biochemical reactions - microbe identification
TRANSCRIPT
OXIDASE TEST
BY
DrMMalathi
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 ndash 1 tetramethyl p
phenylene diamine dihydrochloride
Gordon and Mcleod`s reagent ndash 1
dimethyl-p-phenylene diamine
dihydrochloride
IN REDUCED STATE ndash DYE ndash COLOURLESS
IN OXIDISED STATE ndash DYE ndash 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
CONTINUEDhelliphellip
Technique of flooding the culture with oxidase
reagent ndash rapidly kills the bacteria
Hence if oxidase reagent is used for isolating
Ngonorrhoea 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
CONTINUEDhelliphellip
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 no1 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 gt 60 sec
QUALITY CONTROL
Positive control Pseudomonas
Negative control Ecoli
FALSE POSITIVE REACTIONS
Mac conkey agar ndash a pink violet colour is
due to carry over from the medium
Iron loop Nichrome loop Stainless steel ndash
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 NNNN-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 TMPDendogenous
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 organsimshad 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 oxidaseQ(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 Groumlschel
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 MT 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
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 ndash 1 tetramethyl p
phenylene diamine dihydrochloride
Gordon and Mcleod`s reagent ndash 1
dimethyl-p-phenylene diamine
dihydrochloride
IN REDUCED STATE ndash DYE ndash COLOURLESS
IN OXIDISED STATE ndash DYE ndash 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
CONTINUEDhelliphellip
Technique of flooding the culture with oxidase
reagent ndash rapidly kills the bacteria
Hence if oxidase reagent is used for isolating
Ngonorrhoea 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
CONTINUEDhelliphellip
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 no1 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 gt 60 sec
QUALITY CONTROL
Positive control Pseudomonas
Negative control Ecoli
FALSE POSITIVE REACTIONS
Mac conkey agar ndash a pink violet colour is
due to carry over from the medium
Iron loop Nichrome loop Stainless steel ndash
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 NNNN-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 TMPDendogenous
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 organsimshad 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 oxidaseQ(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 Groumlschel
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 MT 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
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 ndash 1 tetramethyl p
phenylene diamine dihydrochloride
Gordon and Mcleod`s reagent ndash 1
dimethyl-p-phenylene diamine
dihydrochloride
IN REDUCED STATE ndash DYE ndash COLOURLESS
IN OXIDISED STATE ndash DYE ndash 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
CONTINUEDhelliphellip
Technique of flooding the culture with oxidase
reagent ndash rapidly kills the bacteria
Hence if oxidase reagent is used for isolating
Ngonorrhoea 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
CONTINUEDhelliphellip
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 no1 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 gt 60 sec
QUALITY CONTROL
Positive control Pseudomonas
Negative control Ecoli
FALSE POSITIVE REACTIONS
Mac conkey agar ndash a pink violet colour is
due to carry over from the medium
Iron loop Nichrome loop Stainless steel ndash
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 NNNN-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 TMPDendogenous
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 organsimshad 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 oxidaseQ(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 Groumlschel
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 MT 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
REAGENTS
Kovac`s reagent ndash 1 tetramethyl p
phenylene diamine dihydrochloride
Gordon and Mcleod`s reagent ndash 1
dimethyl-p-phenylene diamine
dihydrochloride
IN REDUCED STATE ndash DYE ndash COLOURLESS
IN OXIDISED STATE ndash DYE ndash 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
CONTINUEDhelliphellip
Technique of flooding the culture with oxidase
reagent ndash rapidly kills the bacteria
Hence if oxidase reagent is used for isolating
Ngonorrhoea 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
CONTINUEDhelliphellip
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 no1 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 gt 60 sec
QUALITY CONTROL
Positive control Pseudomonas
Negative control Ecoli
FALSE POSITIVE REACTIONS
Mac conkey agar ndash a pink violet colour is
due to carry over from the medium
Iron loop Nichrome loop Stainless steel ndash
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 NNNN-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 TMPDendogenous
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 organsimshad 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 oxidaseQ(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 Groumlschel
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 MT 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
IN REDUCED STATE ndash DYE ndash COLOURLESS
IN OXIDISED STATE ndash DYE ndash 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
CONTINUEDhelliphellip
Technique of flooding the culture with oxidase
reagent ndash rapidly kills the bacteria
Hence if oxidase reagent is used for isolating
Ngonorrhoea 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
CONTINUEDhelliphellip
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 no1 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 gt 60 sec
QUALITY CONTROL
Positive control Pseudomonas
Negative control Ecoli
FALSE POSITIVE REACTIONS
Mac conkey agar ndash a pink violet colour is
due to carry over from the medium
Iron loop Nichrome loop Stainless steel ndash
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 NNNN-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 TMPDendogenous
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 organsimshad 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 oxidaseQ(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 Groumlschel
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 MT 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
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
CONTINUEDhelliphellip
Technique of flooding the culture with oxidase
reagent ndash rapidly kills the bacteria
Hence if oxidase reagent is used for isolating
Ngonorrhoea 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
CONTINUEDhelliphellip
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 no1 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 gt 60 sec
QUALITY CONTROL
Positive control Pseudomonas
Negative control Ecoli
FALSE POSITIVE REACTIONS
Mac conkey agar ndash a pink violet colour is
due to carry over from the medium
Iron loop Nichrome loop Stainless steel ndash
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 NNNN-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 TMPDendogenous
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 organsimshad 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 oxidaseQ(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 Groumlschel
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 MT 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
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
CONTINUEDhelliphellip
Technique of flooding the culture with oxidase
reagent ndash rapidly kills the bacteria
Hence if oxidase reagent is used for isolating
Ngonorrhoea 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
CONTINUEDhelliphellip
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 no1 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 gt 60 sec
QUALITY CONTROL
Positive control Pseudomonas
Negative control Ecoli
FALSE POSITIVE REACTIONS
Mac conkey agar ndash a pink violet colour is
due to carry over from the medium
Iron loop Nichrome loop Stainless steel ndash
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 NNNN-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 TMPDendogenous
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 organsimshad 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 oxidaseQ(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 Groumlschel
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 MT 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
CONTINUEDhelliphellip
Technique of flooding the culture with oxidase
reagent ndash rapidly kills the bacteria
Hence if oxidase reagent is used for isolating
Ngonorrhoea 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
CONTINUEDhelliphellip
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 no1 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 gt 60 sec
QUALITY CONTROL
Positive control Pseudomonas
Negative control Ecoli
FALSE POSITIVE REACTIONS
Mac conkey agar ndash a pink violet colour is
due to carry over from the medium
Iron loop Nichrome loop Stainless steel ndash
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 NNNN-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 TMPDendogenous
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 organsimshad 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 oxidaseQ(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 Groumlschel
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 MT 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
CONTINUEDhelliphellip
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 no1 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 gt 60 sec
QUALITY CONTROL
Positive control Pseudomonas
Negative control Ecoli
FALSE POSITIVE REACTIONS
Mac conkey agar ndash a pink violet colour is
due to carry over from the medium
Iron loop Nichrome loop Stainless steel ndash
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 NNNN-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 TMPDendogenous
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 organsimshad 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 oxidaseQ(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 Groumlschel
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 MT 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
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 no1 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 gt 60 sec
QUALITY CONTROL
Positive control Pseudomonas
Negative control Ecoli
FALSE POSITIVE REACTIONS
Mac conkey agar ndash a pink violet colour is
due to carry over from the medium
Iron loop Nichrome loop Stainless steel ndash
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 NNNN-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 TMPDendogenous
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 organsimshad 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 oxidaseQ(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 Groumlschel
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 MT 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
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 gt 60 sec
QUALITY CONTROL
Positive control Pseudomonas
Negative control Ecoli
FALSE POSITIVE REACTIONS
Mac conkey agar ndash a pink violet colour is
due to carry over from the medium
Iron loop Nichrome loop Stainless steel ndash
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 NNNN-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 TMPDendogenous
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 organsimshad 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 oxidaseQ(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 Groumlschel
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 MT 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
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 gt 60 sec
QUALITY CONTROL
Positive control Pseudomonas
Negative control Ecoli
FALSE POSITIVE REACTIONS
Mac conkey agar ndash a pink violet colour is
due to carry over from the medium
Iron loop Nichrome loop Stainless steel ndash
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 NNNN-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 TMPDendogenous
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 organsimshad 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 oxidaseQ(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 Groumlschel
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 MT 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
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 gt 60 sec
QUALITY CONTROL
Positive control Pseudomonas
Negative control Ecoli
FALSE POSITIVE REACTIONS
Mac conkey agar ndash a pink violet colour is
due to carry over from the medium
Iron loop Nichrome loop Stainless steel ndash
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 NNNN-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 TMPDendogenous
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 organsimshad 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 oxidaseQ(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 Groumlschel
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 MT 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
INTERPRETATION
Oxidase postive purple blue colour
Oxidase positive 5 to 10 sec
Delayed positive 10 to 60 sec
negative gt 60 sec
QUALITY CONTROL
Positive control Pseudomonas
Negative control Ecoli
FALSE POSITIVE REACTIONS
Mac conkey agar ndash a pink violet colour is
due to carry over from the medium
Iron loop Nichrome loop Stainless steel ndash
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 NNNN-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 TMPDendogenous
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 organsimshad 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 oxidaseQ(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 Groumlschel
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 MT 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
QUALITY CONTROL
Positive control Pseudomonas
Negative control Ecoli
FALSE POSITIVE REACTIONS
Mac conkey agar ndash a pink violet colour is
due to carry over from the medium
Iron loop Nichrome loop Stainless steel ndash
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 NNNN-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 TMPDendogenous
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 organsimshad 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 oxidaseQ(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 Groumlschel
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 MT 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
FALSE POSITIVE REACTIONS
Mac conkey agar ndash a pink violet colour is
due to carry over from the medium
Iron loop Nichrome loop Stainless steel ndash
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 NNNN-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 TMPDendogenous
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 organsimshad 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 oxidaseQ(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 Groumlschel
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 MT 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
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 NNNN-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 TMPDendogenous
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 organsimshad 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 oxidaseQ(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 Groumlschel
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 MT 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
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 NNNN-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 TMPDendogenous
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 organsimshad 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 oxidaseQ(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 Groumlschel
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 MT 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
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 NNNN-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 TMPDendogenous
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 organsimshad 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 oxidaseQ(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 Groumlschel
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 MT 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
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 NNNN-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 TMPDendogenous
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 organsimshad 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 oxidaseQ(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 Groumlschel
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 MT 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
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 TMPDendogenous
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 organsimshad 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 oxidaseQ(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 Groumlschel
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 MT 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
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 organsimshad 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 oxidaseQ(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 Groumlschel
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 MT 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
JOURNAL OF CLINICAL
MICROBIOLOGY
Rapid modified oxidase test for oxidase-variable bacterial
isolates
J J Tarrand and D H Groumlschel
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 MT 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
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 MT 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
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
