Oxidase Test for Bacteria: Introduction, Principle, Procedure, Result Interpretation, and Keynotes

Introduction of Oxidase Test (Kovac’s Method)

Gordon and McLeod introduced the application of a dimethyl-phenylenediamine dihydrochloride solution to test for the presence of oxidase systems in 1928. In particular, they used the test to differentiate N. gonorrhoeae (oxidase test positive) from Staphylococcus species and Streptococcus species (oxidase negative). The sensitivity of the oxidase test was increased when Kovác’s found that a tetramethyl-p-phenylenediamine dihydrochloride solution gave a quicker reaction. Gaby and Hadley developed a modified oxidase test using p-aminodimethylaniline oxalate with α-naphthol to detect oxidase in test tube cultures. 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 a bacterium that contains the cytochrome oxidase enzyme, the reduced colorless reagent becomes an oxidized colored product as shown below image.

Principle

In the presence of atmospheric oxygen, a bacterium’s intra-cellular cytochrome oxidase enzymes oxidize the N, N, N, N-tetramethyl-p phenylenediamine dihydrochloride to indophenol, a dark purple-colored end product. This oxidase test is useful in the initial characterization of gram-negative bacteria.

                                                    

Reaction in brief-

Environmental Oxygen

N, N, N, N-tetramethyl-p phenylenediamine 

dihydrochloride (Kovac’s reagent)     → Indol phenol (Deep blue to purple color)

    Bacterial intracellular cytochrome oxidase

Purpose of the Test

The test is most useful in screening colonies suspected of being one of the family Enterobacteriaceae (all negative except Plesiomonas shigelloides which is positive) and in identifying colonies suspected of belonging to other genera such as Pseudomonas, Aeromonas, Vibrio, Brucella, Haemophilus, Alcaligenes, Neisseria, Campylobacter, and Pasteurella (Positive).

Requirements of the Test

• Moist filter paper with the substrate (1% N, N, N, N-tetramethyl-p-phenylenediamine dihydrochloride), or commercially prepared paper disk,
• Coverslip or glass slide or wooden sticks or platinum wire
• Test Organisms
• Control strains for validation of the test (Positive control-Pseudomonas aeruginosa ATCC 2785 while negative control-Escherichia coli ATCC 25922)

Expected results of the Test

Positive: Development of dark purple color (indophenols) within 10 seconds

Negative: Absence of color

Quality Control

Bacterial species showing positive and negative reactions should be run as controls at frequent intervals. The following are suggested:
A. Positive control: Pseudomonas aeruginosa ATCC 27853
B. Negative control:  Escherichia coli ATCC 25922

Procedure

1. Moisten filter paper with the substrate (1% N, N, N, N-tetramethyl-p-phenylenediamine dihydrochloride) or select a commercially available paper disk that has been impregnated with the substrate.
2. Use a platinum wire or wooden stick to remove a small portion of a bacterial colony (preferably not more than 24 hours old) from the agar surface and rub the sample on the filter paper or commercial disk.
3. Observe inoculated area of paper or disk for a color change to deep blue or purple within 10 seconds.

Observation

Positive test: Development of a dark purple color within 10 seconds.

Negative: Absence of color.

Result Interpretation of Test

Positive Control: Development of a dark purple color within 10 seconds.

Negative Control: Absence of color

Test organism: Positive

List of oxidase-positive bacteria

Gram-negative bacilli/rods-

1. Pseudomonas spp. (except Ps. cepacia)
2. Aeromonas spp.
3. Vibrio spp.
4. Brucella spp. (except B. canis)
5. Haemophilus spp.
6. Alcaligenes spp.
7. Campylobacter spp.
8. Plesiomonas spp.
9. Chromobacterium spp.
10. Bordetella spp. (Except-B. parapertusis)
11. Flavobacterium spp.
12. Achromobacter spp.
13. Pasteurella multicida
14. Eikinella spp.
15. Cardiobacterium spp.
16. Eikinella spp.

Gram-negative cocci-

• Neisseria spp.
• Moraxella spp.

Keynotes

1. Do not use Nickel-base alloy wires containing chromium and iron (nichrome) to pick the colony and make a smear as this may give false-positive results
2. Interpret the results within 10 seconds, timing is critical.
3. The oxidase test must be tested from 5% sheep blood agar (BAP) or another medium without a fermentable sugar i.e. nutrient agar(NA). Fermentation of carbohydrates results in acidification of the medium for example lactose in MacConkey agar (MAC) or Sucrose in Thiosulfate citrate bile salt sucrose (TCBS) agar and a false negative oxidase test may result if the surrounding pH is below 5.1. Therefore Sub inoculation on  Nutrient agar is required before the oxidase test can be performed.
4. All oxidase-positive bacteria are catalase test positive except-Eikenella, Kingella, and Cardiobacterium.
5. A modified oxidase test is applied for Micrococcus species.

Other Methods of Oxidase Test

According to the application of reagents using-

As you know in Kovac’s method- 1% N, N, N, N-tetramethyl-p-phenylenediamine dihydrochloride reagent uses as a substrate, similarly, in Gardon and Mcleod’s method, 1% N, N,-dimethyl p phenylenediamine dihydrochloride reagent is used as a substrate.

In Gaby and Hadley’s method

Indophenol oxidase reagent uses that contains- 1% α naphthol in 95 % ethanol and 1% p-aminodimethylaniline hydrochloric acid.

Bibliography

1. Jean F. Mac Fadden Biochemical tests for Identification of Medical Bacteria
2. Mackie and McCartney Practical Medical Microbiology-14th Edition
3. Bailey’s and Scott’s Diagnostic Microbiology-13th Edition
4. ASM Clinical Microbiology Procedures Handbook-2^nd Edition
5. Monica Cheesbrough Distinct Laboratory Practice in Tropical Countries-2^nd Edition
6. https://asm.org/getattachment/00ce8639-8e76-4acb-8591 0f7b22a347c6/oxidase-test-protocol-3229.pdf