Biochemical tests: No colour change Purple colour

OXIDASE TEST:

The oxidase test identifies organisms that produce the enzyme cytochrome oxidase. Cytochrome oxidase participates in the electron transport chain by transferring electrons from a donor molecule to oxygen. The oxidase reagent contains a chromogenic reducing agent, which is a compound that changes color when it becomes oxidized. If the test organism produces cytochrome oxidase, the oxidase reagent will turn blue or purple within 15 seconds.

PRINCIPLE: The reagent which is used for oxidase test is either N,N,N,N-tetramethyl-p phenylenediamine (TMPD) or N,N-dimethyl-p-phenylenediamine (DMPD). The reagent becomes dark-blue to maroon colour when oxidized, and colourless when reduced. Oxidase-positive bacteria possess cytochrome oxidase or indophenol oxidase. These both catalyze the transport of electrons from donor compounds (NADH) to electron acceptors (usually oxygen). The test reagent, TMPD dihydrochloride acts as an artificial electron donor for the enzyme oxidase. The oxidized reagent forms the coloured compound indophenol blue. The cytochrome system is usually only present in aerobic organisms that are capable of using oxygen as the final hydrogen receptor.

Diagram 1: On the left there is no change in colour of the reagent after addition of the bacteria, whereas on the right a purple colour is seen indicating oxidation of the reagent.

PROCEDURE: Filter paper method

1. Soak a small piece of filter paper with the oxidase reagent and let it dry. 2. Use a sterile loop or stick and pick up a well-isolated colony from the bacterial plate and

rub it onto the treated filter paper.

3. Pseudomonas and E.coli species were used for this practical. 4. Observe for colour change.

Microorganisms are oxidase positive when the colour changes to dark purple within 5 to 15 seconds. Microorganisms are delayed oxidase positive when the colour changes to purple within 60 to 90 seconds. They are oxidase negative if the colour does not change or it takes longer than 2 minutes.

UREASE TEST:

Urease broth is a differential medium that tests the ability of an organism to produce an exoenzyme, called urease. The broth contains two pH buffers, urea, a very small amount of nutrients for the bacteria, and the pH indicator phenol red. PRINCIPLE: Urease is an important microbial enzyme concerned with the decomposition of organic compounds. It can act on the test substrate, urea, contained in the medium resulting in the release of ammonia and carbon dioxide. The release of this alkaline end product results in an up shift in the pH that is detected by the colour indicator phenol red. A positive urease test results in the medium changing from yellowish-orange to bright pink.

Diagram 2: Phenol red turns yellow in an acidic environment and bright pink in an alkaline environment.