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STUDENT OBJECTIVE

Students qualitatively assay the end products of aerobic and anaerobic respiration in yeast cells. They also measure the rate of respiration in normal and azide-treated pea seedlings. Values for the volume of oxygen consumed by the seedlings are corrected to standard temperature and pressure. A procedure is included for demonstrating that cyanide affects cytochrome oxidase in mitochondria.

EQUIPMENT	AMOUNT
	(Class of 24 with 8 groups)
Hot plates Water baths, 4oC and 30oC Balance, electronic or gravimetric Refrigerated centrifuge with rotor, 10,000 x G Barometer (optional, see NOTES) pH meter	2/lab 1/lab 2/lab 1/lab
MATERIALS
Yeast respiration apparatus: Test tube rack Test tubes Flasks, 1000 ml Rubber stopper, one-hole #9 Rubber stopper, two-hole #9 Rubber stopper, #9 Glass tubing, 6 mm x 25 cm bent in middle in U-shape Glass tubing, 6 mm x 30 cm Rubber hose, 50 cm Yeast, dry Aquarium air pump	Demonstration 1/lab 3/lab 3/lab 1/lab 1/lab 1/lab 1/lab 1/lab 2 T/lab 1/lab
Distillation apparatus (see figure 6.2 in lab manual): 1000 ml flask (distillation vessel) Rubber stopper, one-hole #9 Glass tubing, 8 mm x 122 cm bent at 22 cm in V-shape Flask (collection vessel), 125 ml Ring stand and clamp Pipette, 10 ml	3/lab
Ethanol test: Test tubes Test tube rack Beaker, 125 ml Pipettes, 5 ml in 0.1 ml (for distillation samples) Repipetters, 500 ml (for NaOH, ethanol, water, and I2KI) Marking pencil Spot plate Matches Dropper bottle (marked "ETHANOL")	5/group 1/group 1/group 3/group 2/lab 1/group 1/group 1/group 4/lab
Pea respiration test: Beakers, 800 ml (for soaking peas) Peas, two- to four-day old germinating: Sucrose soaked Azide soaked or boiled Forceps, marked "Sucrose" and "Azide" (for dispensing stock peas) Plastic weigh pans, marked "Sucrose" and "Azide" Probes Absorbent and nonabsorbent cotton Pea-tube setup (see lab manual fig. 6.3): Test tubes, 25 mm x 150 mm Pipettes, 1 ml x 0.01 Rubber stoppers, one-hole #3 Disposable syringes, 5 cc Needles, #18	2/lab 8—10/group 8—10/group 2/lab 2/group 2/group box each/lab 3/group
Dye, red food coloring in small dropper bottle Dropper bottle (marked "KOH")	1/group 1/group
Mitochondria test: Peas, two- to four-day germinating Mortar and pestle Sand Beaker, 800 ml Beaker, 250 ml Ice Cheesecloth, 22 cm x 22 cm Graduated cylinder, 50 ml Test tubes Test tube rack Centrifuge tubes, 15 ml (to withstand 15,000 G) Double pan balance with beakers (for balancing centrifuge tubes) Pipettes, 10 ml Suction device, 10 cc disposable syringe with 3 cm rubber tubing attached Wash bottle with distilled water	Demonstration 50 g/lab 1/lab 100 g/lab 1/lab 1/lab 10 pounds/lab 5/lab 1/lab 4/lab 1/lab 8/lab 1/lab 6/lab 1/lab 1/lab

SOLUTIONS

Corn syrup solution
0.1 M barium hydroxide (Ba(OH)₂)
95% ethanol (C₂H₅OH)
1.5 M sodium hydroxide (NaOH)
Iodide solution (I₂KI), strong
0.4 M sucrose (C₁₂H₂₂O₁₁)
0.2 M sucrose + 0.01 M sodium azide (NaN₃)
15% potassium hydroxide (KOH)
0.4 M sucrose + 0.1 M phosphate buffer (KH₂PO₄/K₂HPO₄)
0.2% p-phenylenediamine (C₆H₄(NH₂)₂) (Fisher)*
0.1 M sodium azide (NaN₃)

*Please refer to the Appendix for name and address of supplier.

PREPARATION

Three Weeks before Lab

1. Purchase peas (about 2000 per pound) and find a suitable area in which to germinate the peas. A greenhouse is a good place for maintaining constant temperature; light is not necessary. A minimum temperature of 22^oC is needed. To hasten germination, the temperature can be increased to 27^oC for 24_—48 hours.
2. Make a trial planting of peas to ascertain the number of days needed to obtain a seedling with a root and beginning shoot (no green development). Under ideal conditions, this will take 2—4 days.
3. To germinate peas, fill a large, flat tray with 1/2" sterile moistened soil. Press seeds into the soil and cover with 1/2" vermiculite. Thoroughly moisten with water. Foil over surface will reduce evaporation. Peas also can be germinated on moist paper towels covered with foil. Check the trays every day and water according to need.

One Week before Lab

Yeast Respiration Experiment

1. Yeast culture medium:

   Prepare three 1 liter flasks, one to be used for aerobic respiration, one for anaerobic, and another for a control. For each 1 liter flask, mix 140 ml corn syrup with 280 ml tap water. Cover opening with foil and autoclave for 15 minutes at 15 psi. Store at room temperature until used in the fermentation procedure.

2. Strong iodide solution (I₂KI) preparation:

   Mix 5 g iodine and 10 g potassium iodide in 100 ml distilled water. Store in a brown bottle and put out small amounts in automatic dispenser or beaker for student use.

3. Sodium hydroxide preparation:

   1.5 M NaOH 30 g NaOH/distilled water to make 500 ml

   Completely dissolve NaOH pellets in 400 ml water by stirring; dilute to 500 ml volume. Wear goggles to protect eyes from splashes. Be aware of heat of solution. Store solution in a plastic bottle and put out small amounts in automatic dispenser or beaker for student use.

4. Barium hydroxide preparation:

   0.1 M Ba(OH)₂•8H₂O 7.9 g Ba(OH)₂•8H₂O/distilled water to make 250 ml

   Boil the water to remove the dissolved CO₂. After the water has cooled, add the hydroxide and slowly mix. A crust may form on top of the solution after mixing because CO₂ + Ba(OH)₂ yields BaCO₃ (insoluble). Cover and let sit until a clear solution appears below (4—12 hours). Without agitating the solution, pipette 10 ml of the clear solution into a test tube and cap the tube with a rubber stopper until needed to demonstrate the presence of CO₂.

   Pea Respiration Experiment

5. Plant enough peas so that there will be 20 germinating peas per student group (about 140 g of dry peas per lab).
6. Potassium hydroxide preparation:

   15% KOH 15 g KOH/85 ml distilled water

   Stir hydroxide until dissolved; wear goggles; fill dropper bottles.

7. Sucrose preparation:

   0.2 M sucrose 68.5 g sucrose/add distilled water to 1 liter

   Dissolve sucrose in water and store in refrigerator.

8. Sucrose containing sodium azide preparation:

   0.01 M Azide (NaN₃) in 0.2 M sucrose 0.3g NaN₃/500 ml of 0.2 M sucrose solution

   Warning: In a fume hood, carefully weigh out the azide. Avoid contact with skin and wash immediately if contact occurs. Add to the 0.2 M sucrose solution and stir until dissolved. Immediately clean up any spillage and dispose of in manner approved by institutional safety officer. All glassware should be thoroughly washed. Some instructors substitute boiled peas to eliminate the use of azide.

   Cytochrome oxidase/mitochondria Experiment

9. 0.2% p-phenylenediamine preparation:

   0.1 g C₆H₄(NH₂)₂/50 ml distilled water

   Stir to dissolve the salt; store in the dark.

10. 0.1 M sodium azide preparation:

    0.65 g NaN₃/100 ml distilled water

    In a fume hood, weigh cyanide and dissolve the salt in the water by gently stirring.

    Warning: This is a deadly poison. Use extreme caution in preparation and cleanup.

11. 0.4 M sucrose preparation:

    68.5 g C₁₂H₂₂O₁₁/500 ml distilled water

12. Stock solutions for phosphate buffer preparation:

    "A" solution: 1 M KH₂PO₄ 68 g/500 ml distilled water

    "B" solution: 1 M K₂HPO₄ 87 g/500 ml distilled water

    (If water of crystallizations is present, amounts may need to be changed.)

    0.1 M phosphate preparation:

    Mix 4.9 ml "A" with 30.4 ml "B." Check the pH with a pH meter and adjust the pH to 6.8 by adding "A" to decrease the pH or by adding "B" to increase pH.

    Dilute: 1 buffer: 2 distilled water

13. 0.4 M sucrose with 0.1 M phosphate buffer preparation:
    Mix together 9.5 ml "A" buffer, 40.5 ml "B" buffer, and 450 ml 0.4 M sucrose. Adjust pH to 6.8 by adding "A" or "B."
    
    
14. Make test runs in a refrigerated centrifuge with the appropriate rotor. Keep the centrifuge "on" until the lab is completed. On high-speed centrifuges, it is a good idea to keep a log book. All rotors should be washed with distilled water and rinsed with alcohol. Centrifuge maintenance usually involves lubrication and replacement of brushes. See manufacturer’s instruction booklet.
15. Fill the water bath with distilled water and adjust to 30^oC. Provide adequate support to accommodate the beakers of pea seedlings. Set aside a space in the refrigerator (4^oC) for solutions and such used in the demonstration of electron transport system.
16. Bend and insert glass tubing into stoppers for the distillation flasks (2), respiration flasks (2), and student pea tube setups (24). Protect hands and use glycerin as a lubricant when inserting glass.

24 Hours before Lab

Add 1 tablespoon dry granular yeast (baker’s) to the yeast respiration flasks. Seal with the rubber stopper/tubing assemblage and vent the gas into a water-filled test tube. Connect aerobic flask aeration tube to aquarium pump. Aerate for 24 hours at room temperature.

Morning of Lab

1. Fill 800 ml beakers: 200—300 ml of 0.2 M sucrose in "Sucrose" container
   200—300 ml of 0.2 M sucrose—0.01 M azide in container clearly
   labeled "AZIDE-POISON"

   Use clearly labeled disposable dispensers. Add washed, germinated peas to each container and place in water bath. Provide large forceps with matching labels for each container. Optimum results are achieved if the peas are soaked 45—90 minutes before students fill tubes. Timing is important. After 90 minutes, respiration falls quickly for both kinds of pea treatments.

2. Wrap 50 g germinated peas in plastic wrap and place in the refrigerator for use in mitochondria preparation.
3. Put mortar and pestle and graduated cylinder in the refrigerator.
4. Obtain the ice and store in a styrofoam container.
5. Containers (large glass jars with lid, etc.) clearly labeled should be provided for disposing of azide-soaked peas and cotton. This will insure the safe use of azide. A reminder should be posted in the lab to wash hands thoroughly before leaving.

24 Hours after Lab

All azide solutions and azide-soaked materials should be properly packaged and discarded according to the requirements of the lab and community.

NOTES

1. The construction of the respirometer is similar to the assemblage used in "Photosynthesis" (see Exercise #22). However, the bend in the pipette should be at the 0.75 ml mark with the delivery end of the pipette projecting 1.5 cm through stopper.
2. Students can get barometric readings for their STP calculations from local airports or radio stations if no barometer is available.

CLASSROOM SUGGESTIONS

1. Because of student error in pipetting, a yellow color may not develop in the iodoform test. Addition of more iodine solution and a 5—10 minute reaction period after mixing will give a positive reaction. Extra iodine solution should be added to all tubes so that results are comparable.
2. Students must make certain that the potassium hydroxide is dropped directly onto the cotton and does not contaminate the wall of the tube and the pea seedlings; otherwise, little or no respiration will be observed.
3. To get a drop of dye in the pipette, students can dip the end of the pipette into the dye bottle and draw up a drop with the plunger of the syringe. Caution students that touching the test tube after temperature equilibration will raise the temperature and then, as the tube cools, it will appear as if peas are using oxygen.
4. Students can complete the exercise in 2 1/2 hours if they set up the respirometers and work the other laboratory procedures in between the equilibration periods. The mitochondrial cytochrome oxidase experiment can be done as a demonstration during the lab, interrupting students’ work to show how homogenate is made, how centrifugations are done, and how to assay cytochrome oxidase.
5. A minimum homework assignment would be to construct graphs of pea seedling oxygen consumption versus time. The percent inhibition of respiration by cyanide should be calculated.
6. Lab takes a full three hours if mitochondrial experiment is done.
7. Check out the links for this lab topic at http://auth.mhhe.com/biosci/genbio/dolphin/ You will find useful materials for developing your lab introduction or summary, and in some cases, you may want to tell students to connect to a particular site for further information.

ANSWERS TO CRITICAL THINKING QUESTIONS

1. The atmospheric pressure in Denver, at the elevation above 5,000 feet, is much less than in New Orleans which is at sea level. However, by converting both sets of results to standard temperature and pressure, the results could be compared.
2. Anaerobic organisms can live in environments without oxygen such as in swamps, deep soils, and deep lakes. They are able to escape competition with aerobic organisms in doing so.
3. The data obtained in this experiment clearly show that plants, at least in the form of germinating peas, consume oxygen. The supplemental demonstration shows they have mitochondria. Therefore, plants not only photosynthesize, they also respire.
4. Cells lacking mitochondria may be prokaryotic or eukaryotic. If eukaryotic cells lack mitochondria, they cannot perform aerobic metabolism and have only anaerobic metabolism.

SUPPLEMENTAL MATERIALS