The Cell: Division (Mitosis & Cytokinesis) and Cellular Respiration Exercise 4 (begins page 39 in 9th 10th 11th and 12th editions) and Supplemental Experiment Handout “Anaerobic Metabolism: Fermentation” Lab 3 Objectives Read lab Exercise 4 Supplement: do cellular respiration experiment For Exercise 4: do Activity 6 Begin with the supplemental experiment handout and get all experiments set up first

before beginning slide and model observations in Exercise 4. Supplemental Experiment Handout: Anaerobic Metabolism: Fermentation Work in groups of 4 people and divide up the work: Follow the directions to set up the experiment. Exercise 4: The Cell: Anatomy and Division (Activity 6) Each student will need to use: whitefish blastula slide Identify states and features of mitosis on diagrams and slides (Activity 6, Fig. 4.4): Interphase nuclear envelope mitotic spindle Prophase chromatin metaphase plate Metaphase centrosomes/centrioles Anaphase chromosomes Telophase chromatid Cytokinesis centromere For study: Review Sheet Exercise 4 pages 49-52 in 9th 10th 11th and 12th editions Answers in the Instructors Manual at the Eastern Campus Library on reserve

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ATP Production in Cells Anaerobic Metabolism: Fermentation

Cells require energy to drive cellular functions, maintenance, repair, and division. Cells

acquire this energy by performing oxidation and reduction reactions on food molecules such as glucose. This energy is then stored in the form of ATP to be used as needed by the cell. The reactions necessary to produce ATP involve enzymes, biological catalysts, that facilitate the reactions to occur in a step-wise fashion to insure that the energy production and release is controlled and does not damage the cell. Additionally, enzymes speed up chemical reactions insuring efficient energy production at temperatures and pressures compatible with life. Enzymes however require optimal conditions to catalyze reactions. The cell, and even the physiology of a whole person, must maintain homeostasis at the optimal conditions for the catalytic power of the cellular enzymes or the chemical reactions necessary for life will cease.

In this experiment, we will investigate the cellular process of anaerobic metabolism called Glycolysis, a nearly universal pathways utilized by almost all cells from humans to bacteria. Glycolysis (literally translated as “sugar-breaking”) is the ten-step process by which a six-carbon glucose molecule is oxidized into two three-carbon pyruvate molecules resulting in the production of two ATP molecules for use by the cell (see Figure 1). During this process, two cofactor molecules of NAD+ are reduced into NADH. The resulting NADH will need to be oxidized back into NAD+ to complete the process. When oxygen is available, this is usually done in the Electron Transport Chain resulting in the production of additional ATP by oxidative phosphorylation in a process called aerobic cellular respiration. When oxygen is not available, such as in skeletal muscles cells during rigorous activity, the oxidation of NADH will be done in conjunction with reduction of pyruvate in a fermentation process resulting in the formation of waste products such as lactic acid (see Figure 2).

Since it is difficult to study the activity of human cells in the laboratory, we will investigate a similar process in yeast. Yeasts are eukaryotes like humans and perform many of the same chemical reactions as human cells. During anaerobic metabolism, yeasts will perform the same Glycolysis reactions as human cells, however a different set of enzymes will convert the NADH and pyruvate into ethanol and carbon dioxide instead of lactic acid at the end (see Figure 3). As with human cells, the enzymes catalyzing a reaction at each step in this pathway function best at the optimal temperature of 37°C, at a nearly neutral pH, and in the presence of the cofactor Mg2+. Using glucose as the substrate, the efficiency of enzyme function can be quantified by measuring the accumulation of the waste product carbon dioxide as the glycolysis reactions progress.

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Figure 1: Glycolysis Pathway

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Figure 2: Cellular Respiration and Fermentation for ATP Production

Figure 3: Fermentation Reactions

Amy Warenda Czura, Ph.D. 4 SCCC BIO130 Lab 3 Mitosis & Cellular Respiration

Experimental Procedure: Set up and incubate respirometer tubes as follows

1. Organize in numerical order in the test tube rack the 6 plastic conical 15 ml tubes labeled 1-6 on the bases. Label the six large (25 x 150 mm) glass tubes #1-#6 with a wax pencil and include group initials to identify the tube later.

2. Using squeezable transfer pipettes for dispensing and the milliliter lines on the plastic conical tubes for volumetric measuring, add reagents to the 15 ml conical tubes according to the table below. Be sure to use different transfer pipettes for each different reagent and do not contaminate the stock containers when acquiring and dispensing fluids.

a. Shake to mix up the yeast suspension before removing a sample to insure high and equal cell concentration gets delivered to each tube. Remove the appropriate quantity of yeast cells immediately after resuspension of the stock container.

b. Add yeast first filling to the 5 ml line on the conical tube. Tubes 1-5 all contain the same live yeast, however do note that tube 6 contains a different yeast sample that was boiled for 10 minutes prior to the start of the experiment.

c. Add glucose second, 5 ml, to tubes 2-6, filling to the 10 ml line. d. Add Magnesium third, 1 ml, to tubes 3-6, filling to the 11 ml line. e. Add acid fourth, 2 ml, to tube 5, filling to the 13 ml line.

Anaerobic Metabolism Respirometer Tubes

SOLUTION TUBE 1 TUBE 2 TUBE 3 TUBE 4 TUBE 5 TUBE 6 4% Live Yeast 5 ml 5 ml 5 ml 5 ml 5 ml 0 4% Boiled Yeast 0 0 0 0 0 5 ml 2.5% Glucose 0 5 ml 5 ml 5 ml 5 ml 5 ml 0.1M MgSO4 0 0 1 ml 1 ml 1 ml 1 ml 1M HCl 0 0 0 0 2 ml 0

Incubation Temperature

37°C

37°C

37°C

0°C

37°C

37°C

3. Once all the tubes contain the appropriate solutions, fill each conical tube to the clear,

unmarked 15 ml line with distilled water using the squeeze bottle. (The clear 15 ml line is above the blue 14 ml line but below the threads for the screw cap.)

4. Cover each conical tube with the screw cap and mix by inverting several times to insure the ingredients are uniformly distributed. Remove the cap.

5. To assemble the respirometer, hold the filled conical tube upright and slip the corresponding large glass tube over it. Keep one finger at the tip of the filled conical tube and press it firmly against the bottom of the larger outer tube. (See the figure 4 on the next page)

6. Quickly invert the respirometer and measure the height of the air bubble at the top of the small conical tube in milliliters using the blue labeled lines. Record this number in the data chart. (If you were careful and have no spillage, the bubble should be about even with the 2 ml line.)

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7. Repeat steps 5 & 6 for all six tubes. 8. Place each respirometer in the appropriate water bath for the incubation temperature indicated

in the chart. (Tubes 1, 2, 3, 5, 6 @ 37°C, Tube 4 @ 0°C). Be certain you have the initial bubble height recorded in the data chart for each tube.

9. Check the progress of the respirometers every 30 minutes. After about 2 hours (or when large bubbles have formed in the most active tubes), remove the respirometers and record the final height of the bubble. It is normal for fluid to have been forced out of the small tubes during incubation and for this fluid to pool in the bottom of the large tubes.

10. Place all materials and equipment in the appropriate discard area and analyze your results.

Data Table Start time: End Time: Total incubation time (hr): TUBE Initial height of

bubble (ml) Final height of bubble (ml)

Total height of CO2 produced (ml)*

Rate of CO2 production (ml/hr)**

1

2

3

4

5

6

*Total height of CO2 produced is calculated by subtracting the initial height from the final height. **Rate of CO2 production (an indicator of reaction rate) is calculated by dividing the total height of CO2 produced in milliliters by the total time of incubation.

Figure 4: Respirometer Set-up

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Understanding the Experiment Thought Questions:

1. Which tube would you expect to have a greater reaction rate, tube 3 or tube 4?

Why? Was this observed in the experimental results?

2. Do you expect tube 6 to produce as much CO2 as tube 3?

Why or why not? Was this observed in the experimental results?

3. Why would you predict tube 1 to show little to no reaction occurring?

Was this observed in the experimental results?

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4. Would you expect the reaction rate to be faster in tube 2 or tube 3?

Why? Was this observed in the experimental results?

5. Which tube has an acidic environment?

How do you expect this to impact enzyme activity? Was this observed in the experimental results?

6. Based on the results you observed for your experiment, what are the necessary conditions for optimal glycolysis enzyme activity?

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