Microlab example pictures practical 1 (part 2)

  1. Which letters on this petri dish shows a positive result of proteolytic enzyme production for casein hydrolysis?
    Image Upload 1
    • Positive: A & B
    • Proteolytic enzyme production shows a clearing around colonies, while a negative result is cloudy around colonies.
    • Here is another example:
    • Image Upload 2
  2. What is being tested for in this sample picture?
    What do the tubes on the left and right mean?Image Upload 3
    • This is testing for hydrolysis of gelatin
    • If gelatin is hydrolyzed by enzyme gelatinase, it does not gel when cooled but remains liquid, which is indicated on the right tube.
    • The left tube is a solid gelatin and does not flow when tube is tilted.
  3. What is the biochemistry of gelatin hydrolysis? (Include the enzyme used and end products)
    • 1. Substrate: Gelatin (semi solid gel) + H2O  
    • 2. Enzyme used: Gelatinase(enzyme)
    • 3. Further hydrolyzation: Polypeptides + H2O
    • 4. Enzyme used again: Gelatinase
    • 5. End products: Amino acids (liquid medium after chilling)
  4. In these sample slide, a test was done to show catalase production. Which slide represents a positive result?
    Image Upload 4
    Top slide: appearance of gas bubbles indicates a positive test.
  5. What is catalase and how is it used? (Show the biochemistry, including substrate and its end product)
    • It is used to turn hydrogen peroxide into water and O2.
    • 2 Hydrogen peroxide(H2O2) + Catalase --> 2 Water + O2
  6. What is catalase and how is it used? (Show the biochemistry, including substrate and its end product)
    • Bacteria produce this enzyme to form a fibrin clot around themselves as protection against host defenses. 
    • It is detected by any degree of clotting in the test tube of animal plasma that is added by the bacteria. 
    • Image Upload 5
  7. What is DNase and how is it detected?
    • It is a nuclease enzyme used by bacteria. 
    • It degrades host DNA 
    • It is detected by using DNase agar cotaining methyl green, which binds to intact DNA only.
    • After incubation, a positive test will show a pink area around the culture.
  8. In this DNase test, which side is the positive result for DNase production?
    Image Upload 6
    The colony on the right is surrounded by a pink zone, indicating a positive DNase test.
  9. What is the biochemistry of the oxidase enzyme?
    • Cytochrome c oxidase uses O2 as an electron acceptor during oxidation of reduced cytochrome c to form water and oxidized cytochrome c.
    • Reduced cytochrome c + O2 --oxidase--> oxidized cytochrome c + H2O
  10. In the oxidase test, what is the tetramehtyl-p-phenylenediamine used for?
    • Production of oxidase can be determined by the use of oxidase test reagent (tetramethyl-p-phenylenediamine). 
    • Oxidation results in the reagent turning to dark blue then dark purple in the presence of free O2 and oxidase.
    • Image Upload 7
  11. What are the roles for oxidase enzymes for bacteria?
    • They play an important role in the operation of the electron transport system during aerobic respiration.
    • It uses O2 as an electron acceptor during the oxidation of reduced cytochrome c.
  12. What is the biochemistry of Urease enzymes? (List substrate and end products)
    Urea + H2O --Urease--> ammonia + CO2 +H2O
  13. T or F: Some bacteria can use urea as a source of nitrogen with the enzyme urease.
    True: urease attacks the nitrogen and carbon bond in amide compounds such as urea
  14. What pH indicator is added to the urease test that will help indicate a positive result and how does it work?
    • Phenol Red: It is a pH indicator that will turn pink when the medium turns alkaline due to urea being hydrolyzed.
    • Urea hydrolyzation creates ammonia, thus increasing pH of medium.
  15. In this urea agar slants, explain the results of each tube:
    Image Upload 8
    • A: yellow color is a negative result for urease production
    • B: slight pink on the slant means a weakly positive reaction
    • C: deep pink equals urease production
    • D: yellow tube means no urease production
  16. What is decarboxylation and show the biochemistry of it.
    • Decarboxylation is the removal of a carboxyl group from an organic molecule (amino acid). This results in the production of an amine and CO2.
    • Image Upload 9
  17. What condition is optimal for decarboxylation of amino acids? (What pH and O2 levels)
    Bacteria growing in liquid media decarboxylate most rapidly when conditions are anaerobic and slightly acidic.
  18. What is the purpose of the mineral oil in the decarboxylase exercise?
    • It is layered onto the broth to prevent oxygen from reaching the bacteria.
    • Optimal conditions for decarboxylation are anaerobic and slightly acidic
  19. How is decarboxylation initially initiated?
    • When glucose is fermented, it lowers the pH of the medium. 
    • The acid pH activates the enzyme, decarboxylase, and neutralizes the medium.
  20. What is the pH indicator used for the lysine decarboxylase test and which one of these tubes shows a positive result?
    Image Upload 10
    • Bromcresol purple: this pH indicator turns purple when the pH is at 6.8 or higher, and yellow below 5.2
    •  the appearance of a purple color throughout the broth after a yellow color is a positive test for decarboxylase.
  21. What were the ODC tubes in the decarboxylase exercise used to detect?
    Decarboxylation of the amino acids, ornithine
  22. Interpret the results of these ODC tubes. (A is a control tube)
    Image Upload 11
    • B: this tube is negative for ornbithine decarboxylase due to the yellow color. (Even though there was a acid fermentation, it was a weak acid because the bacterium was only capable of glucose fermentation, so the tube remained yellow).
    • C: The light purple meant a slight positivity for ortnithine decarboxylase
    • D: More positive for the enzyme indicated by the darker purple
    • E: same result from D
  23. Phenylalanine deaminase is an enzyme that catalyzes the removal of the amino group from phenylalanine. Show the biochemistry of this. (Substrate and end products)
    • Phenylalanine (substrate) + Phenylalanine deaminase (enzyme) --> removes NH3 --> (End products) Phenylpyruvic acid + NH4 + H2O
    • The end products included are organic acids, water, and ammonium ion (NH4)
  24. In the Phenylalanine deamination test, what is ferril chloride used for and how does it work?
    When it is added to the phenylalanine agar and there is deamination of phenylalanine producing phenylpyruvic acid, it reacts with the acid, forming a green compound.
  25. Which of these test tubes is a positive result of phenylalanine deamninase?
    Image Upload 12
    • Tube B: Ferric chloride is added to these tubes, and if there is phenylalanine deaminase production, an end product is phenylpyruvic acid.
    • The ferric chloride reacts with the acid, forming a green compound.
  26. Chemoorganoheterotrophs can use ____ as a terminal electron acceptor during anaerobic respiration:
    A. Carbon dioxide
    B. Nitrate
    B. Nitrate
  27. Show the biochemistry of nitrate reductase. (Substrate + end products)
    **Remember that the substrate is reduced and serves as terminal electron acceptor.**
    Nitrate + 2 Hydrogen + 2 Electrons --nitrate reductase--> Nitrite + Water
  28. T or F: bacteria have other enzymes, apart from nitrate reductase, to reduce nitrite further.
    True: Nitrite is reduced to either ammonium ion or molecular nitrogen.
  29. What is used to detect nitrite ions in the nitrate reduction test? Which one of these tubes show a positive result?
    Image Upload 13
    • Sulfanilic acid and N,N-dimethyl-1-naphthylamine
    • The tube on the right has turned red due to nitrate reduction and is a positive result.
  30. In the nitrate test, after sulfanilic acid and N,N are added, what are some possible expalantions if there are no color changes to pink red? (3 possible)
    • 1. The bacteria actually did have nitrate reductase enzymes, but also reduced nitrite further to ammonia or molecular nitrogen, so it was not detected by the sulfanilic acid.
    • 2. They posses other enzymes that reduce nitrate to ammonia
    • 3. Nitrates were not reduced by the bacteria.
  31. When would you use zinc powder in the nitrate reduction test? What does a color change mean?
    • To determine if nitrates were reduced past nitrate.
    • When zinc is added, it will react with nitrate and turn to pink or red. 
    • If there is a color change, this will mean that the bacteria did not reduce the nitrates and is a negative result for nitrate reduction.
Card Set
Microlab example pictures practical 1 (part 2)
Lab result pictures examples