Catalase Test

Principle:
Catalase catalyzes the breakdown H202 into water and oxygen. When a
small amount of an organism produces catalase is introduced into hydrogen
peroxide, rapid elaboration of bubbles of oxygen, the gaseous product of the
enzyme’s activity, is produced.

Notes:

Spot Oxidase Test

Principle:
The cytochrome oxidase enzyme is able to oxidize the substrate
tetramethyl-p-phenylenediamine dihydrochloride, forming a colored end
product, indophenol. The dark- purple end product will be visible if a small
amount of growth from a strain that produces the enzyme is rubbed on a
substrate- impregnated filter paper.

Notes:
 Slide Coagulase Test
Principle:
The presence of a cell surface- associated substance that binds
fibrinogen and thus allows aggregation of organisms in plasma containing
fibrinogen is detected by observation of clumping of cells.

Notes:

Tube Coagulase Test

Principle:
The production of the enzyme coagulase of some organisms lead to
clotting of rabbit plasma.

Notes:
 Bile Solubility Test
Principle:
S. pneumoniae will rapidly autocatalyze in the presence of the surfactant
sodium deoxycholate. The “melting” or dissolution of a colony within 30
minutes after exposure to the detergent is indicative of this species.

Notes:

Optochin Susceptibility Test

Principle:
S. pneumoniae is susceptible to the antibacterial agent
ethylhydrocupreine hydrochloride (optochin) which is impregnated in a filter
paper disk.

Notes:
 PYR test
Principle:
S. pyogenes and Enterococcus sp. possess the enzyme L- pyroglutamyl
aminopeptidase, which which hydrolyzes an amide substrate with formation
of the free- β-naphthylamine, which combines with a cinnamaldehyde reagent
to form a bright-red end product.

Notes:

Rapid Hippurate Hydrolysis

Principle:
The end products of hydrolysis of hippuric acid include glycine and
benzoic acid. Glycine is deaminated by the oxidizing agent, ninhydrin, which
becomes reduced during the process. The end products of the ninhydrin
oxidation react to form a purple colored dye.

Notes:
 CAMP Test
Principle:
Group B streptococci produce a protein-like compound called the
“CAMP factor” that is able to act synergistically with the beta toxin
produced by some strains of S. aureus to produce even more potent
hemolysis.

Notes:

Methyl Red and Voges- Proskauer Tests

Principle:
The presence of certain metabolic enzymes can be used to
differentiate organisms based on end products of glucose metabolism
detected with various color indicator reagents. Acid products produced by
enzymes of the mixed acid fermentation pathways are detected by
development of a red color by the methyl red indicator. Acetone and
butanediol, products of the butanediol fermentation pathways, yiel a red or
pink color in the presence of α- naphthol in the relatively alkaline
environment.

Notes:
 Phenylalanine Deaminase Test
Principle:
Deamination of phenylalanine yields phenylpyruvic acid.
Deamination of tryptophan yields indolepyruvic acid. These alpha keto
acids react with ferric chloride to form a green end product (phenylpyruvic
acid) or an orange-brown end product.

Notes:

Conventional Nitrate Reduction Test

Principle:
Organisms that possess nitrate reductase can reduce nitrate to nitrite.
Nitrite combines with an acidified naphthylamine substrate to form a red-
colored end product. If the organism has further reduced nitrite to nitrogen
gas, the test will yield a negative (colorless) result. An additional test for the
presence of unreacted nitrate must be performed to validate such a colorless
result. Metallic zinc catalyzes the reduction of nitrate to nitrite; thus with the
addition of zinc, a negative test will yield a red color indicating the presence
of unreacted nitrate.