Pracitcal

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Principal of OF carbs: Differentiates fermentors and oxidizers of carbs
How is an OF carb test set up?: Inoculate 2 tubes with bug;
-one overlaid, anaerobic environment;
-one is left open for aerobic;

Incubate, wait to see which turn acid
Results of OF: -Fermentor only of carb -Fermentor and Oxidizer of carb -Oxidizer of carb -Neither: 1. Only tube overlaid will turn yellow
2. Both tubes turn yellow
3. Only open tube turns yellow.
4. Both stay neutral or blue due to peptone use
What's in an OF carb tube?: -1% carb
-Brom thymol blue
Semisolid agar
what type of dye is in DNAse plates?: -methyl green
or
-toluidine blue
what reagent is used for catalase?: 3% H2O2
How can you mess up a catalase?: 1. Dropping H2O2 and then adding bug; touching needle to H2O2 causes false +.
2. Blood agar in peroxide -> false+
3. Old cultures
4. Exposing H2O2 to light.
What does coagulase test differentiate?: S. aureus from coag-neg staphs.
what isbound coagulase also known as?: clumping factor
Which type of coagulase does the tube coag test detect?: Both free and bound.
Which type of coagulase does the slide test detect?: Bound (to the cell) coagulase
What is the action of bound coagulase?: Converting fibrinogen rapidly to fibrin.
What is the action of free coagulase?: Reacts w/ a plasma factor to form substance like thrombin which eventually activates formation of FIBRIN.
Process of a Slide coag test:: 1. Heat slide; draw 2 circles, label TEST and CONTROL
2. drop of water in each circle.
3. emulsify w/ organism
4. to test add drop of plasma
5. rotate/look for clumps in 30 sec.
how long should a tube coag test be incubated before calling negative?: 4 hours
What is the reagent for a coag test?: Plasma
How can you mess up a coag test?: 1. Using cold plasma -> false neg
2. Failing to run control; maybe it was a false neg.
3. Using a mixed culture can give false pos (even tho its not coag)
4. Observe every 30 min; fibrinolysin may break down clot b4 you see it.
5. Don't shake tube or you'll ruin it.
What does a DNAse differentiate?: 1. S. aureus from coag neg staphs

2. Serratia(+) from Enterobacter(-)

3. M. catarrhalis(+) from Neisseria(-)
Which produces DNAse, S. aureus or S. epidermidis?: BOTH; but s. epi produces less - not enough to make a positive test.
2 types of DNA agar:: -methyl green
-toluidine blue
whats teh problem with toluidine blue?: inhibits growth of enteric bacteria
What are results of a DNase test: positive negative: pos = zone of clearing around inoculum
neg = growth right up to edge
How can you mess up a DNAse: not enough inoculum - not enough DNAse activity to cause clearing
What's in a Mannitol salt agar?: -7.5% salt
-Phenol red
-Mannitol
Purpose of Mannitol salt: to preliminary id. S. aureus from coag neg s. aureus.

-isolate S. aureus from a clinical specimen
Results of a Mannitol Salt agar test: pos neg: Pos = growth; yellow - mannitol ferm.
Neg = no growth; red - nonfermentor.
How can you mess up a mannitol salt agar?: Too much inoculum - the mannitol runs out, Positive bugs revert to peptone use and it appears negative falsely.
What is a BACTi staph? What does it detect?: Quick slid latex agglutination test kit.

Both protein A and coagulase from S. aureus
Why are acid fast organisms called that?: they don't decolorize with acid alcohol
What dye is used in a cold acid fast stain?: Kinyoun's carbol fuchsin
What is the counterstain in a cold acid fast stain?: methlene blue
What stains are used in a cold acid fast stain?: Primary - Kinyoun's carbol fuchsin
Counter - Methylene blue or brilliant green
What are 2 alternates to an acid fast stain?: -Ziehl-neelson
Truant flourescent
What is -beading -cording: beading - the rods don't take up the stain evenly.

cording - cells grow lying end-end; make long strands when stained.
What do Acid fast stain results look like? -Pos -Neg: pos = red
neg = blue or green
What is a bacitracin test for, what are -Pos -Neg results?: Strep group A;
Pos = inhibition, susceptible.
Neg = no inhibition, growth right up to disc
how can you mess up a bacitracin test?: -use bugs other than beta-strep; can give false +

-think it's a definitive test - some Beta-streps other than Strep A can give false +
What do you run a Bile Solubility test for?: To differentiate Strep pneumonia from other alpha-hemolytic Streps. Alternative to optichin
What are the active factors in a bile solubility test?: Bile salts - sodium deoxycholate, sodium taurocholate

Streptolysin produced by the strep pneumoniae itself.
What do the active ingredients of the Bile solubility test do?: Bile salts and autolysin cause cell-memrane derangement, and destruction of the pneumococcal cell.
What are results of a bile solubility -Pos -Neg: Pos:
-Cleared (lysed) test, Turbid control

Neg:
-Turbid test AND control
What reagent is added to a broth culture for a bile solubility test?: Sodium deoxycholate - the bile salt
How to report Bile solubility results?: Positive, bile soluble

Negative, bile insoluble.
Camp test identifies what? Alternate?: Strep group B
Hippurate hydrolysis
What happens in a CAMP test?: CAMP factor produced by Strep B bugs acts synergistically with Staph A's beta hemolysin to lyse sheep RBCs
What is a hippurate hydrolysis test for? Alternate to:: I.D. of Strep group B; an alternate for the CAMP test.
What chemical reaction occurs in the hippurate hydrolysis?: Hippuricase hydrolyzes Sodium hippurate into benzoid acid plus glycine.
What reagent is used in the Hippurate Hydrolysis test; what product does it check for? What color forms?: Ninhydrin; checks for glycine produced from hippurate hydrolysis.

Forms a purple color
what do results of a hippurate hydrolysis test look like?: Positive = deep purple
Negative = faint purple
What error can occur in hippurate hydrolysis?: Incubating too long = results beyond thirty min after adding the ninhydrin can be false positive since everything will be positive after a while.
6.5% NACL is for: Error can be from:: Enterococcus differentiation from Strep group D nonentero.

Contamination
optichin test is for: error can be from:: -strep pneumo from viridans

-presumptive only; using non-alpha hemolytic bugs
Chemical ingredient of Optochin:: ethylhydrocupreine hydrochloride
What is the alternate test for a Bacitracin test? What does it identify?: PYR hydrolysis;

Identifies Strep group A
What enzyme hydrolyzies PYR, and what bugs produce it? What is produced from the hydrolysis?: L-pyroglutamyl aminopeptidase.

Enterococci and Strep group A.

Produces Beta-naphthylamine
What reagent do you add run a PYR test?: Cinnamaldehyde
What are PYR test results -Pos -Neg What does the test indication result from?: Pos = bright red color w/in 5 min
Neg = no color or orange.

Red is from Cinnemaldehyde reacting with Beta-naphthylamine.
What tests are used to identify/differentiate Neisserias and Moraxella catarrhalis?: -CTA carb tubes
-Oxidase
-APT NH
what are CTA carbs for?: differentiating Neisserias.
What is CTA? What does it show?: Cystine Trypticase Agar;
carbs added to it;

Shows organism's ability to oxidize carbs.
what is the pH indicator in CTA tubes: phenol red
CTA results for positive negative: pos = yellow; oxidizer of carb
neg = red/orange.
how can you mess up a CTA carb?: Too small of inoculum gives = false neg
Incubating not long enough = false neg
Yellow color all the way down indicates a contaminant
What is OXIDASE essentially for differentiating?: gram negative cocci and bacilli
What is oxidase?: an enzyme necessary for some bugs, for oxidation of carbs.
What is the reagent used for oxidase testing?: tetramethylparaphenylendiamine dihydrochloride.
What happens when colonies are rubbed into filter paper soaked in Oxidase reagent? Pos Neg: Pos = purple color forms when reagent reacts with oxidase.

Neg = no color appears.
How can you mess up an oxidase test?: -Exposing Oxidase reagent to light causes auto-oxidation; discard.
-Reading beyond 10 seconds.
-using a metal inoculating needle - false positive
-getting culture media in the reagent.
-false neg for organisms growing on high-glucose media; fermentation inhibits oxidase activity.
when testing a gram neg cocci on a nutrient agar plate, how do you incubate it?: in a candle jar. they're fastidious organisms.
what is the endproduct that is purple in the Oxidase reaction?: indophenol
what is the staining reaction of Listeria?: very short gram positive coccobacilli
How can gram positive bacilli be identified?: -various catalase reactions
-various lengths on gram stain
-all oxidase positive

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