Purpose
- The oxidase test is a biochemical reaction that assays for the presence of cytochrome oxidase, an enzyme sometimes called indophenol oxidase. In the presence of an organism that contains the cytochrome oxidase enzyme, the reduced colorless reagent becomes an oxidized colored product.
Protocols
- There are many method variations to the oxidase test. These include, but are not limited to, the filter paper test, filter paper spot test, direct plate method, and test tube method. All times and concentrations are based upon the original authors’ recommendations.
Filter Paper Test Method
- Soak a small piece of filter paper in 1% Kovács oxidase reagent and let dry.
- Use a loop and pick a well-isolated colony from a fresh (18- to 24-hour culture) bacterial plate and rub onto treated filter paper.
- Observe for color changes.
- Microorganisms are oxidase positive when the color changes to dark purple within 5 to 10 seconds. Microorganisms are delayed oxidase positive when the color changes to purple within 60 to 90 seconds. Microorganisms are oxidase negative if the color does not change or it takes longer than 2 minutes.
Kovács oxidase reagent :
- 1% tetra-methyl-p-phenylenediamine dihydrochloride, in water
- Store refrigerated in a dark bottle no longer than 1 week.
Oxidase Test – Filter Paper Method
Filter Paper Spot Method :
- Use a loop and pick a well-isolated colony from a fresh (18- to 24-hour culture) bacterial plate and rub onto a small piece of filter paper (please see Comments and Tips section for notes on recommended media and loops).
- Place 1 or 2 drops of 1% Kovács oxidase reagent on the organism smear.
- Observe for color changes.
- Microorganisms are oxidase positive when the color changes to dark purple within 5 to 10 seconds. Microorganisms are delayed oxidase positive when the color changes to purple within 60 to 90 seconds. Microorganisms are oxidase negative if the color does not change or it takes longer than 2 minutes.
Oxidase Test – Filter Paper Spot Method
Direct Plate Method :
- Grow a fresh culture (18 to 24 hours) of bacteria on nutrient agar using the streak plate method so that well-isolated colonies are present (please see Comments and Tips section for notes on recommended media).
- Place 1 or 2 drops of 1% Kovács oxidase reagent or 1% Gordon and McLeod reagent on the organisms. Do not invert or flood plate.
- Observe for color changes.
- When using Kovács oxidase reagent, microorganisms are oxidase positive when the color changes to dark purple within 5 to 10 seconds. Microorganisms are delayed oxidase positive when the color changes to purple within 60 to 90 seconds. Microorganisms are oxidase negative if the color does not change or it takes longer than 2 minutes.
- When using Gordon and McLeod reagent, microorganisms are oxidase positive when the color changes to red within 10 to 30 minutes or to black within 60 minutes. Microorganisms are oxidase negative if the color does not change.
Gordon and McLeod reagent :
- 1% dimethyl-p-phenylenediamine dihydrochloride, in water
- Store refrigerated in a dark bottle no longer than 1 week.
Oxdiase Test – Direct Plate Method
Oxidase Test Direct Plate Method
Test Tube Method
- Grow a fresh culture (18 to 24 hours) of bacteria in 4.5 ml of nutrient broth (or standard media that does not contain a high concentration of sugar, please see Comments and Tips section for notes on recommended media).
- Add 0.2 ml of 1% α-naphthol, then add 0.3 ml of 1% p-aminodimethylaniline oxalate (Gaby and Hadley reagents).
- Observe for color changes.
- Microorganisms are oxidase positive when the color changes to blue within 15 to 30 seconds. Microorganisms are delayed oxidase positive when the color changes to purple within 2 to 3 minutes. Microorganisms are oxidase negative if the color does not change.
Oxidase Test – Tube Method
References: http://www.microbelibrary.org/library/laboratory-test/3229-oxidase-test-protocol
