Oxidase Test

Objective:

•    To demonstrate the ability of bacteria that produces the enzyme cytochrome oxidase.
•   To distinguish the bacterial groups based on oxidase activity.

Principle:

            In aerobic respiration, oxidase enzyme plays a vital role in operation of electron transport chain. Cytochrome oxidase transfers the electrons from donor to molecular oxygen, during the oxidation of reduced cytochrome c to form water (or) hydrogen peroxide and oxidized cytochrome c.

            The ability of bacteria produce cytochrome oxidase enzyme can be determined by using oxidase disc impregnated with N, N, N΄, N΄ tetramethyl p-phenylene diamine dihydrochloride (TMPD) reagent. This reducing dye become oxidized and turns into deep purple color from colorless by oxidase enzyme, the colored end product is called indophenol. Pseudomonas is the only species which gives positive reaction in enterobacteriaceae family.

Materials required:

- Bacterial cultures (Pseudomonas sp., and E.coli )

- Oxidase disc (Readymade)
(Preparation of Reagent:  1% solution of N, N, N΄, N΄ tetramethyl p-phenylene diamine dihydrochloride (5g in 5ml in distilled water).  Disc preparation: Soak the sterile filter paper disc in few drops of reagents)

Procedure :
1.      Pick the 24 hours old bacterial cultures by platinum loop/plastic loop and smear over the oxidase    disc.
2.      After 10 min. observe the color change on disc.

Observation :
-   Deep purple color change of the disc with   Pseudomonas sp.,
-   No color change in the disc with E.coli

      

Result :
-  Pseudomonas sp., gives oxidase positive
- E.coli shows oxidase negative.

                             

       

Interpretation:

            Pseudomonas sp., gives positive result because it oxidizes the reagent N, N, N΄, N΄ tetramethyl p-phenylene diamine dihydrochloride and turns into deep purple color by oxidase enzyme. The purple colored compound known as indophenol. E.coli gives negative reaction because it doesn’t produces oxidase enzyme.

Oxidase positive organisms:

Pseudomonas sp., Vivrio chlorae, Nisseriae  sp., Camphylobacter sp., Alcalgenes sp., and Helicobacter sp.,

Precautions:

1.      The regent is to be prepared fresh.

2.      Nichrome wire is not to be used take culture. Trace of iron gives false positive result

3.      Should not take culture from selective medium

4.      Observe the result within the prescribed time.