The oxidase test is performed to identify the bacteria if it produces cytochrome c oxidase and to detect the presence of an enzyme called cytochrome c oxidase that catalyses the transport of electrons between electron donors that are present in the bacteria and a redox dye- tetramethyl-p-phenylene-diamine. In case of the positive result the dye is reduced to purple colour. This test is used in identification of all the bacterial species that produce the enzyme cytochrome oxidase, for example, Pseudomonas, Neisseria, Alcaligens, Aeromonas, Campylobacter, Vibrio, Brucella and Pasteurella. It is also used to differentiate pseudomonads from related species.
All bacteria that give positive oxidase result are aerobic and are capable of using oxygen as a terminal electron acceptor during respiration, but this does not imply that the bacteria is a strict aerobe. Whereas, those bacteria that show a negative result in an oxidative test can be categorized as anaerobic, aerobic or facultative, this shows that the negative oxidase result is the indication of the organism not producing or having the cytochrome c oxidase enzyme that gives the positive result by oxidizing the reagent. These bacteria respire using other oxidases enzymes that are present in their electron transport systems.
One of the following reagents is used to perform the oxidase test:
- Kovacs (Oxidase) Reagent: 1% tetra-methyl-p-phenylenediamine dihydrochloride, (composition is made in distilled water).
- Gaby and Hadley (indophenol oxidase) Reagent: 1% α-naphthol in 95% ethanol.
- Gordon and McLeod’s Reagent: 1% dimethyl-p-phenylenediamine dihydrochloride, (composition is made in distilled water).
A filter paper that is dipped in the substrate i.e. 1% tetramethyl-p-phenylenediamine dihydrochloride or we can also get one commercially prepared paper disk can be used for this test. After this the colony or the microbe that is to be tested is put on the substrate soaked filter paper and the color change is observed within 10 to 30 seconds.
The bacteria that contain cytochrome produces an enzyme called oxidase that catalyze the oxidation of cytochrome c. the organism are said to be oxidase positive when they contain cytochrome c as a part of their respiratory chain and hence they turn the reagent used while performing oxidase test to blue/purple. Whereas, the organisms that lack the cytochrome c are unable to oxidize the reagent and hence they leave it colourless and are called as oxidase negative.
Usually aerobic organisms that are capable of utilizing oxygen as a final hydrogen receptor have this cytochrome system, which give rise to end products like water or hydrogen peroxide which is then broken down by catalase.
The procedures of oxidase test includes filter paper test, in which the filter paper is soaked in the reagent and then a stripe of bacterial culture broth is put on that filter paper and a color change is observed within 15 to 20 seconds. Other methods include swab method, impregnated oxidase test strip method and test tube method.
Bacterial genera characterized as oxidase positive include Neisseria and Pseudomonas. Genera of the Enterobacteriaceae family are characterized as oxidase negative.
Indole test lets us determine if an organism has the ability to split amino acid tryptophan to form indole. This tryptophan hydrolysis is catalyzed by tryptophanase to produce indole as an end product. The indole production is detected by the appearance of red colour that is a resultant of the reaction of the reagent used and indole. Indole test is used to differentiate Enterobacteriaceae from other genera.
There are two methods to perform Indole test
- Tube method which requires an overnight incubation. This test is ideal for the identification of weak indole producing organisms
- Spot indole test. This test detects rapid indole producing organisms.
Tryptophanase breaks down tryptophan to release indole, which when reacts with cinnamaldehyde produces a blue-green compound. The absence of enzyme results in no color production.
Procedure of Spot Indole Test
- Soak a piece of filter paper in the 1% paradimethylaminocinnamaldehyde reagent.
- Use a sterilized wooden stick or inoculating loop to remove a small portion of a bacterial colony from the agar surface and rub the sample on the filter paper.
If a blue colour is developed within 30 seconds of inoculation of the colony on the soaked filter paper the organism is considered to be indole positive and if there is no change in colour the organism is considered as indole negative.
Procedure of Tube method for Indole Test
The tryptophan broth is inoculated with broth culture or we can emulsify isolated colony of the test organism in tryptophan broth. Now this is incubated at 37°C for 24-28 hours in ambient air. After incubation period 0.5 ml of Kovac’s reagent is added to the broth culture.
If the pink colored ring is appeared after the addition of the reagent the microbe is considered to be indole positive otherwise negative.