Gram Stain

Blood Agar Plate

Mannitol Salt Agar

Trypticase Soy Agar

Catalase

Coagulase

Rabbit Plasma.

Staphaurex: Protein "A" binds to Ig which is complexed with Latex to maximize visibility.

DNase

TNase

Heat tolerant form of DNAse.

Sample is heated, cooled, and placed into agar wells.

Oxidative-Fermentative Glucose Tubes

1) Low peptone media mixed with sterile ~0.5-1.0% glucose suspension.

2) Inoculate a pair of tubes with organism to be tested. Then cover surface of one tube with vaspar. Incubate for 24-78+ hours and observe for color change.

Most staphs are fermenters.

(Can be done with other CHO besides Glucose.)

Polymyxin

300 U Disk

Intermediate = 10

S. Aureus and S. Epidermidis: R

The rest: S

Modified Oxidase

Tetramethyl-p-phenylenediamine dihydrochloride (TMPD) in dimethyl sulfoxide (DMSO).

Examine after two minutes to observe for dark blue color change.

Novobiocin

Intermediate: 12-16

S. Saprophyticus, S. Xylosus, S. Sciuri, S. Cohnii: R

The rest: S

5 ug Novobiocin disk

MacConkey Agar

Chocolate Agar

Thioglycollate Broth

Colistin Nalidixic Acid Agar

Phenyl Ethyl Alcohol Agar

Bacitracin

0.04 U Disk

When used to ID Staph:

Staph: R

Micrococcus: S

Intermediate: 9-12

When used to ID Group A Strep. (S. Pyogenes):

Group A Strep: +

Most other Beta Strep: -

When testing for Strep, ANY zone of inhibition=Susceptible

Lysostaphin

1) "Lyses" staph.

2) Specifically breaks pentaglycine linkages in Staph cell walls.

3) 200 ug/mL Lysostaphin suspension is inoculated with diluted suspension of organism. Then sample is incubated at 35-37^o C. After 30 minutes, sample is examined for lysis.

Tellurite Reduction

1) Tests for presence of Tellurite Reductase

2) Tellurite Reductase reduces K Tellurite (in broth or media) to Metallic Tellurium.

3) Inoculate, incubate, and observe for black precipitate to indicate reduction.

(CoNS typically inhibited by K Tellurite and do not grow.)

Phosphotase

1) Broth test.

2) Inoculate substrate suspension with organism, incubate, add indicator, and observe for color reaction.

S. Aureus & S. Epidermidis: +

PYR Disk Test

Tests for Pyroglutamyl aminopeptidase

Substrate: L-Pyrrolidonyl-Beta-Naphthylamide (Pyr)

Reagent: N,N-Dimethyl-aminocinnamaldehyde (DMACA) in detergent.

Hydrolosis of Pyr releases B-Naphthlamide which reacts with DMACA within 2 minutes, creating a cherry red color.

S. Aureus, Group A Strep, and Enterococcus sp.: +

CAMP Test

1) Christie, Atkins, Munch-Peterson.

2) Detects CAMP factor that acts synergistically with Staph Beta Hemolysin.

3) Streak S. Aureus down the center of the BAP. Then streak the known positive and negative controls perpendicular to the S. Aureus streak on one side of the BAP. Then streak the organism to be tested on the other side of the BAP.

4) Common + Control: S. Agalactiae

Common - Control: S. Pyogenes

Hippurate Hydrolysis

1) Tests for the presence of hippurcase.

2) Hippurcase hydrolyzes Hippurate to Benzoic Acid and Glycine. Ninhydrin is then added to detect Glycine.

3) Add Hippurate disk to 0.4 mL DI water. Add heavy inoculum of organism to be tested. Incubate at 35-37^oC for two hours. Then add 0.2 mL of Ninhydrin reagent, shake, and observe for color change. Deep purple color = Positive

4) Group B Strep: +, Group A Strep: -

LAP

Bile Esculin

1) Detects bile tolerance & ability to hydrolyze Esculin. 

2) When Esculin is hydrolyzed, 6,7 dehydroxy coumarin (DHC) is produced, which reacts with Fe Salts to produce black or brown color change.

3) Inoculate, incubate for 18-24 hrs at 35-37 C.

4) All Group D: +

Most Betas: -

Optochin

1) Optochin = ethylhydrocupreine HCl (5ug/mL)

2) Incubate at 35-37^oC for 18-24 hours.

3) 6 mm disk: > 14 mm is S

4) S. Pneumoniae: S

5) The rest: R