This is the preferred reagent. Use freshly prepared. However, reagent can be used up to 7 days if stored in a dark glass bottle under refrigeration.

Apply freshly prepared solution directly to young culture (24 h) on either agar plate or slant. Oxidase-positive colonies develop a pink color and progressively turn dark purple. If cultures are to be preserved, complete the transfer from plates to which reagent has been added within 3 min, since reagent is toxic to organisms.

Optional method: Transfer small amount of culture to reagent-impregnated filter paper. Dark purple color within 10 s indicates positive test. Use platinum wire or sterile wooden stick (toothpicks or applicator sticks), since wire containing iron (e.g., nichrome wire in ordinary inoculating needles and loops) gives false-positive reactions.

Alternatively, the test may be performed with a 1% solution of N,N-dimethyl-p-phenylenediamine hydrochloride. Apply solution directly to culture plate or slant.

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