36 STUDIES IN MICROBIOLOGY (1) 



Exercise VI 



tubes. Replace the stopper, making sure that 

 the side-arm, as also that of the thermobarom- 

 eter, is horizontal. Place a drop of kerosene in 

 the end of each side-arm. Unclamp the escape 

 tube, and with the aid of a medicine dropper in- 

 serted into the rubber tubing withdraw enough 

 air to pull this indicator drop back to the 

 proximal end of the scale on the side-arm. 

 When all is in order, add one drop of 3% hydro- 

 gen peroxide solution to the test tube containing 

 bacteria through the escape tube. Immediately 

 clamp it shut, and read the positions of the in- 

 dicator drops in both tubes. From now on re- 

 read both tubes every minute, agitating the 

 whole volumeter back and forth for 15 seconds 

 before each reading to hasten the escape of 

 oxygen. When the indicator drop has ceased to 

 move, add a second drop of hydrogen peroxide 

 to the bacterial suspension, and repeat the entire 

 performance. {Note: The position of the in- 

 dicator drop can be moved back to the proximal 

 end of the scale just before adding the second 

 drop of hydrogen peroxide.) 



Subtract all changes recorded in the thermo- 

 barometer from those measured in the experi- 

 mental tube. Plot a graph showing the volume 

 of oxygen emitted vs. time. (To turn your 

 measurements of the distance the indicator drop 

 has traveled into gas volumes you must of course 

 measure the internal diameter of the side-arm. 

 Better still, calibrate the volume of the side-arm 

 by injecting known volumes of air through the 

 escape tube with a 1-cc syringe.) Does the 

 evolved gas account for all the oxygen you would 



expect to obtain from one drop (0.05 ml) of 3% 

 hydrogen peroxide? [The equation for this 

 change is: H2O2 "'^'"") H2O + ^Oj. Starting 

 with the fraction of a mole of H2O2 added, 

 knowing from this the fraction of a mole of 

 oxygen that should be evolved, turn this into a 

 gas volume through the relationship: 1 mole of 

 gas at 273°K (0°C) and 1 atmosphere pressure 

 (as here) occupies 22,400 ml. At room tem- 

 perature on the absolute scale (r°K), this vol- 

 ume is increased by the factor 7"°/273.] 



Add two drops of hydroxylamine solution to 

 the test tube containing the bacteria. After one 

 minute, add two more drops of hydrogen 

 peroxide. Take readings until sure of the result. 

 Now add 10 more drops of hydrogen peroxide, 

 and again follow the reaction. Describe your 

 observations. How does the "noncompetitive" 

 inhibition of catalase by hydroxylamine differ 

 from the competitive inhibition of succinic 

 dehydrogenase by malonate studied in Exercise 

 V? 



An experiment in comparative biochemistry 



Cut off a 5 inch cube of potato. Mash it up 

 with a glass rod in 1 ml of water in a 4-inch 

 test tube. Set up three 4-inch test tubes con- 

 taining (1) two drops of Serratia culture, (2) two 

 drops of horse blood, (3) two drops of potato 

 extract. Add a drop of hydrogen peroxide to 

 each. Observe. After foaming has subsided add 

 a drop of hydroxylamine to each. Swirl to mix. 

 Add a drop of hydrogen peroxide. Is there a 

 reaction in any of the tubes? 



EQUIPMENT 



Per student 



5-ml pipet, sterile 



10 dropper pipets, sterile 



test tube, sterile 



2 test tubes, nonsterile 



2 wide tubes, sterile 



aerator assembly, sterile 



bacteriological loop 

 2 nutrient agar plates 

 bunsen burner 

 wood splint 

 marking pencil 



Per 8 students 



nutrient broth in 250-ml Erlenmeyer flask (20 ml) 



