It
is a biochemical reaction that assays for the presence of cytochrome oxidase enzyme.
The cytochromes are iron containing hemoproteins that act as the last link in
the chain of aerobic respiration by
transferring electrons (hydrogen) to oxygen, with formation of water. This cytochrome
system is found in aerobic, microaerophilic and facultatively anaerobic
organism.
Oxidase
reagent:
TMPD
(Tetramethyl-p-phenylenediamine dihydrochloride) is a electron rich colorless dye
Theory
In
bacteria, that contains enzyme cytochrome oxidase, an electron from each
cytochrome C is temporarily transferred to the enzymes resulting in four
electron poor cytochrome C and electron rich cytochrome oxidase. Instead of acquiring
an electron from another component of electron transport chain, an electron
rich TMPD molecule passes an electron to electron poor cytochrome C. Now, the
electron poor TMPD radical has a dark blue colour.
The
organism can be oxidase positive, negative, variable or delayed positive.
Recipes:
- Kovac’s reagent: 1%TMPD (Tetramethyl-p-phenylenediamine dihydrochloride)
- Gorden and Mcleod’s reagent : 1%DMPD (Dimethyl-p-phenylenediamine dihydrochloride)
- Gaby and Hadley’s oxidase test: 1% alpha napthol in 95% ethanol + 1% p-aminodimethylaniline oxalate
- Rapid, modified oxidase test for oxidase variable organism: 1%TMPD in dimethyl sulfoxide (DMSO) or 6% TMPD in DMSO (differentiate Stah and micrococci). DMSO help penetrate gram positive cell wall and increase from 1% to 6% gives more exposure)
Protocols
for oxidase testing
- Filter
paper test method:
Few
drops of reagent is added in the filter paper strip. With the help of glass rod
the colony to be tested is rubbed over the filter paper containing the reagent.
- Filter
paper spot method/ Disk method
Bacterial
colonies are streaked on the commercial oxidase test disc or dried out filter
paper strip with the reagnt
- Direct
plate method
Two
or three drops of reagent is added directly in isolated bacterial colonies
growing on plate medium
- Test
tube method
Colonies
are picked with cotton swab and dropped in test tube containing the reagent.
Quality control
Positive
control: Pseudomonas aeruginosa ATCC 27853
Negative
control: Escherichia coli ATCC 25923
Interpret
the result within 10 seconds.
Comments and Tips:
- Reagents autooxidise, so use fresh reagent. Do not use beyond 1 week
- Auto-oxidation can be slowed by adding 1% ascorbic acid.
- Using nickel, steel or other wires may result false positive reaction
- Bacteria grown on sugar rich media shows inhibited oxidase activity, so use colony from nutrient ager for this test.
- When testing colonies from MacConkey medium, a pink violet color is due to carry over from the medium and is not a true oxidase reaction.
Oxidase positive organism
Neisseria, Areomonas, Vibrio, Campylobacter, Pseudomonas, Alcaligens
Oxidase negative organism
Enterobctericecae family.
.
