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Carb fermentation (setup, purpose)
- Fermentation media and durham tube (upside down tube in medium)
- medium contains peptones, ONE carb, and a pH indicator (colormetric)
- durham tube determines gas production
- red is neg, yellow is pos, gas is present or not
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Citrate utilization (diff, purpose, protocol)
- +: Klebsiella, Salmonella enteritidis, citrobacter, Enterobacter, Serratia
- -: E. coli, Salmonella typhi, Shigella, Yersinia enterocolitica
- IDs which bacteria can utilize citrate as carbon source and inorganic ammonium salts as nitrogen source
- Synthetic medium (slant) w/ bromthymol blue pH indicator
- *positive result is growth REGARDLESS of color change (color change to blue indicates citrate breakdown)
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Gelatin hydrolysis (diff, purpose, protocol)
- +: Proteus vulgaris AND P. mirabilis (and Serratia odorifera biotype 2)
- -: everything else
- Determines ability of bacteria to produce gelatinase (liquify gelatin)
- stab inoculate in gelatin deep OR add 4-5 drops of broth culture
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Indole test (diff, purpose, protocol)
- +: E. coli, Proteus vulgaris
- -: other enterics, Proteus mirabilis
- IDs bacteria that produce tryptophanase by detecting end product
- add Kovac's reagant (entero) or Ehrlich's reagant (anaerobes/nonfermentors) - produces red color
- Test often combined with other biochemical test (SIM or MIO agar)
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MR/VP (diff, purpose, mechanism, protocol)
- Methyl red/Voges-Proskauer test
- diff between many gram- rods (E. coli MR+/VP- | Enterobacter MR-/VP+)
- Determines metabolic pathway for glucose ferm (mixed acid = acidic pH, acetoin = neutral pH)
- MR coloration - 4.4 red, 7 orange, 9 yellow
- VP coloration - pink-red complex
- protocol: innoculate broth, incubate, then aliquot.
- add methyl red to tube 1, look for immediate change (red is +, no change is -)
- add a-naphthol to tube 2, shake well, observe for 5 minutes (red = +, no change is -)
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Moeller Decarboxylase medium (diff, purpose, mechanism, protocol)
- Arginine - E. cloacae+, E. aerogenes-
- Lysine - E. aerogenes+, E. cloacae-
- Ornithine - Enerobacter+, Klebsiella-
- Differentiates by measuring ability to decarboxylate an amino acid, raising pH (indicator becomes purple)
- *anaerobic process, requires mineral oil overlay
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Motility medium (purpose, protocol)
- Differentiates bacteria on ability to move through semi-solid medium
- stab innoculate into agar, check growth daily
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MUG test (diff, purpose)
- Diff between commensal E. coli (fluorescence) and O157:H7 (no fluorescence)
- Looks for β-D-glucuronidase enzyme, end product in medium fluroesces blue under UV
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Nitrate reduction (purpose, mechanism, protocol)
- Determines if nitrate is final e- acceptor in aerobic resp
- All enterics use nitrate reductase, some use nitrite reductase
- Reagant A + Reagant B = pink color if Nitrite is present
- no color could be no nitrite OR nitrite further reduced
- Zinc dust (after) = pink color means zinc reduced nitrate (∴ bacteria doesn't reduce nitrate)
- if no color change then bacteria reduces nitrate AND nitrite
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ONPG test (diff, QC, purpose, protocol)
- O-nitrophenyl-B-D-galactopyranoside
- diff between late-lactose-fermentors and non-lactose fermenters
- *ONPG is like lactose
- Shigella is + (color change) and salmonella is -
- place ONPG disc in suspension and incubate, look for color change
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Oxidase test (diff, purpose, protocol)
- Diff between gram- nonfermentors (pos) and gram- enterics (neg)
- Tests for cytochrome oxidase
- innoculate filter paper, add drop of oxidase reagent. Purple = +, no color = -
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Oxidation-fermentation tubes (diff, purpose, protocol)
- Diff between G- nonfermentors (P. aeruginosa = oxidizer / E. coli = fermentor / Alcaligenes faecalis = asaccharolytic)
- 2 tubes are used - one left open to O2, one overlaid with mineral oil
- Stab innoculated
- both yellow = fermentation
- aerobic yellow, oil overlay red = oxidation
- both red = no utilization of glucose
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Phenylalanine deamination (diff, purpose, protocol)
- +: Proteus, Morganella, Providencia
- -: everything else
- Determines ability to oxidatively deaminate phenylalanine
- 1 drop broth on slant, leave caps loose, add 4-5 drops FeCl3 (Green = + / no color change = -)
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TSI agar (diff, purpose, protocol, important results to know)
- Triple Sugar Iron agar
- Diff G- based on ferm of glu, lac, and suc AND H2S production
- Tube uses 2 areas - aerobic slant and anaerobic butt (stab, then streak as you remove needle)
- results are given "slant/butt" yellow = acid, red = alkaline, black = H2S (can only happen in acid), gas = gap at bottom of slant
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TSI important results to know (E. coli, S. typhi, Shigella, Plesiomonas, Pseudomonas, Alcaligenes, Salmonella serotypes)
- E. coli: A/(A)
- S. typhi: K/A H2S
- Shigella: K/A
- Plesiomonas: K/A
- Pseudomonas: K/K (K/NC)
- Alcaligenes: K/K (K/NC)
- Salmonella serotypes: A/(A) H2S
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Urea hydrolysis (diff, purpose, protocol)
- + (rapid): Proteus
- + (weak): Klebsiella pneumoniae, some Enterobacter sp
- enzyme used to break down urea
- slant or broth
- pink = +, colorless = -
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