The cytochrome are iron containing hemoproteins that act as the last link in the chain of aerobic respiration by transferring electrons to oxygen with the formation of water. Oxidase test is supreme supportive in the screening colonies so-called of being one of the Enterobacteriaceae group (All gram negative bacteria).
Purpose or Uses:
The oxidase test is helpful to contribute in the identification of Neisseria, Vibrio, Pseudomonas, Brucella, and Pasteurella spp, all of which produce the oxidase enzyme.
Principle:
- A small piece of filter paper is soaking with some drops of oxidase solution. On the piece of filter paper a colony of the test micro-organism is smeared. If the test organism is oxidase producer, the phenylene-diamine in the solution will be oxidized and produce a deep purple color.
Requirement:
- Oxidase reagent
- Freshly prepared (Tetramethyl-p-phenylenediamine dihydrochloride).
- Inoculating loop
- Swabs
- collection containers
- Incubators
- Supplemental media
- Whatman (No.1) filter paper or oxidase test strip
- Platinum loop or needle, or wooden applicator stick
Method:
- Take 2 to 3 drops of freshly prepared oxidase reagent and place the filter paper in a clean and dry Petri dish.
- With the help of glass rod remove a colony of the test organism from culture plate and smeared it on the filter paper and wait.
- Observe for the production of a deep purple-blue color within a few seconds.
Results:
- Blue-purple color within 10 seconds ….. Positive oxidase test
- No blue-purple color within 10 seconds ….. Negative oxidase test
Limitations of Oxidase Test:
- Always use freshly prepared oxidase reagent because the reagent has shown auto oxidizing properties.
- It is recommended that oxidase reagents shouldn’t older than 1 week.
- It is important to test colonies grown on culture media without excess sugar because both bacteria and yeast on culture media consisting of high concentrations of glucose which inhibited oxidase activity such as Nutrient agar. Tryptic soy agar is also a tremendous media.
- Bacteria which grow on media holding dyes that can produce unusual results.
- The reagents will kill the organisms, so adding any further reagent in an active culture plate it should be necessary to sub culture the micro-organisms.
- Oxidase test may be used in the possible identification of Neisseria and can also for the differentiation and identification of Gram-negative bacilli. Oxidase positive test micro-organisms must be examined by doing gram stain to see or check their morphology and gram reaction.
- Nichrome made or other iron containing wireloop can produce false positive results. Platinum wireloops are best and recommended.
- Less sensitive strips or reagents can give false negative reaction.
- Oxidase reactions of gram-negative bacilli should be determined on non-selective and non-differential media to ensure valid results.
- Colonies which are taken from culture media consisting of high concentration of glucose might give false negative results.
- It is important to use colonies that are 18 to 24h older because old colonies might give weaker reactions.
- After 20 seconds any color changes production must be discarded.
