Oxidase Test

Key Points to Remember

• Always use fresh colonies grown on non-sugar media.
• Observe the color change within seconds — late color changes can mislead results.
• Store reagent away from light and use it within a week.
• Include both positive and negative controls for accuracy.

Keywords

Oxidase test, Cytochrome c oxidase, TMPD, Indophenol blue, Kovács reagent, Pseudomonas aeruginosa, Neisseria, Microbiology test.

Introduction

The oxidase test is a simple yet powerful microbiological test used to detect the presence of the cytochrome c oxidase enzyme in bacteria. This enzyme plays a vital role in the bacterial electron transport chain, helping cells produce energy through respiration. The test is particularly useful for distinguishing between different groups of bacteria, such as Pseudomonas (oxidase-positive) and E. coli (oxidase-negative).

Purpose of the Oxidase Test

The main purpose of the oxidase test is to identify bacteria that produce the enzyme cytochrome c oxidase.
It helps differentiate:

• Oxidase-positive bacteria → Pseudomonas, Neisseria, Vibrio, Campylobacter
• Oxidase-negative bacteria → Escherichia coli, Klebsiella, Salmonella, Shigella

This test is fast, inexpensive, and routinely used in clinical, food, and environmental microbiology labs.

Principle of the Oxidase Test

• The test is based on the oxidation of a chemical reagent known as tetramethyl-p-phenylenediamine (TMPD).
• If cytochrome c oxidase is present in the bacterial cells, it transfers electrons from TMPD to oxygen.
• This reaction oxidizes TMPD and produces a dark purple-blue compound called indophenol blue.
• If the enzyme is absent, no color change occurs.

Color Reaction:

• Positive: Blue or purple color within 10 seconds
• Negative: No color change within 2 minutes

This visual change makes the test quick and easy to interpret.

Reagents Used

Kovács Oxidase Reagent

• 1% TMPD (tetramethyl-p-phenylenediamine dihydrochloride) in distilled water
• Store in a dark bottle at 2–8°C; use within one week

Alternative Reagents (less commonly used):

• Gordon & McLeod reagent: Produces slower red-black reaction (>10 minutes)
• Gaby & Hadley reagents: Used for test-tube methods

Tip: Always use freshly prepared reagent. If it turns blue before use, it has auto-oxidized and should be discarded.

Materials Required

• Fresh bacterial culture (18–24 hours old)
• Non-sugar media (like nutrient agar or blood agar)
• Sterile swabs or wooden sticks (avoid metal loops)
• Whatman filter paper or oxidase test disks
• Pipette/dropper for reagent application
• Timer or stopwatch
• Safety gear (gloves, lab coat, eye protection)

Controls:

• Positive control: Pseudomonas aeruginosa
• Negative control: Escherichia coli

Oxidase Test Methods

Filter Paper (Direct/Drop) Method

• Place a piece of filter paper on a clean surface.
• Moisten it with 1% oxidase reagent.
• Pick a small bacterial colony using a wooden stick and rub it on the paper.
• Observe for purple-blue color within 5–10 seconds.
• Dispose of the paper safely — the reagent kills bacteria on contact.

Positive: Blue/purple within 10 seconds
Negative: No color change within 2 minutes

Swab or Plate Method

• Dip a sterile cotton swab into 1% oxidase reagent.
• Touch a fresh colony on an agar plate with the swab.
• Watch for an immediate colour change.
• Alternatively, place a few drops of reagent directly on colonies on the agar plate (avoid flooding).

Precautions:

• Read results quickly — the reagent oxidizes in air and can give false positives.
• Do not use colonies from sugar-containing media like MacConkey agar.
• Avoid metal loops (can cause false-positive reactions).

Interpretation of Results

Result Type	Observation	Interpretation	Examples
Positive	Blue or purple within 5–10 sec	Bacteria produce cytochrome c oxidase	Pseudomonas, Vibrio, Neisseria, Campylobacter
Negative	No color change	No cytochrome c oxidase enzyme	E. coli, Klebsiella, Salmonella, Shigella
Weak Positive	Light purple after 30–90 sec	Low enzyme activity	Some Aeromonas species

Diagnostic Importance

The oxidase test plays a key role in the identification of Gram-negative bacteria:

• Oxidase-positive organisms are usually aerobic (require oxygen for growth).
• Oxidase-negative organisms are often facultative anaerobes (can grow with or without oxygen).

In clinical microbiology, this test helps distinguish:

• Pseudomonas (oxidase-positive) from Enterobacteriaceae (oxidase-negative).
• Neisseria species from other Gram-negative cocci.

Conclusion

The oxidase test is a quick, reliable, and essential biochemical test in microbiology.
By identifying bacteria that possess cytochrome c oxidase, it helps differentiate between aerobic and facultative anaerobic organisms — a critical step in clinical diagnosis, environmental microbiology, and food safety testing.