Oxidase Test

Oxidase enzymes play a vital role in the operation of the electron transport system during aerobic respiration.  Cytochrome oxidase catalyses the oxidation of a reduced cytochrome by molecular oxygen (O₂), resulting in the formation of H₂O or H₂O_2.  Aerobic bacteria as well as some facultative anaerobes and microaerophiles, exhibit oxidase activity.  The oxidase test aids in differentiation among members of the genera Neisseria and Pseudomonas, which are oxidase positive, and Enterobacteriaceae, which are oxidase negative.

Aim

To distinguish among groups of bacteria on the basis of cytochrome oxidase activity.

Principle

The ability of bacteria to produce cytochrome oxidase can be determined by the addition of the test reagent, p-aminodimethylaniline oxalate, a reagent which serves as an artificial substrate, donating electrons and thereby becoming oxidized to a blackish compound in the presence of the oxidase and free oxygen.  The dark coloration is indicative of cytochrome oxidase production and represents a positive test.  No color change is indicative of the absence of oxidase activity and is a negative test.

Requirements

24 hr nutrient broth cultures of species Escherichia coli, Proteus, Pseudomonas, Staphylococcus, Streptococcus, and Bacillus.

Oxidase strips containing p-aminodimethylaniline oxalate, Bunsen burner, glass rod, glass marking pencil etc.

Procedure

1. Remove oxidase strips from the container and place in a petridish.
2. With the help of a clean glass rod / plastic loop or platinum wire pick a colony from 24 hrs growth of      the test organism and rub over the filter paper.  Do not use nichrome wire.
3. Observe the color change within 5-10 sec. 

Observations

Pseudomonas sp. produced an intense deep blue color within 5-10 sec indicating a positive result.  Escherichia coli, Proteus, Staphylococcus, Streptococcus, and Bacillus sp. produced no change in color.

Result

Pseudomonas sp. is oxidase positive whereas Escherichia coli, Proteus, Staphylococcus, Streptococcus, and Bacillus sp. are oxidase negative.

(Image courtesy: orbitbiotech.com)