BI 073 SEMO Anaerobic Respiration Lab

Description

Please read the Lab handout to understand the setup and structure of this experiment, which your instructors carried out for you. The following file contains the data we generated for you and all of the information you will need to complete the lab. The following videos are here to provide some helpful background that I hope you find useful and interesting. You do not need to view them unless you want to. There is a 9-minute intro from Amoeba Sisters and a slightly more detailed, 15-minute version from Khan Academy.https://youtu.be/eJ9Zjc-jdys https://youtu.be/2f7YwCtHcgkName
BI073 Post Lab 12
Please read the Lab handout to understand the set up and structure of this experiment, which your
instructors carried out for you. In this lab we used CO2 concentration and sugar consumption to assay
anaerobic respiration under three conditions: in the absence of sugar, in the presence of sugar, and in
the presence of magnesium (Mg) an important cofactor in glycolysis. We have listed the results you
might have generated from conducting the experiments in this lab.
Please recall that your bicarbonate solution will change color as the CO2 concentration (and therefore
pH) changes and this change in color will therefore change the absorbance at 550nm. A reminder of
how bicarbonate solution changes in response to CO2 concentration and pH is shown in Figure 1.
Figure 1.
The data you will need to answer the following questions are listed in Table 1.
Table 1: Color, estimated pH and absorption every 10 min from the bicarbonate solution.
Time
W Syringe
G Syringe
(min) Color
Estimated pH Absorption Å Color
Estimated pH
0
Red/Purple
8.8
0.779
Red/Purple
8.8
Absorption Å
0.761
10
Red/Purple
8.8
0.734
Red
8.6
0.624
20
Red/Purple
8.8
0.751
Red
8.6
0.603
30
Red/Purple
8.8
0.753
Dark 0range
8.4
0.583
40
Red/Purple
8.8
0.753
Dark orange
8.4
0.504
50
Red/Purple
8.8
0.751
Orange
8.2
0.433
Time
(min)
Mg Syringe
Estimated pH
Color
0
Red/Purple
8.8
10
Red
8.6
20
Dark 0range
8.4
30
Orange
8.2
Absorption Å
0.729
0.607
0.529
0.407
40
Orange/Yellow
8.2
0.334
50
Orange/Yellow
7.8
0.308
2) (2 pnts) Calculate the change in absorption using the formula ABS0 – ABST (Absorption at time 0 min –
the Absorption at time T min) and place the values in Table 2.
Table 2: Change in absorption over time for the bicarbonate solutions.
Time (Min)
W Syringe
G Syringe
0
0
0
Mg Syringe
0
10
20
30
40
50
3) (4pnts) Graph (use scatter plot) the change in absorption over time for all 3 treatments (W, G, Mg)
and paste the graph below. Be sure to label the axes.
4) (3pnts) Which reaction produced CO2 the fastest? Use the rate of color change and the change in
absorption to answer the question.
Data collected from conducting the Benedicts test are listed in Table 3.
Table 3: Results of Benedicts test
Treatment
Time 0 Absorption
Å
W
0.021
Time 0 Color
Time 50 Color
Blue
Time 50 Absorption
Å
0.022
Blue
G
0.593
Orange/Red
0.379
Yellow/Orange
Mg
0.589
Orange/Red
0.182
Green/yellow
6) (1pnts) Calculate the change in absorbance between Time 0 and Time 50 from the Benedicts test and
place the results in Table 4.
Table 4: Change in absorbance in the Benedicts test
Treatment
Time 0 – Time 50 Å
W
G
Mg
7) (4pnts) Which reaction showed the loss of most of the glucose? Justify your answer with the data you
collected.
8) (4pnts) Based on the both data sets (bicarbonate and Benedicts) what affect did Mg have on the rate
of the reaction? Justify your answer with both data sets. (Note: if you know there was an issue with
some of your data you can explain what you expected to happen and why your results do not match the
expectation).
BI 073 – Lab 12:
Cellular Respiration II
Required: Lab Coat, and Gloves
Introduction
In the previous lab, we examined aerobic respiration. This lab will examine the process of
anaerobic respiration. There are a number of types of anaerobic respiration such as lactic acid
fermentation, which is used by animals and alcohol fermentation which is used by yeast and plants. In
alcohol fermentation, glucose (C6H12O6) is broken down into carbon dioxide (CO2) and ethanol (C2H5OH).
The equation that summarizes the process of aerobic respiration is below.
C6H12O6  2CO2 + 2C2H5OH
This lab will have one major experiment in which we will run several fermentation reactions
with yeast and then test for CO2 and glucose to determine the rate of the reactions. We will also be
examining the effect of magnesium (Mg) of the fermentation process. Mg is a cofactor for the enzymes
in glycolysis that involve the use or production of ATP. (If you wish to see which enzymes these are you
can look at page 170-171 in your textbook). Think about how the addition of Mg will affect the rate of
anaerobic respiration.
You will be given
3 10cc syringes inserted through caps of 50 ml centrifuge tubes
3 50 ml centrifuge tubes with the bottoms removed
2 Stands
4 Specimen cups
6 2.0ml tubes
Setup1) Take the three syringes and label them the following: Mg, G, W
For each syringe
1) Fill the syringe with the amounts shown in Table 1 that correspond to its label. Be sure to use the fluid
levels, not the plunger for your measurements. Fill it in the order the solutions appear in Table 1.
Table 1: Contents of each Syringe
Syringe Contents
W
6cc Water + 2cc Yeast suspension
G
2cc Glucose + 4cc Water + 2cc Yeast suspension
Mg
2cc Glucose + 4cc 0.1M MgSO4 + 2cc Yeast suspension
2) Shake the contents of the syringe so that it is mixed well and then fill a 2.0 ml microcentrifuge tube so
that it is ¾ full. Cap and label the tubes and then centrifuge the tubes for 1 min (Remember to balance
the centrifuge). Use the samples from the 2.0 ml microcentrifuge tubes to run the Benedicts Assay (see
box on next page) for time 0 and record you initial values in Table 2.
Table 3: Results of Benedicts test
Treatment
Time 0 Absorption Å
W
Time 50 Absorption Å
G
Mg
Benedict’s Assay Procedure
For each sample:
1) Add 1ml (1000 µl) sample in a 1.5 ml microcentrifuge tube
2) Add 200 µl Benedict’s Reagent
3) Cap the tube and shake
4) Heat the sample at 95°C for 5 min
5) Centrifuge the tube for 2 min
5) Determine the absorbance at 735nm
Dispose of waste in appropriate hazardous waste container
3) Immediately after filling the initial 2.0ml microcentrifuge tube, flip the syringe over so the top is
facing up and pull the plunger to the 9 cc mark to make a small air pocket in the syringe.
4) Screw on the bottomless centrifuge tube (See Figure 1).
5) Clamp the apparatus to the stand with the centrifuge portion facing up (See Figure 1).
Figure 1: Apparatus
6) Pour bicarbonate solution into centrifuge tube to the 15ml mark.
7) Take a 1 ml sample from the bicarbonate solution and measure the absorbance at 550 nm (after
blanking it of course). Record the color, estimated pH and absorption in Table 3 (on the next page).
8) Repeat step 6 every 10 minutes for 50 min.
9) After the last measurement (at 50 min) is taken pour the reagent into the waste bottle. Unscrew the
centrifuge tube from the cap.
10) For each Syringe fill a 2.0ml tube with the contents so that it is ¾ full. Cap and label the tubes and
then centrifuge the tubes for 1 min (Remember to balance the centrifuge).
11) For each treatment, run the Benedicts assay and record your results in Table 2.
Table 3: Color, estimated pH and absorption every 10 min.
Time
W Syringe
(min) Color
Estimated pH Absorption °Color
Å
0
10
20
30
40
50
Time
(min)
0
10
20
30
40
50
Color
Mg Syringe
Estimated pH
Absorption
Å
G Syringe
Estimated pH
Absorption Å

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