The document describes the oxidase test, which is used to identify bacteria. It works by detecting cytochrome c, an iron-containing protein in the respiratory chain, which causes an oxidase reagent to turn purple in oxidase-positive bacteria within 5-10 seconds. The document outlines several methods for performing the test, such as using filter paper or commercial strips soaked in reagents like tetramethyl-p-phenylenediamine. It lists both oxidase-positive and -negative bacteria and notes the test is useful for bacterial classification. Modifications to the standard method are presented to more rapidly and accurately detect weakly oxidase-positive organisms.

1. OXIDASE TEST
BY,
Dr.M.Malathi

2. INTRODUCTION;
 It is a biochemical test
 Used for the identification of bacteria

3. 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.

4. REAGENTS:
Kovac`s reagent – 1% tetramethyl p
phenylene diamine dihydrochloride
Gordon and Mcleod`s reagent – 1%
dimethyl-p-phenylene diamine
dihydrochloride

6. IN REDUCED STATE – DYE – COLOURLESS
IN OXIDISED STATE – DYE – PURPLE BLUE

7. PROCEDURE:
Plate method
Dry filter paper method
Wet filter paper method

8. 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.

9. 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.

10. 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 .

13. 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.

14. 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

16. 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.

18. COMMERCIAL STRIPS
 Stable oxidase reagent strips are available
commercially.
 50 strips / pack
 Shelf life = 5 years
 Storage 2 to 8 deg C

20. INTERPRETATION:
 Oxidase postive: purple blue colour
Oxidase positive 5 to 10 sec
Delayed positive 10 to 60 sec
negative > 60 sec

21. QUALITY CONTROL:
 Positive control : Pseudomonas
 Negative control : E.coli

22. 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.

23. OXIDASE POSITIVE ORGANISMS:
 Pseudomonas
 Neisseria
 Vibrio
 Campylobacter
 Aeromonas
 Alcaligenes
 Brucella
 Pasturella
 Eikenella
 Kingella
 Moraxella
 Legionella
 Helicobacter
 Chromobacter
(oxidase variable)

24. OXIDASE NEGATIVE
 All enterobacteriaceae are OXIDASE
NEGATIVE
 Acinetobacter
 Staphylococci
 streptococci

25. APPLIED AND ENVIRONMENTAL
MICROBIOLOGY:
USE OF A QUANTITATIVE OXIDASE TEST
FOR CHARACTERIZING OXIDATIVE
METABOLISM IN BACTERIA.
P Jurtshuk, Jr and D N McQuitty

26.  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.

27.  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.

28.  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.

29. 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.

30. 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.

31.  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.