34 STUDIES IN MICROBIOLOGY (1] 



Exercise Vi 



+ O2). Catalase is a red iron-porphyrin-protein 

 closely related chemically to the blood pigment 

 hemoglobin. The oxygen which is produced can 

 be measured with a volumeter; thus the en- 

 zyme action is easily followed. In particular, we 

 shall study the behavior of the enzyme when 

 different amounts of the substrate (hydrogen 

 peroxide) are added. We shall also investigate 

 the inhibition of catalase activity by hydroxyl- 

 amine (NH2OH). This chemical interferes with 

 the enzyme by attaching to and hence block- 

 ing the iron atoms upon which its action 

 depends. 



Catalase is found in many organisms. We 

 shall study it in bacteria, the horse, and potato 

 plants. In all of them it possesses the same type 

 of enzyme activity, and, as can be judged from 

 inactivation by hydroxylamine, this activity is 

 based on the same active group. (Actually these 

 catalases do vary somewhat in their properties, 

 owing to differences in the amino acid composi- 

 tion of their protein components.) How do you 

 suppose organisms so different from one another 

 come to possess such similar enzymes? 



EXPERIMENTS 



A note on sterile procedure. In working with 

 bacteria it is necessary to minimize the possi- 

 bility of stray microorganisms from the air 

 entering the cultures and agar plates. Sterilized 

 glassware and pipets should be used whenever 

 possible. Containers should be opened for as 

 brief a time as possible when material is being 

 transferred. The wire loop which is used to 

 sample a suspension and spread it on agar must 

 be heated in a flame before use. If necessary, 

 dropper pipets and tubes may be sterilized by 

 heating them in a boiling water bath for five 

 minutes. However, always allow an instrument 

 to cool before using it, or else the heat will kill 

 the bacteria with which you are working. Avoid 

 touching pipet tips or the sterile part of cotton 

 plugs with your fingers; also do not place them 

 on the bench. 



Bacterial growth 



Into a sterile 4-inch test tube pour with sterile 

 precautions 2 ml (^ inch) of the young Serratia 

 culture, and into a wide 6-inch tube pour 20 ml 

 of nutrient broth (about 3 inches). Also obtain 

 nine sterile dropper pipets; wrap these in a clean 

 paper towel before placing them on your bench. 

 With a sterile 5-ml pipet, transfer 5 ml of broth 

 to a wide tube, and without putting down the 

 pipet, deliver 0.9-ml portions of broth to eight 

 4-inch tubes which will be used for dilutions. 

 With a dropper pipet, inoculate the tube con- 

 taining 5 ml of broth with 2 drops of Serratia 

 culture. Swirl to suspend the bacteria evenly. 

 Label the tube with your name; this will be 

 your culture tube. 



Set up a dilution series A with four small 

 tubes, labeled Al, A2, A3, A4. Transfer 2 drops 

 from your culture tube into tube No. 1. Swirl 

 contents to mix, and with a fresh pipet add 2 

 drops of No. 1 to No. 2. Continue in this man- 

 ner so that you have a series of four tubes each 

 of which has only -^ as many cells as the one 

 before it. (A drop contains 0.05 ml.) Be careful 

 to add just the right number of drops, since 

 your calculation of the number of cells present 

 depends on the accuracy of the dilutions. 



Now, insert a sterile aerator into your culture 

 tube in place of the cotton plug. Attach the 

 long arm of the aerator to the air hose adjacent 

 to the water bath. Adjust the flow of air so 

 that it bubbles gently through your culture. 

 Incubate the tube in the bath at 37°C. 



Take an agar plate. Turn the petri dish up- 

 side down, and with a wax pencil divide the 

 bottom into four quadrants, labeled Al, A2, 

 etc. Pass a wire loop through the flame, wait 

 a moment for it to cool, then dip it into the 

 No. 4 tube. Make sure you have a loopful of 

 liquid. (A loopful contains just about 0.001 ml.) 

 Gently spread the contents of the loop on the 

 agar in the appropriate quadrant. Repeat with 

 the other tubes. It is not necessary to flame the 

 loop each time when going from lower to higher 

 concentrations. Why? 



After your culture has grown for two hours, 

 repeat this procedure to determine the number 



