Oxidase test negative and positive as shown above picture. Gordon and McLeod introduced the use of a dimethyl-phenylenediamine dihydrochloride solution to test for the presence of oxidase systems in 1928. In particular, they used the test to distinguish N. gonorrhoeae (oxidase test positive) from Staphylococcus species and Streptococcus species (oxidase negative). The sensitivity of the oxidase test was increased when Kovács 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 an organism that contains the cytochrome
oxidase enzyme, the reduced colorless reagent becomes an oxidized
colored product as shown above image.
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.
Oxidase test is most helpful 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).
Positive: Development of dark purple color (indophenols) within 10 seconds
Negative: Absence of color
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
Positive test: Development of a dark purple color within 10 seconds.
Negative: Absence of color.
Positive Control: Development of a dark purple color within 10 seconds.
Negative Control: Absence of color
Test organism: Positive
Note- as shown above figure.
Gram-negative bacilli/rods-
Gram-negative cocci-
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 method
Indophenol oxidase reagent uses that contains- 1% α naphthol in 95 % ethanol and 1% p-aminodimethylaniline HCl.