Oxidase test

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Pre-prepared reagent
Positive result

Contents

Introduction

The Oxidase test (also known as the Cytochrome Oxidase test) is used to look for oxidase enzymes produced by certain bacteria. Oxidases catalyse electron transport between substrates acting as electron donors in the bacterium and tetramethyl-p-phenylenediamine OR dimethyl-p-phenylenediamine - a redox dye present as the hydrochloride or oxalate salt (the latter has a longer shelf-life). The dye is reduced to a deep violet-blue colour in the presence of oxidase enzymes.

Methods

Solutions of the dye are unstable and will spontaneously change from its colourless state to its purple state in a matter of hours. Thus solutions must be freshly prepared using the dried dye and distilled water. All the methods below use a 1% solution. There are stabilised pre-prepared reagents available which tend to have a much longer shelf-life.

Dry filter paper method

Soak strips of filter paper in a fresh dye solution, drain and freeze dry. Strips should be stored in an air-tight bottle and kept in a cool dark environment. Strips prepared in this manner will keep for several months, and have a faint pastel-violet colour. To use, take a strip and soak in distilled water. Pick the colony to be tested with a loop and rub onto moistened strip. A colour change within 10 seconds indicates a positive reaction.

Wet filter paper method

Wet a strip of filter paper with a fresh dye solution, and use as for the dry filter paper method. Strips prepared in this way must be used immediately.

Cotton-tipped stick method

Dip a sterile cotton-tipped wooden stick into a fresh dye solution. Remove excess liquid from the tip by pressing against the side of the dye container. Press the cotton tip against the colony to be tested. A colour change within 10 seconds indicated a positive result.

Plate method 1

Culture the organism on suitable solid agar medium overnight. Pour a freshly prepared dye solution over the plate, covering the surface, and then pour off. Colonies which rapidly change in appearance to a deep violet colour may be regarded as indicating a positive result.

Plate method 2

Culture the organism for 24-48 hours on a nutrient agar plate. Pour a few drops of a freshly prepared solution of 1% aqueous p-aminodimethylaniline oxalate and 1% α-napthol in ethanol over the colony to be tested. Colonies which change to a deep blue colour indicate a positive reaction.

Pitfalls

  • When transferring colonies to filter paper, use either a glass rod, a clean platinum loop or a single-use plastic loop. Dirty or nichrome loops may give false positive results due to the presence of iron.
  • Colonies picked from MacConkey agar may exhibit a violet-pink colour on testing. This is due to the ingredients of the agar and do not represent a positive reaction - only a deep violet colour is sufficient to indicate a positive result.

Control organisms

Typical results

A summary of typical results seen with some commonly encountered organisms.

Oxidase positive Oxidase negative
Alcaligenes spp. Acinetobacter spp.
Bordatella pertussis Bordatella parapertussis
Campylobacter spp. Francisella tularensis
Brucella spp. Brucella canis
Kingella kingae Flavimonas oryzihabitans
Moraxella spp. Stenotrophomonas maltophilia
Neisseria spp. Enterobacteriaceae
Pseudomonas spp.  
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