Filter Paper Method

• First, a small strip of filter paper is placed on a sterile petri plate and soaked with 1% Kovacs’ oxidase reagent and kept to dry. 
• A well-isolated colony of test bacteria from a fresh 18-24 hour old culture is picked up using a sterile platinum or inert inoculation loop and smeared on the reagent-soaked filter paper piece. 
• The color change and time taken for the color change are observed. 

Filter Paper Spot Method

• A well-isolated colony of test bacteria from a fresh bacterial plate is picked up using a sterile platinum or inert inoculation loop and rubbed onto a small piece of filter paper. 
• 1 or 2 drops of 1% Kovacs’ oxidase reagent is placed on the organism smear. 
• The color change and time taken for the color change are observed. 

Direct Plate Method

• A few drops of Kovacs’ oxidase reagent are added over well-isolated (pure culture) colonies of test bacteria from fresh culture in a plate. 
• The plate is tilted and shaken gently to expose the colonies to oxygen. 
• The color change and time taken for the color change over the reagent-moistened colonies are observed. 

Test Tube Method (Gaby-Hadley Oxidase Test)

• A nutrient broth medium is inoculated with sample bacteria and incubated aerobically for 18-24 hours at 35±2°C. 
• 0.2 ml of 1% α-naphthol (Gaby-Hadley Reagent A) is added to the culture followed by the addition of 0.3 ml of 1% paminodimethylaniline oxalate (Gaby-Hadley Reagent B) and shaken to ensure they are well mixed.  
• The color change and time taken for the color change are observed.