Exercise 1

1
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.) Fermenta
tion 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.
2
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. Whi
ch one releases CO2 causing an increase and which
one uses up CO2 which will cause a decrease?
3
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.
4
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.
5
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.
6
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.
7
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.
8
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.
9
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 yeast and sucrose. This flask contains sucrose alone.

10
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.
11
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 blue to clear.
12
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 clear as FADH2 is
produced. The methylene blue will start out blue.
13
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.
14
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.
15
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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