Microbiological and biochemical method for identification
Theoxidase test is used to determine whether an organism possesses thecytochrome c oxidase enzyme. The test is used as an aid for the differentiation ofNeisseria,Moraxella,Campylobacter andPasteurella species (oxidase positive). It is also used to differentiate pseudomonads from related species.[1]
OX+ normally means the bacterium contains cytochrome c oxidase (also known as Complex IV) and can therefore useoxygen for energy production by converting O2 to H2O2 or H2O with anelectron transfer chain.
OX− normally means the bacterium does not contain cytochrome c oxidase and, therefore, either cannot useoxygen for energy production with anelectron transfer chain or employs a different cytochrome for transferring electrons to oxygen.
The test uses disks impregnated withreagents such asN,N,N′,N′-tetramethyl-p-phenylenediamine, TMPD (orN,N-dimethyl-p-phenylenediamine, DMPD, which is anotherredox indicator). The reagent is a dark-blue tomaroon color when oxidized, and colorless when reduced. Oxidase-positive bacteria possess cytochrome oxidase orindophenol oxidase (an iron-containing hemoprotein).[5] These both catalyze the transport of electrons from donor compounds (NADH) to electron acceptors (usually oxygen). The test reagent TMPD acts as an artificial electron donor for the enzyme oxidase. The oxidized reagent forms the colored compoundindophenol 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 bycatalase).[1]
Wet each disk with about four inoculating loops of deionized water.
Use a loop to aseptically transfer a large mass of pure bacteria to the disk.
Observe the disk for up to three minutes. If the area of inoculation turns dark-blue to maroon to almost black, then the result is positive. If a color change does not occur within three minutes, the result is negative.
In alternative manner, live bacteria cultivated ontrypticase soy agar plates may be prepared usingsterile technique with a single-line streak inoculation. The inoculated plates are incubated at 37 °C for 24–48 hours to establish colonies. Fresh bacterial preparations should be used. After colonies have grown on the medium, 2-3 drops of the reagent DMPD are added to the surface of each organism to be tested.
A positive test (OX+) will result in a color change violet to purple, within 10–30 seconds.
A negative test (OX-) will result in a light-pink or absence of coloration.
^abcMacFaddin JF, editor. Biochemical Tests for Identification of Medical Bacteria. 3rd ed. Philadelphia:Lippincott Williams and Wilkins; 2000. p. 363-7
^S. T., Cowan; Steel, K.J. (1993).Cowan and Steel's Manual for the Identification of Medical Bacteria (3rd ed.). Cambridge: Cambridge University Press.ISBN9780511527104.
^Isenberg HD, editor. Clinical Microbiology Procedures Handbook. American Society for Microbiology; 2004. p. 3.3.2-3.3.2.13.
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