillation apparatus to collect alcohol from both flasks. The
flasks will be heated to separate the alcohol from the water. The
distillate will be tested for the presence of alcohol as described in the lab
manual.
This flask contains
sucrose alone.
=====>
This flask contains
yeast and sucrose.
<=====
CELL RESPIRATION
Experimental procedure for fermentation (cont.)
Tests performed by the Student:
Controls for alcohol production:
You will prepare two tubes
(Tube 1 and Tube 2) that will be used as controls for the presence of
alcohol.
Tube 1 will be a negative control because it will not contain
alcohol. This tube will give a negative result for the presence of alcohol.
Tube 2 will contain alcohol and will give a positive result for the
presence of alcohol.
You will use these tubes to interpret the results of the alcohol
tests performed by the instructor.
CELL RESPIRATION
Introduction to the Krebs Cycle and the Action of Succinic (Acid)
Dehydrogenase.
Succinic acid dehydrogenase is and enzyme that oxidizes
succinic acid into fumaric acid as part of the Krebs cycle. During this
reaction the coenzyme FAD is reduced to FADH2. This reaction is
shown as Figure 7.4 in your lab manual.
This reaction represents a typical enzymatic reaction that involves
a substrate (succinic acid), an enzyme (succinic acid dehydrogenase),
and a product (fumaric acid).
We will use methylene blue as an indicator that this reaction is
taking place. Methylene blue will remove the electrons from FADH2 and
turn from blue to colorless. Thus we will be able to determine the rate of
the reaction by noting how long it takes the methylene blue to turn from
CELL RESPIRATION
Introduction to the Krebs Cycle and the Action of Succinic (Acid)
Dehydrogenase.
We will use methylene blue as an indicator that this reaction is
taking place. Methylene blue will remove the electrons from FADH2 and
turn from blue to colorless. Thus we will be able to determine the rate of
the reaction by noting how long it takes the methylene blue to turn from
blue to clear.
The methylene blue
will start out blue.
===>
The methylene blue
will clear as FADH2
is produced.
<====
CELL RESPIRATION
Experimental procedure for The Krebs Cycle and the Action of
Succinic (Acid) Dehydrogenase.
Tube 1: Place approximately 2 cm of liquid hamburger into a test
tube. Then add 10 drops of succinic acid and 3 drops of methylene blue
to the hamburger and mix. Note the time as you mix. This is a timed
experiment and it is important to know the time the reaction started.
Once the reagents are mixed do not mix them again (Your instructor will
explain why). Not the time when the only blue remaining is a thin band at
the surface of the reagents.
Tube 2: Place approximately 2 cm of liquid hamburger into a test
tube. Then add 10 drops of distilled water instead of succinic acid and 3
drops of methylene blue to the hamburger and mix. Note the time as
before and watch for the color to change.
CELL RESPIRATION
Experimental procedure for The Krebs Cycle and the Action of
Succinic (Acid) Dehydrogenase (cont.).
Tube 3: Place approximately 2 cm of liquid hamburger into a test
tube. Then add 10 drops of malonic acid instead of succinic acid and 3
drops of methylene blue to the hamburger and mix. Note the time as
before and watch for the color to change.
Tube 4: Add 5 ml of water to the test tube instead of liquid
hamburger. Then add 10 drops of succinic acid and 3 drops of
methylene blue to the hamburger and mix. Note the time as before and
watch for the color to change.
Suggestions: Rinse the mixing rod between tubes.
Mix all the tubes at the same time. Do wait for tube 1 to
finish reacting before starting tube 2, tube 3, or tube 4.
CELL RESPIRATION
Experimental procedure for The Krebs Cycle and the Action of
Succinic (Acid) Dehydrogenase (cont.).
Results: Three of the four tubes should clear. The time required
for each of the three tubes should be different. To determine why the
times are different you should consider the only variable used in this part
of the experiment. One of the tubes should not change. Can you guess
which one and why? Your results should look like this picture.
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