OXIDASE TEST

BY,Dr.M.Malathi

INTRODUCTION;

It is a biochemical test

Used for the identification of bacteria

PRINCIPLE: Cytochromes are the iron containing

hemoproteins .

These act as last link in the chain of aerobic respiration transferring electrons (H+) to oxygen in the formation of water

Cytochrome system is seen in aerobic, microaerophilic and facultive anaerobic organisms.

REAGENTS:

Kovac`s reagent – 1% tetramethyl p phenylene diamine dihydrochloride

Gordon and Mcleod`s reagent – 1% dimethyl-p-phenylene diamine dihydrochloride

IN REDUCED STATE – DYE – COLOURLESSIN OXIDISED STATE – DYE – PURPLE BLUE

PROCEDURE:Plate method

Dry filter paper method

Wet filter paper method

PLATE METHOD Cultures are made on a suitable solid growth

medium A freshly prepared 1% solution of

tetramethyl-p-phenylene-diamine dihydrochloride (TMPDD) is poured on to the plate so as to cover the surface, and is then decanted.

The colonies of oxidase positive organisms rapidly develop purple colour.

If subcultures are required from the colonies, they should be made immediately.

CONTINUED…….. Technique of flooding the culture with

oxidase reagent – rapidly kills the bacteria.

Hence if oxidase reagent is used for isolating N.gonorrhoea colonies from mixed cultures in the absence of selective medium, the oxidase positive colonies must be removed and subcultured within 30 seconds of flooding the plate.

CONTINUED…….. Acidity inhibits the oxidase enzyme activity.

Therfore it must not be performed on colonies that produce fermentation of carbohydrates.

Eg. TCBS, Macconkey agar

Colonies tested from a medium that contains nitrate may give unreliable oxidase test.

Hence the best plate is nutrient agar .

DRY FILTER PAPER METHOD:

Since the oxidase reagent is unstable and has to be freshly prepared for use, this method is used.

Strips of Whatman`s no.1 filter paper are soaked in a freshly prepared 1% TMPDD. After draining for about 30 seconds, the strips are freeze dried and stored in a dark bottle tightly sealed with a screw cap.

CONTINUED.. For use, a strip is removed and kept in petri

dish, moistened with distilled water.

The colony to be tested should be picked up with a platinum loop or glass rod and smeared over the moist area.

a positive reaction is indicated by intense deep purple hue appearing within 5 to 10 seconds.

Rusted iron loop should not be used as it interferes with reaction

WET FILTER PAPER METHOD:

A Strip of filter paper is soaked with a little freshly made 1% reagent and then a speck of culture with the help of platinum loop is rubbed on it.

COMMERCIAL STRIPS Stable oxidase reagent strips are

available commercially.

50 strips / pack

Shelf life = 5 years

Storage 2 to 8 deg C

INTERPRETATION: Oxidase postive: purple blue colour

Oxidase positive 5 to 10 sec

Delayed positive 10 to 60 sec

negative > 60 sec

QUALITY CONTROL:

Positive control : Pseudomonas

Negative control : E.coli

FALSE POSITIVE REACTIONS: Mac conkey agar – a pink violet colour

is due to carry over from the medium.

Iron loop, Nichrome loop, Stainless steel – surface oxidation products formed while doing flame sterilising gives false positive reactions.

OXIDASE POSITIVE ORGANISMS: Pseudomonas Neisseria Vibrio Campylobacter Aeromonas Alcaligenes Brucella Pasturella

Eikenella Kingella Moraxella Legionella Helicobacter Chromobacter

(oxidase variable)

OXIDASE NEGATIVE All enterobacteriaceae are OXIDASE

NEGATIVE

Acinetobacter

Staphylococci

streptococci

APPLIED AND ENVIRONMENTAL MICROBIOLOGY:

USE OF A QUANTITATIVE OXIDASE TEST FOR CHARACTERIZING OXIDATIVE METABOLISM IN BACTERIA.

P Jurtshuk, Jr and D N McQuitty

It was possible to quantitate the terminal oxidase(s) reaction using bacterial resting-cell suspensions and demonstrate the usefulness of this reaction for taxonomic purposes. Resting-cell suspensions of physiologically diverse bacteria were examined for their capabilities of oxidizing N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) using a manometric assay. For organisms having this capability, it was possible to calculate the conventional TMPD oxidase Q(O2) value (microliters of O2 consumed per hour per milligram [dry weight]). All cultures were grown heterotrophically at 30 C, under identical nutritional conditions, and were harvested at the late-logarithmic growth phase.

The TMPD oxidase Q(O2) values showed perfect correlation with the Kovacs oxidase test and, in addition, it was possible to define quantitatively that point which separated oxidase-positive from oxidase-negative bacteria. Oxidase-negative bacteria exhibited a TMPD oxidase Q(O2) value (after correcting for the endogenous by substraction) of less than or equal 33 and had an uncorrected TMPD/endogenous ratio of less than or equal 5.

The TMPD oxidase Q(O2) values were also correlated with the data obtained for the Hugh-Leifson Oxferm test. In general, bacteria that exhibited a respiratory mechanism had high TMPD oxidase values, whereas fermentative organsims had low TMPD oxidase activity. All exceptions to this are noted. This quantitative study also demonstrated that organisms that (i) lack a type c cytochrome, or (ii) lack a cytochrome-containing electron transport system, like the lactic acid bacteria, exhibited low or negligible TMPD oxidase Q(O2) values. From the 79 bacterial species (36 genera) examined, it appears that this quantitative oxidase test has taxonomic value that can differentiate the oxidative relationships between bacteria at the subspecies, species, and genera levels.

JOURNAL OF CLINICAL MICROBIOLOGY:

Rapid, modified oxidase test for oxidase-variable bacterial isolates.J J Tarrand and D H Gröschel

ABSTRACT

A modified oxidase reagent, 1% tetramethyl-p-phenylenediamine in dimethyl sulfoxide, proved superior to the routinely used 1% aqueous tetramethyl-p-phenylenediamine dihydrochloride in detecting weakly oxidase-positive gram-negative bacteria after 24 h of growth on agar media (40 of 40 positive versus 22 of 40 positive). The bacterial inoculum was obtained with a cotton-tipped swab instead of a loop or wooden applicator, and the reaction required less than 15 s.

JOURNAL OF APPLIED MICROBIOLOGY:

A ONE-MINUTE OXIDASE TEST TO DETECT VIBRIO STRAINS ISOLATED FROM CULTURES ON THIOSULPHATE-CITRATE-BILE SALTS-SUCROSE (TCBS) MEDIUM

J. VILA, S. ABDALLA, J. GONZALEZ, C. GARCIA, J. A. BOMBI AND M.T. JIMENEZ. 1992. 

Vibrio cholerae is oxidase positive, a primary characteristic used to differentiate it from Enterobacteriaceae. But false negative oxidase test results have been obtained with colonies from thiosulphate-citrate-bile salts-sucrose (TCBS) agar medium. A rapid oxidase test procedure is described here. This takes 1 min, avoids false negative results and the necessity to grow the bacteria in a general-purpose medium. The bacteria may be recovered after the test and used for further investigations.