Lab quiz 5

  1. Nitrate reduction test

    Use of Nitrate broth can determine
    the ability of an organism to reduce nitrate to nitrite using the enzyme nitrate reductase
  2. Nitrate reduction test can also determine
    the ability of the organism to perform nitrification on nitrate and nitrite to produce molecular nitrogen and or ammonia
  3. nitrate reduction test will be testing for
    the presence or absence of nitrate reductase and/or nitrite reductase
  4. If the bacteria produces nitrate reductase
    the test tube will contain nitrite
  5. if the bacteria produces both enzymes
    the test tube will contain ammonia and/or nitrogen gas
  6. if the bacteria does not produce either enzyme,
    the test tube will continue to contain nitrate, thus the bacteria did not produce the necessary enzymes to reduce the nitrate
  7. What is added to the broth to determine which enzyme if any is produced by the bacteria
    • Reagent A - sulfanilic acid
    • Reagent B - a-naphthylamine
  8. If broth turns dark red after adding Reagent a and b it means
    the bacteria has reduced nitrate to nitrite. the bacteria produces the nitrate reductase enzyme
  9. The addition of ZINC
    reduces nitrate to nitrite
  10. if the broth turns red after adding zinc it means
    nitrate is present and the bacteria lacks nitrate reductase and nitrite reductase
  11. If the medium does not turn red after addition of zinc it means
    ammonia or molecular nitrogen were formed, thus the bacteria produces both nitrate reductase and nitrite reductase
  12. Nitrate Reduction test
    • P. aeruginosa = produces both enzymes
    • E. coli = nitrate   nitrite
    • M. luteus = nitrate - nitrite -
  13. Nitrate reduction test procedures
    Inoculate nitrate broth and incubate.

    • After incubation add reagent a and b
    • if a and b do not work add zinc
  14. IMViC test was developed as a means of
    separating members of the Enterobacteriaceae
  15. E. coli and E. aerogenes
    can be differentiated by the test because they give opposite reactions
  16. I
    Indole production from tryptophan

    inoculate Tryptone broth
  17. M
    methyl red

    tests for acid production from glucose (mixed acid fermentation)
  18. V

    tests for production of acetoin from glucose (butanediol fermentation)
  19. C
    citrate test

    tests for utilization of citrate as sole carbon source
  20. IMViC test results
    E. coli

    • I +
    • M +
    • V -
    • C -
  21. IMViC test results 2
    E. aerogenes

    • I -
    • M -
    • V +
    • C +
  22. Multitest System test

    The Enterotube multitest system is used for
    the rapid identification of Enterobacteriaceae
  23. The Enterotube II system is used for
    Gram negative, oxidase negate organisms
  24. If the Gram negative tests positive for oxidase
    the Oxi/Ferm tube is used
  25. The Enterotube OO system incorporates
    12 different compartments with 15 biochemical tests
  26. Multitest system procedure
    Take off the blue cap, pick up bacteria with needle part of wire, inoculate and incubate

    a numerical code is used to determine the bacteria
  27. Multitest system

    Puncture holes in
    adonitol, lactose, arabinose, sorbitol, V-P, dulcitol/PA, urea, citrate
  28. Lysine and Ornithine Decarboxylase test

    Decarboxylation is
    the removal of the carboxyl group
  29. Bacterial decarboxylation of lysine, an amino acid, results in
    the formation of an alkaline end product
  30. Lysine and Ornithine Decarboxylase test

    ph indicator
    Bromcresol purple
  31. Lysine and Ornithine Decarboxylase test

    positve and negative reaction
    • purple at alkaline pH - positive reaction
    • yellow as acidic pH - negative reaction

    color change = negative result
  32. Lysine and Ornithine Decarboxylase test

    Lysine and ornithine decarboxylase broth
  33. Lysine and Ornithine Decarboxylase test

    inoculate both broths, add 1 mL of sterile mineral oil to the top of the broths, tighten screw caps to create anaerobic environment which enhances decarboxylase activity
  34. Phenylalanine Deaminase test

    Which bacterias produce the enzyme phenylalanine deaminase?
    Proteus, Morganella, and Providencia
  35. What does the enzyme phenylalanine deaminase do?
    removes the amino (NH2) group from the amino acid phenylalanine to produce phenylpyruvic acid (PPA)
  36. Phenylalanine Deaminase tests

    organisms from other genera of the Enterobacteriaceae
  37. Phenylalanine Deaminase tests

    Detection of PPA is achieved by the addition of
    acidified ferric chloride (FeCL3) to the surface of the medium
  38. Phenylalanine Deaminase tests

    positive and negative reaction
    • green = positive
    • yellow = negative
  39. Phenylalanine Deaminase tests

    • use phenylalanine deaminase agar slant
    • fishtail it
    • second lab period - add acidified ferric chloride solution (FeCl3 + HCl) to slant
    • roll tube to loosen growth
  40. Amylase
    hydrolyses starch (complex carb) into simple sugars
  41. Gelatinase
    enzyme that degrades the protein gelatin
  42. The presence or absence of amylase and gelatinase can be helpful
    in the identification of bacterial species
  43. Phenylalanine Deaminase tests and lysine and ornithine decarboxylase test

    bacteria used
    • S. marcescens
    • P. vulgaris
  44. Digestive Exoenzymes test

    • b. subtilis = amylase +     gelatinase +
    • e. coli = amylase -     gelatinase -
    • p. aeruginosa = amylase -     gelatinase +
  45. gram - rod bacterias
    e. coli

    p. aeruginosa

    S. marcescens

    P. vulgaris
  46. gram +
    • m. luteus - cocci
    • tetrad

    • b subtilis - rod
    • chains or single
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Lab quiz 5