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Describe catalase test and its reaction time.
- Detects catalase enzyme
- - converts H2O2 to H2O and O2
- - uncommon in anaerobes
Rapid detection (within minutes)
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Describe the coagulase test and its reaction time.
Two main types: agglutination and tube
- Agglutination: rapid, done by a kit to detect bound coagulase
- - microparticles with bound fibrinogen and IgG
- - agglutinate in presence of coagulase
Tube: detects free coagulase; overnight reaction
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Define Free and Bound Coagulase
Free: reacts with coagulase-reactin factor to form staphylothrombin, which catalyzes the breakdown of fibrinogen to fibrin
Bound: attached to bacterial cell wall and enzymatically converts fibrinogen to fibrin
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How is fermentation used to differentiate microbes?
Fermentation converts sugars in the medium to alcohol/acids, which lowers the pH.
Different mediums have different indicators that change color based on these pH changes, assisting in identification.
These reactions occur overnight.
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Describe the oxidase test.
The microbes are dabbed directly on a wet piece of paper that will react in the presence of cytochrome oxidase.
A positive reaction will occur within 3 minutes, yielding a purplish-maroon spot.
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Describe how the urease test works.
Microbes with urease enzyme break down amides in the medium to ammonia, lowering the pH.
A pH indicator in the medium changes color in response; test happens overnight.
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What is thioglycollate, and how is it used?
Thioglycollate is added to a semisolid medium to remove all O2 from it.
A tube containing the medium is innoculated, and based on the pattern of growth, we can determine if the microbe is a faculative or obligate aerobe, oxygen tolerant, or an obligate anaerobe.
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What staining is this?
Gram Positive
Thick peptidoglycan cell wall retains crystal violet staining after alcohol wash
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What staining is this?
Gram Negative
- Thin peptidoglycan cell wall with outer membrane
- Alcohol wash removes outer membrane and crystal violet, counterstain with pink saffranin
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What staining is this? (Look at small rods)
- Acid-Fast Staining
- Some bacteria have unusual cell walls (contain mycolic acid, giving waxy surface) and resist Gram staining
Used especially for microbes like Mycobacterium tuberculosis, leprae, and avium; also Nocardia and Corynebacterium (partial acid-fast)
Stain using hot carbolfuschin (pink color), and counterstain with methylene blue (gets everything else)
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What genera stain poorly?
Obligate Intracellulars (Chlamydia, Chlamydiophila, Rickettsia, Ehrlichia, Anaplasma, and Coxiella)
Spirochetes (Treponema and Borrelia)
Mycoplasma (do NOT have cell wall!)
Mycobacteria
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