Microlab exercises 31-34

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Microlab exercises 31-34
2015-10-13 00:44:16
Microbiology Lab Exercises
Microlab practical 1
Reading and questions
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  1. T or F: When H. Pylori produces high urease enzymes in the mucous layer of the stomach, it creates a localized alkaline environment.
    True: the increased pH may protect the bacterium from the gastric acid in the stomach.
  2. Some bacteria can use urea as a source of ___.
  3. What are the end products when urea attacks the nitrogen and arbon bonds in urea?
    Urea + Water --Urease--> CO2 + H20 + Ammonia
  4. What colors indicate a positive test for Urea hydrolysis?
    Orange-red to deep pink or purplish red
  5. How is urease activity detected in lab?
    • By growing bacteria in a medium (urea agar slants) which contains urea, and a pH indicator
    • If the bacteria produces the urease enzymes, the urea in the medium will become hydrolyzed, and ammonia accumlates in the medium, which makes it alkaline.
    • This is then detected by the pH indicator.
  6. T or F: In the preperation of the urea agar slants, they cannot be heated above 50 degrees celcius.
    True: Also, the agar medium must be slanted before the medium solidifies to avoid the necessity of remelting the agar and causing hydrolysis of urea.
  7. What color is cerise?
    A deep, reddish pink
  8. This term means the removal of a carboxyl group from an organic molecule.
  9. Bacteria growing in liquid media decarboxylate amino acids most rapidly when conditions are:
    A. Aerobic and slightly acidic
    B. Aerobic and slightly alkaline
    C. Anaerobic and slightly alkaline
    D. Anaerobic and slightly acidic
    D. Anaerobic and slightly acidic
    (this multiple choice question has been scrambled)
  10. T or F: Many decarboxylases are activated by a high pH.
    False: low pH, they remove the acid groups from amino acids, producing lkaline amines, which raise the pH of medium
  11. Why is sterile mineral oil used in the decarboxylase test?
    To prevent oxygen from reaching the bacteria and inhibiting the reaction (decarboxylation happens most rapidly when conditions are anaerobic)
  12. In the decarboxylation test, why is there a color change in the pH indicator from purple to yellow, then yellow back to purple? What does the final color change indicate?
    • Acids produced by the bacteria from fermentation of glucose will initially lower pH of medium to yellow.
    • The low pH activates the enzyme, decarboxylase, which eventually raises the pH again, making the medium turn to purple.
    • A color change back to purple indicates a positive test.
  13. What was the LIA tubes used to test for? What does a color change of purple mean in the LIA tube? Black color change?
    • For the cultivation and differentiation of bacteria based on their ability to decarboxylate lysine and to form H2S.
    • Purple color: Indicates bacteria that decarboxylate lysine
    • Black colonies: indicates that H2s was produced
  14. Why is the lysine decarboxylse test negative if both LDC and DC broths turn purple?
    • DC broth (control) is supposed to be a negative result because it contained no lysine for the bacteria to hydrolyze with deoxycarbolase. This means the broth is supposed to stay a yellow color (no raising of the pH). 
    • If it turns purple, this could mean the incorrect tube was taken for the control.
  15. What does production of H2S mean in the LIA tubes?
    It shows a positive test for the bacteria to decarboxylate lysine, which meant the medium would have also turned purple from yellow.
  16. What does the phenylalanine deaminase test for?
    • Phenylalinine agar is used to detect the deamination of the amino acid, phenylalanine. 
    • An enzyme,,, phenylalanine deaminase catalyzes the removal of the amino group from phenylalanine.
  17. What are the end products from the phenylalanine deaminase enzymes? Why is ferric chloride added to the deamination test?
    • An acid (phenylpyruvic acid), ammonium ion, and water.
    • When bacteria produce the deaminase enzyme and create the phenylpyruvic acid, it reacts with the ferric chloride and forms a green compound.
  18. What does a positive test for phenylalanine show?
    A positive reaction with ferric chloride, which is a green color produced in the tube.
  19. Why must the phenylalanine test be determined within 5 minutes?
    After 5 minutes the ferric chloride reaction occurs, the green color will disappear.
  20. How is the enzyme, nitrate reductase, used by bacteria?
    • A class of bacteria, chemoorganoheterotrophs, require organic compounds for growth. This includes using nitrate as a terminal electron acceptor.
    • In this process, the enzyme reduces nitrate to nitrite (NO3- to NO2-)
  21. In the nitrate test, what does the gas bubble in the durham tubes indicate?
    The gas is a mixture of CO2 and N2, which is released from the reduction of nitrate.
  22. In the nitrate test, if there is no color change from the nitrate reagents to show that nitrate was reduced, how can the use of zinc show that there was still a possibility of nitrate reduction?
    • Some bacteria can reduce nitrate only to nitrite, while others can reduce it past nitrite to molecular nitrogen. 
    •   - By using the nitrate reagents, they will look for any nitrites that were reduced from nitrate, showing a red color. However, if the nitrates were reduced past nitrite, it will present a negative result since there are no nitrites for it to react with.
    •  - If this occurs, zinc is then used to make sure that the test really is negative. If there is a color change, after using the zinc, this solidifies the negative result that nitrate was not initially reduced.
    •  - If there is NO color change, this shows that there was nitrate reduction because the nitrates were reduced past nitrites and could not be detected by the nitrate reagents.