Oxidase Test is an important differential procedure which should be performed on all gram-negative bacteria that are to be identified.

Composition: (1 package contains 50 discs) ; discs contains N,N-dimethyl-p-phenylenediamine oxalate and alpha-naphthol

Directions: Take aninoculating loop or a toothpick. Then touch and spread a well isolated colony on a oxidase disc. The reaction is observed within 2 minutes at 25-30°C.

Principle and Interpretation:
Gordon and McLeod (1) introduced oxidase test for identifying Gonococci based upon the ability of certain bacteria to produce indophenol blue from the oxidation of dimethyl-p-phenylenediamine and alpha-naphthol. Gaby and Hadley (2) introduced a more sensitive method by using N,N-dimethyl-p-phenylenediamine oxalate where all Staphylococci were oxidase negative.
In presence of the enzyme cytochrome oxidase (gram-negative bacteria) the N,N-dimethyl-p-phenylenediamine oxalate and alpha-naphthol react to indophenol blue.
Oxidase test is mainly used to differentiate:
1) Oxidase positive Neisseriafrom other gram-negative diplococci.
2) Oxidas positive Aeromonas hydrophilafrom Escherichia coli(gram-negative)
3) Oxidase positive Plesiomonas shigelloidsfrom Shigella sonnei(gram-negative)

Note: Cytochrome oxidase production may be inhibited by acid production and false negative reaction may be given by Vibrio, Aeromonas, and Plesimonasspecies when grow on a medium containing fermentable carbohydrate e.g. MacConkey Agar. Colonies taken from media containing nitrate may give unreliable results. The loss of activity of the oxidase reagent is caused by auto oxidation which may be avoided by adding 0.1% ascorbic acid

Reaction within 2 minutes at 25-35ºC.

Organisms (ATCC^®) / Reaction / Color :
- Pseudomonas aeruginosa (ATCC^® 27853) / Positive / Deep purple blue
- Staphylococcus aureus (ATCC^® 25923) / Negative
- Neisseria gonorrhoeae (ATCC^® 19424) / Positive / Deep purple blue
- Escherichia coli (ATCC^® 25922) / Negative