Introduction
Oxidase test is most helpful in screening colonies suspected of being one of the Enterobacteriaceae (all negative) and in identifying colonies suspected of belonging to other genera such as Aeromonas, Pseudomonas, Neisseria, Campylobacter, and Pasteurella (positive). Oxidase test uses disks impregnated with a reagent such as N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) or N,N-dimethyl-p-phenylenediamine (DMPD), which is also a redox indicator. The reagent is a dark-blue to maroon color when oxidized, and colorless when reduced.
Oxidase-positive bacteria possess cytochrome oxidase or indophenol oxidase (an iron-containing hemoprotein).These both catalyze the transport of electrons from donor compounds (NADH) to electron acceptors (usually oxygen). The test reagent, TMPD dihydrochloride acts as an artificial electron donor for the enzyme oxidase. The oxidized reagent forms the colored compound indophenol blue. The cytochrome system is usually only present in aerobic organisms that are capable of using oxygen as the terminal electron acceptor. The end-product of this metabolism is either water or hydrogen peroxide (broken down by catalase).
Objective
- To check for presence of terminal enzyme Cytochrome C oxidase or Cytochrome a3.
Principle
Cytochrome containing organisms produce an intracellular oxidase enzyme. This oxidase enzyme catalyzes the oxidation of cytochrome c. Cytochrome C oxidase is the terminal or last H2 electron acceptor in aerobic respiratory mechanism which is composed of a number of enzymes which alternatively oxidize and reduce each other by donating or accepting electrons derived from H2. Organisms which contain cytochrome c as part of their respiratory chain are oxidase-positive and turn the reagent blue/purple. Organisms lacking cytochrome c as part of their respiratory chain do not oxidize the reagent, leaving it colorless within the limits of the test, and are oxidase-negative.
The capability of a bacteria to produce the cytochrome C oxidase can be discovered by using the reagent tetramethyl-p-phenylenediamine dihydrochloride impregnated in filter disk. The reagent serves as an artificial substrate donating electrons and thereby becoming oxidized to a deep purple compound in the presence of the enzyme oxidase and free oxygen. The appearance of pink, then maroon and finally dark purple color after rubbing the organism in the oxidase disc containing the reagent indicates positive test result. The positive reaction involve the conversion of colorless, reduced tetramethyl-p-phenylenediamine to oxidized form into deep purple color in presence of Cytochrome C oxidase. If there is No color change then the inference is negative test result.
Experiment
Requirements
- 24 hours Culture of E.coli and Pseudomonas spp
- Filter paper soaked with 1% tetra methyl-p-Phenylenediamine dihydrochloride
- Glass rod
Procedure
- Place a piece of filter paper in a clean petridish and add 2-3 drops of freshly prepared oxidase reagent (1% tetra methyl-p-Phenylenediamine dihydrochloride)
- Place a small portion of culture on the filter paper with the help of a sterile glass rod and make a smear on it.
- Examine for immediate color change to blue purple within 10 seconds.
NOTE: Oxidase test can also be performed by flooding the culture plate with oxidase reagent but it is not recommended because the reagent rapidly kills the bacteria.
Observation And Result Interpretation of Oxidase Test
Positive Result
Development of a deep purple-blue/blue colour indicates oxidase production within 5-10 seconds.
Negative Result
No purple-blue colour/No colour change.
Examples Of Oxidase Positive Organisms
Pseudomonas, Neisseria, Alcaligens, Aeromonas, Campylobacter, Vibrio, Brucella, Pasteurella, Moraxella, Helicobacter pylori, Legionella pneumophila, etc.
Examples Of Oxidase Negative Organisms
Enterobacteriaceae (e.g. E. coli).
Precautions Of Oxidase Test
- The reagents used in the oxidase test have been shown to autooxidize, and hence false positive result may be obtained.
- Nickel, steel, and other wire loops may give false-positive results, so it is important to use only platinum or inert transfer loops, such as sterile wood sticks commonly used in teaching laboratories.
- Observe the result within 10 seconds as Atmospheric Oxygen may react with reagent to give false positive result.
- Bacteria grown on media containing high concentrations of glucose show inhibited oxidase activity, so it is recommended to test colonies grown on media without excess sugar, such as nutrient agar.
- Culture should not be more than 24 hours old.Older colonies will produce weaker reactions.
- Any color changes appearing after 20 seconds should be disregarded.
- Oxidase reactions of gram-negative bacilli should be determined on non-selective and non-differential media to ensure valid results. Also, colonies taken from media containing high levels of glucose may give false-negative reactions.
- Bacteria grown on media containing dyes may give aberrant results.