EXERCISE 4

 

Biochemical Activities of Microorganisms: Enzymology

 

Supplies Required: 3 T.S. agar plate; 2 triple sugar iron (T.S.I) slants; 2 citrate slants; 3 T.S. deeps; cultures of Escherichia. coli, Enterobacter aerogenes, Pseudomonas aeruginosa, Enterococcus faecalis, Proteus vulgaris, Clostridium perfringens

 

Protocol:

 

PERIOD 1:

 

1.         Pick up a plate of T.S. agar and label it “catalase”. Using your marking pen, divide the plate in half (on the bottom). On one half of the plate streak for isolated colonies with P. aeruginosa and label. Streak the other half with E. faecalis and label.

 

2.         Pick up 2 T.S.I. agar slants and label them T.S.I. Aseptically inoculate the tubes as follows: Take a loopful of E. coli, enter the tube and stab the inoculating loop into the butt of the slant. The goal here is to get the organisms deep into the agar where conditions will be anaerobic. You do NOT want to tear up the agar significantly. As you remove your loop, gently brush it over the slant surface, flame the tube, cap it loosely, flame the loop and set it down. Label the tube. Inoculate the second slant using P. vulgaris and label it.

 

3.         Pick up two Simmon's citrate slants and label them “citrate”. Aseptically inoculate with E. coli by gently dragging the loop up from the bottom across the surface of the slant--you do NOT want to stab into the butt here as was done in step 8. Flame and close the tube in the usual manner. Label the tube. Inoculate the second tube with E. aerogenes and label.

 

4.         Pick up 3 T.S. deeps and label each one with an organism: Pseudomonas aeruginosa,

E. coli, or Clostridium perfringens. Using an inoculating needle aseptically transfer each organism into a deep, stabbing the needle well down into the agar. Again, the goal is to get the organisms deep into the agar where conditions will be anaerobic. Flame the tube, cap it loosely, flame the needle.

 

            Incubate all tubes and plates at 32oC. In 48 hours your plates and slants will be

refrigerated.

 

PERIOD 2

 

This experiment has been designed such that for each test, one positive and one negative result should be obtained..

 

1.         On your catalase/oxidase plate, add a few drops of H2O2 to an isolated colony on each half of the plate and look for gas bubbles effervescing off the colony. If this occurs, the organism is catalase positive.

 

2.         Examine your T.S.I. agar slant. Look for a darkening within the butt of the slant. This is a positive reaction for H2S, since the H2S formed has combined with iron to form iron sulfide. Compare the results obtained with P. vulgaris with those of E. coli.

 

3.         Examine your citrate tubes. If the agar slant has become a bright blue in color(the original color was green), the organism is citrate positive. Compare the results obtained with E. aerogenes with those obtained with E. coli.

 

4.         Examine your deeps. Is there growth throughout the agar, only at the top or only near the

bottom of the agar?