Oxidase Test in Microbiology: Everything You Need to Know

 

Video Lecture

Table of Contents

• Introduction to Oxidase Test
• Objectives
• Principle of Oxidase Test
• Requirements for Oxidase Test
• Procedure of Oxidase Test
• Result and Interpretation of Oxidase Test
• Quality Control
• Precautions during Oxidase Test
• Applications of Oxidase Test
• Limitations of Oxidase Test

Introduction to Oxidase Test

• The oxidase test is a biochemical testing module used to assay the ability of bacteria to synthesize cytochrome c oxidase enzymes.
• This test was first introduced by Gordon and McLeod in 1928 to distinguish Neisseria gonorrhoeae from Staphylococcus spp. and Streptococcus spp.
• It was later modified by Kovacs, who used Kovacs’ oxidase reagent (tetra-methyl-p-phenylenediamine dihydrochloride) for the identification of the cytochrome oxidase enzyme.
• Gaby and Hadley further modified the test, using p-amino dimethylaniline oxalate with α-naphthol as a reagent to detect the cytochrome oxidase enzyme in tube culture.
• Cytochrome c oxidase is a large transmembrane protein acting as the terminal enzyme in the electron transport chain of aerobic bacterial and mitochondrial respiration systems.
• This enzyme catalyzes the final electron transfer from cytochrome c to the oxygen molecule.
• Some bacteria contain cytochrome c oxidase and can transfer a terminal electron to molecular oxygen.
• Other bacteria lack this enzyme and either fail to transfer a terminal electron to molecular oxygen or use a different cytochrome for this function.
• Differentiating bacteria that contain cytochrome c oxidase from those that do not is very important for characterizing and identifying bacteria biochemically.

Objectives

• To assess the presence of the cytochrome c oxidase enzyme within the bacterial electron transport chain system.
• To biochemically characterize bacteria and aid in their identification.

Principle of Oxidase Test

In the presence of molecular oxygen, the cytochrome c oxidase enzyme in bacteria oxidizes phenylenediamine in the colorless reagent to produce a deep purple to blue-colored compound known as indophenol blue. Therefore, if bacteria possess the cytochrome c oxidase enzyme, a deep purple/blue color will develop. If the bacteria lack this enzyme, no color change will occur.

Requirements for Oxidase Test

Culture Media

• When performing the oxidase test using the disc, filter paper, swab, or direct plate method, there is no need for specific culture media. Bacteria grown on any selective medium (or pure colonies from any medium) can be used for the test.
• For the tube method (Gaby and Hadley method), nutrient broth medium (or any standard broth medium with low glucose content) is required. In this guide, nutrient broth will be used.

Composition of Nutrient Broth per 1000 mL

• Peptone: 5.00 grams
• HM Peptone B (Beef Extract): 1.50 grams
• Yeast Extract: 1.50 grams
• Sodium Chloride: 5.00 grams
• Final pH: 7.4 ± 0.2 at 25°C

Preparation of Nutrient Broth

• Measure the appropriate amount of nutrient broth powder (or the media components) and mix in the required volume of water in a conical flask (or glass bottle) according to the manufacturer's instructions.
• Stir well using a magnetic stirrer or manually and heat to boiling if necessary to dissolve all components completely.
• Dispense 5 mL of broth into each test tube and loosely cap (or use a cotton plug to cover the opening).
• Autoclave the tubes with nutrient broth at 121°C and 15 lbs pressure for 15 minutes. Let cool to room temperature before inoculation.

Reagents

Kovacs’ Oxidase Reagent (for disc, filter paper, swab, or direct plate method)

1% N, N, N, N-tetramethyl-p-phenylenediamine dihydrochloride

Preparation of Kovacs’ oxidase reagent:

• Dissolve 1.0 grams of N, N, N, N-tetramethyl-p-phenylenediamine dihydrochloride in 100 mL of sterile distilled water and mix well.

Gordon and McLeod Oxidase Reagent (for disc, filter paper, swab, or direct plate method)

1% dimethyl-p-phenylenediamine dihydrochloride

Preparation of Gordon and McLeod Reagent

• Dissolve 1.0 grams of dimethyl-p-phenylenediamine dihydrochloride in 100 mL of sterile distilled water and mix well. 

Gaby-Hadley Reagents (for tube method)

Reagent A (1% α-naphthol)

• Add 1.0 grams of α-naphthol to 100 mL of 98% ethanol.

Reagent B (1% p-amino dimethylaniline oxalate)

• Add 1.0 grams of p-amino dimethylaniline oxalate to 100 mL of distilled water. 

Impregnated Oxidase Disc/Test Strip 

Equipment

• Petri plates
• Whatman no.1 filter paper (disc or strip)
• Weighing machine
• Autoclave
• Bunsen burner
• Test tubes
• Dropper
• Inoculating loop
• Cotton swab
• PPE
• Other laboratory materials

Test Bacteria (Sample bacteria with well-isolated colonies)

• Positive control: Pseudomonas aeruginosa ATCC 2783
• Negative control: E. coli ATCC 25922

Procedure of Oxidase Test

(Kovacs’ oxidase reagent is preferred for the oxidase test due to its higher sensitivity and faster, clearer results compared to Gordon and McLeod reagent. However, either reagent can be used for the filter paper method, swab method, or direct plate method.)

Filter Paper Method

• Place a strip/disc of Whatman no. 1 filter paper in a sterile petri plate. Soak the filter paper with 1% Kovacs’ oxidase reagent and let it dry.
• Using a sterile inoculating loop, pick a well-isolated colony of test bacteria from a fresh (18 to 24 hours old) culture and smear it on the reagent-soaked filter paper piece.
• Observe for color change and note the time required for color change for up to 60 seconds.
• Alternatively, pick a well-isolated colony of test bacteria from a fresh culture with a sterile inoculating loop and smear it over the Whatman no. 1 filter paper strip/disc. Add 1 to 2 drops of Kovacs’ oxidase reagent over the smear.
• Observe for color change and note the time required for color change for up to 60 seconds.

Swab Method

• Moisten a sterile swab with 1% Kovacs’ oxidase reagent.
• Touch a well-isolated colony from a fresh culture with the swab.
• Observe the development of color on the swab and note the time required for color change for up to 60 seconds.

Direct Plate Method

• Add a few drops of Kovacs’ oxidase reagent over well-isolated (pure culture) colonies of test bacteria from a fresh culture.
• Tilt the plate and shake it gently so that the colonies are exposed to oxygen.
• Observe for the formation of purple (deep blue) color over the reagent-moistened colonies and note the time required for color change for up to 60 seconds.

Impregnated Disc/Strip (Oxidase Disc) Method

• Place the impregnated oxidase disc or strip on a clean petri plate (or glass slide) and moisten it with sterile deionized water. (Some discs may not need to be moistened; check the manufacturer’s instructions.)
• Using a sterile inoculating loop, pick a well-isolated colony of test bacteria from a fresh culture and smear it on the oxidase disc/strip.
• Observe for color change and note the time required for color change for up to 60 seconds.

Tube Method (Gaby-Hadley Oxidase Test)

• Inoculate a nutrient broth medium with sample bacteria and incubate aerobically at 35±2°C for 18 to 24 hours.
• Add 0.2 mL of Gaby-Hadley Reagent A (1% α-naphthol) and 0.3 mL of Gaby-Hadley Reagent B (1% p-amino dimethylaniline oxalate) and mix well by shaking the medium.
• Observe for color change and note the time required for color change for up to 3 minutes.

Result and Interpretation of Oxidase Test

Using Kovacs’ oxidase reagent 

• Positive Test: Development of purple to deep blue color within 10 to 30 seconds indicates a positive oxidase test. If the color develops within 30 to 60 seconds, it indicates a weak oxidase positive reaction or delayed oxidase positive.
• Negative Test: No development of purple to deep blue color within 60 seconds. If the color develops after 60 seconds, it is considered negative.

Using Gaby-Hadley Reagents (For Tube Method)

• Positive Test: Development of purple to deep blue color within 15 to 30 seconds indicates a positive oxidase test. If the color develops within 2 to 3 minutes, it indicates a weak oxidase positive reaction or delayed oxidase positive.
• Negative Test: No development of purple to deep blue color within 3 minutes. If the color develops after 3 minutes, it is considered negative.

Oxidase Positive Bacteria

Neisseria gonorrhoeae, Neisseria spp., Pseudomonas aeruginosa, Aeromonas spp., Vibrio spp., Brucella spp., Moraxella spp., Micrococcus spp., Bordetella pertussis, Campylobacter spp., etc.

Oxidase Negative Bacteria

E. coli and all Enterobacteriaceae except Plesiomonas shigelloides, Staphylococcus spp., Streptococcus spp., Pseudomonas maltophilia, Mycoplasma spp., Bordetella parapertussis, Listeria spp., etc.

Variable Oxidase Result Showing Bacteria

Haemophilus spp., Brucella spp., Pasteurella spp.

Quality Control

• Positive Control: Pseudomonas aeruginosa ATCC 2783 demonstrates rapid development of deep blue or purple color within 10 to 30 seconds.
• Negative Control: E. coli ATCC 25922 does not produce deep blue or purple color within 60 seconds.

Precautions during Oxidase Test

• Ensure the use of fresh bacterial cultures for testing.
• Avoid testing bacteria grown on media containing dyes (e.g., EMB, MAC medium).
• Avoid testing bacteria grown on glucose-rich culture media.
• Do not test strict anaerobes.
• Use freshly prepared oxidase reagent.
• Store the oxidase reagent in a dark place at -20°C.
• Ensure samples are taken from well-isolated colonies. Do not perform the direct plate method on mixed cultures; use cultures from selective media to ensure purity.
• Avoid overflooding the plate with oxidase reagent.
• Record results within 60 seconds when using Kovacs’ reagent and within 3 minutes when using Gaby-Hadley reagents to differentiate between rapid oxidase-positive, delayed oxidase-positive, and oxidase-negative bacteria.
• Use non-nichrome wire loops to prevent false-positive results.

Applications of Oxidase Test

• Assessing the ability of bacteria to synthesize cytochrome c oxidase enzyme.
• Distinguishing Neisseria spp. (oxidase-positive cocci) from Staphylococcus spp. and Streptococcus spp. (oxidase-negative cocci).
• Differentiating Enterobacteriaceae from other Gram-negative bacilli.
• Identifying Pseudomonas aeruginosa from Enterobacteriaceae. 

Limitations of Oxidase Test

• The oxidase test does not provide a definitive identification and typically requires additional biochemical tests for complete bacterial identification.
• Fresh preparation of reagents is crucial as they are prone to auto-oxidation and are sensitive to light; therefore, reagents should be prepared daily.
• Performing the direct plate method can lead to rapid nonviability of reagent-soaked colonies, necessitating immediate subculture.
• Bacteria grown on glucose-rich media may yield false-negative results.
• Older bacterial cultures may also result in false-negative outcomes.
• Nichrome or iron inoculating loops commonly used in laboratories may produce false-positive results, necessitating the use of plastic or platinum loops.
• Testing requires pure cultures or well-isolated colonies for accurate results.
• Accurate recording of the time taken for color change development is essential for interpretation.