WEEK 7 - Staphylococcus Micrococcus Identification Methods

What does the oxidation-fermentation (OF) test determine?

Oxidation-fermentation test

What is another name for the oxidation-fermentation test?

Oxferm test

The OF test differentiates between which bacterial groups?

Gram negative rods, Staphylococcus, Micrococcus

What does the oxidative-fermentative test determine about bacteria?

Carbohydrate metabolism (oxidative, fermentative, or non-saccharolytic)

Which genus ferments glucose?

Staphylococci

Which genus is negative for glucose fermentation in the OF test?

Micrococci

What enzyme does the Microdase (Modified Oxidase) test detect?

Oxidase enzyme

What reagent is used in the Microdase test?

Tetramethyl-p-phenylenediamine dihydrochloride

What solvent is the oxidase reagent dissolved in for the Microdase test?

Dimethyl sulfoxide (DMSO)

What compound forms in the presence of oxidase enzyme and oxygen in the Microdase test?

Indophenol

What is the positive result for the Microdase test?

Blue to purple blue color

What is the negative result for the Microdase test?

No color change

What does bacitracin inhibit in bacteria?

Peptidoglycan synthesis

Which bacterial groups are distinguished by the Bacitracin Susceptibility Test?

Staphylococci (resistant) and Micrococci (susceptible)

What is the positive result for the Bacitracin test?

Zone of inhibition > 10 mm (susceptible)

What is the negative result for the Bacitracin test?

No zone of inhibition (resistant)

What enzyme does the catalase test detect?

Catalase

Catalase test differentiates catalase-positive Micrococcaceae and Staphylococcaceae from what group?

Catalase-negative Streptococcaceae

What concentration of hydrogen peroxide is used for Neisseria in the catalase test?

30% H2O2

What concentration of hydrogen peroxide is used for anaerobes in the catalase test?

15% H2O2

What concentration of hydrogen peroxide is used for other bacteria in the catalase test?

3% H2O2

What must be avoided when picking colonies for the catalase test, especially from blood agar?

Agar

What reagent is dropped onto the organism in the catalase test?

3% H2O2

What indicates a positive catalase test?

With bubbles

What indicates a negative catalase test?

Without bubbles

What type of coagulase is detected in the slide test?

Bound coagulase

What is another name for bound coagulase?

Clumping factor

Which Staphylococcus species usually produces bound coagulase?

Staphylococcus aureus

Which other Staphylococcus species may also produce clumping factor?

Staphylococcus lugdunensis, Staphylococcus schleiferi

What is mixed with rabbit plasma in the slide coagulase test?

Bacterial colony

What is used as a negative control in the slide coagulase test?

Sterile saline or sterile water

What indicates a positive slide coagulase test?

Agglutination with white fibrin clots within 1 minute

What does the conversion of fibrinogen to fibrin indicate in the slide coagulase test?

Positive for Staphylocoagulase or clumping factor

What does agglutination in both test and control in the slide coagulase test indicate?

Autoagglutination (invalid test)

What result indicates a positive bound coagulase test?

White clumpings

What type of coagulase is detected in the tube test?

Free coagulase

What must be done if the slide coagulase test is negative?

Perform tube coagulase test

What is free coagulase?

Extracellular toxin

What does free coagulase react with in plasma?

Coagulase-reacting factor (CRF)

What complex does free coagulase form with CRF?

Coagulase–CRF complex

The coagulase–CRF complex resembles which enzyme?

Thrombin

What does the coagulase–CRF complex convert fibrinogen into?

Fibrin

How much rabbit plasma is inoculated in the tube coagulase test?

0.5 mL

At what temperature is the tube coagulase test incubated?

35–37°C water bath

At what intervals is clot formation checked in the tube coagulase test?

Every 30 minutes up to 4 hours

Which enzyme may cause clot lysis in some S. aureus strains, leading to false negatives?

Fibrinolysin (staphylokinase)

At what temperature does fibrinolysin have increased activity?

35–37°C

If no clot is formed after 4 hours, how should the tube coagulase test be continued?

Incubate at room temperature for 16–18 hours

What indicates a positive free coagulase test?

Clot formation

What indicates a negative free coagulase test?

Fluid only

What does the DNase test determine?

Ability to hydrolyze DNA

Which Staphylococcus species gives a positive DNase test?

Staphylococcus aureus

What dye is incorporated into DNase agar medium?

Toluidine blue

What indicates a positive DNase test with toluidine blue?

Clearing of dye around growth

What modification of the DNase test involves flooding the plate after incubation?

Dilute hydrochloric acid

What color change indicates positive hydrolysis of DNA with HCl flooding?

Blue green to pink

At what temperature and time is the DNase agar incubated?

35–37°C for 13–24 hours

What indicates a positive DNase test with methyl green?

Release of methyl green and colorless area around growth

What indicates a negative DNase test with methyl green?

No degradation, medium remains green

What do chromogenic agar media contain as a nutrient source?

Peptone

What types of organisms are inhibited by chromogenic agar?

Gram-negative bacteria, yeast, some gram-positive bacteria

What is incorporated into chromogenic agar to produce colored compounds?

Chromogens (artificial substrates)

What color do Staphylococcus aureus colonies appear on HardyCHROM Staph aureus agar?

Deep pink to fuchsia

What color do Staphylococcus saprophyticus colonies appear on HardyCHROM Staph aureus agar?

Turquoise

What is the typical growth of Staphylococcus epidermidis on HardyCHROM Staph aureus agar?

Partially or totally inhibited

Is chromogenic agar commonly used?

Not commonly used

What type of medium does chromogenic agar utilize?

Agar

What is tested in the novobiocin susceptibility test?

Novobiocin susceptibility

What antibiotic disk strength is used in the novobiocin susceptibility test?

5 µg novobiocin disk

Which organism is resistant to novobiocin?

Staphylococcus saprophyticus

Which organisms are usually susceptible to novobiocin?

Most other coagulase-negative staphylococci

What agar is used in the Hebert method of the novobiocin test?

Blood agar plate (BAP)

What agar is used in the Kirby-Bauer method of the novobiocin test?

Mueller-Hinton agar (MHA)

What McFarland standard is used for BAP suspension in the novobiocin test?

Standard 1

What McFarland standard is used for MHA suspension in the novobiocin test?

Standard 0.5

What incubation temperature is used for the novobiocin susceptibility test?

35±2°C

How long is incubation for novobiocin susceptibility on MHA?

18 hours

How long is incubation for novobiocin susceptibility on BAP?

24 hours

What zone size on BAP indicates novobiocin sensitivity?

≥12 mm

What zone size on BAP indicates novobiocin resistance?

What zone size on MHA indicates novobiocin sensitivity (CLSI)?

>16 mm

What zone size on MHA indicates novobiocin resistance (CLSI)?

≤16 mm

What is the most commonly used agar for novobiocin testing?

MHA

If a coagulase-negative Staphylococcus isolated from urine is novobiocin-resistant, how should it be reported?

Staphylococcus saprophyticus

Can novobiocin resistance alone confirm S. saprophyticus from non-urine sources?

No

What does the PYR test detect?

Pyrrolidonyl arylamidase enzyme

What enzyme hydrolyzes the PYR substrate?

L-pyrrolidonyl arylamidase

What is the substrate used in the PYR test?

L-pyrrolidonyl-β-naphthylamide

What compound is produced when the PYR substrate is hydrolyzed?

β-naphthylamine

What reagent detects β-naphthylamine in the PYR test?

N,N-dimethylaminocinnamaldehyde

What color indicates a positive PYR test?

Bright red

What incubation time is required for the PYR broth method?

4 hours

At what temperature is the PYR broth incubated?

35–37°C

How quickly should red color appear after reagent addition in the PYR test?

Within 1–2 minutes

What is the rapid version of the PYR test?

Disk method

What result indicates a negative PYR test?

No color change

What does the ornithine decarboxylase test detect?

Ability to decarboxylate amino acids

Which amino acids are tested in decarboxylase media?

Arginine, lysine, ornithine

What type of product is formed when amino acids are decarboxylated?

Amine (alkaline pH)

At what temperature is ornithine decarboxylase broth incubated?

35–37°C