OXIDASE TEST

 

 

 

AIM:

            To identify whether the bacteria produces the enzyme Cytochrome C oxidase.

PRINCIPLE:

            Cytochrome C oxidase is a iron-containing heme protein, which plays an important role in Electron Transport chain (ETC) of aerobic respiration. Hence, the cytochrome system is found in aerobic, microaerophilic and facultative anaerobic organisms and are absent in obligate anaerobes.

 Generally the enzyme oxidizes cytochrome C and reduces O₂ to form H₂O in aerobic respiration (final step of ETC).

            In this test, if the organism produces the enzyme cytochrome C oxidase, in the presence of atmospheric oxygen, it will oxidizes the substrate Tetramethyl p-phenylenediamine dihydrochloride (TMPD) to Indophenol blue [oxidation].

 

                        TMPD + O₂    ---------^{cytochrome oxidase}-----------------> Indophenol blue

           

TMPD remains colourless in reduced state, when it gets oxidized it produces the deep blue/violet colour.

REAGENTS:

Kovac’s Reagent:

            1% Tetramethyl p-phenylenediamine dihydrochloride (TMPD)

Gordon and Mcleod’s Reagent:

            1% Dimethyl p-phenylenediamine dihydrochloride

Gaby and Hadley’s Reagent:

            1% α-napthol in 95% ethanol

            1% p-aminodimethyl aniline oxalate

Modified oxidase test reagent:

            1% tetramethyl p-phenylenediamine dihydrochloride in 6% Dimethyl sulfoxide (DMSO) 

PROCEDURE: (Disk method)

1.     Take the commercially available oxidase disk impregnated with dried TMPD reagent and place it over a clean glass slide.

2.     Using wooden stick/tooth stick pick the test colony from the 18-24hr culture plate and smear it over the oxidase disk along with positive and negative controls.

3.     Check for deep blue/violet colour change immediately within 10secs and interpret the results.

INTERPRETATION:

Positive control (ATCC 27853 Ps.aeruginosa) – Development of deep blue/violet colour at the inoculation site within 10secs.

Negative control (ATCC 25922 E.coli) – No colour change at the inoculation site.

If the controls are satisfactory, we can interpret the results for test organisms.

Test – Development of deep blue/violet colour at the inoculation site within 10 secs.

EXAMPLES:

 

OXIDASE POSITIVE ORGANISMS	OXIDASE NEGATIVE ORGANISMS
Pseudomonas sp.	Enterobacteriaceae family
Neisseria sp. Etc.,	Acinetobacter sp. Etc.,

 

OTHER METHODS:

1.     Modified oxidase test (for differentiating Micrococcus from Staphylococcus)

2.     Filter paper method

3.     Direct plate method

 

NOTE:

·       Any organism producing blue colour in 10-60 secs period is considered as Negative.

·       Nichrome, nickel steel loops should not be used as it may produce false positive results.

·       Old cultures, organisms grown on media containing dye should not be used.