How to

Identify Bacteria by
Chart
4 Characteristics of Gram positive cocci ADDIN EN.CITE 9,50,51
Gram positive cocci

*
Some
Anaerobic growth only Aerobic or facultative growth

Catalase

Peptostreptococcus
Gemella morbillorum

Negative Positive

Streptococcus * Staphylococcus
Enterococcus Micrococcus
Abiotrophia Rothia***
Aerococcus **
Gemella
Helcococcus
Leuconostoc
Pediococcus
Rothia ***

species may be anaerobic

** May be weak catalase positive
*** This organism is pleomorphic (with a variation in the size and shape of cells) catalase variable, catalase test may not be helpful for differentiation
The flowchart is for guidance only.

4 Identification of Corynebacterium species

 Clinical Specimen

Screening, diagnosis/investigation of cases, contacts/carriers

Blood agar- skin swabs incubated in5-10% CO2 at 35-37C for 40-48hr and throat
swabs incubated anaerobically at35-37C for 16-24hr.

Tellurite agar – incubated in air at35-37°C for 16-46hr.

Grey/black colonies
Blood agar
Gram stain : Gram positive rods

Nitrate Catalase Pyrazinamidase Urease Pure culture

4hr 4hr 4hr 4hr

Positive Negative Positive Negative Positive Negative Positive Negative

C. striatum C. diphtheriae C. ulcerans C. diphtheriae var mitis

C. diphtheriae var C. diphtheriae var All Corynebacterium sp Gardnerella vaginalis
C. jeikium C. pseudotuberculoisis C. pseudotuberculoisis
gravis belfanti (formerly known as C. diphtheriae var gravis
C. ulcerans C. diphtheriae var
C. diphtheriae var mitis C. ulcerans Corynebacterium
C. diphtheriae var C. pseudotuberculoisis vaginalis) intermedius
intermedius var ovis C. diphtheriae var belfanti
C. striatum C. striatum C. striatum
C. pseudotuberculoisis C. jeikium C. jeikium
var equi

For further identification, if clinically indicated, refer to the Reference Laboratory

4 Identification of Listeria species and other Non-Sporing Gram Positive Rods
(except Corynebacterium)
Clinical specimens

Listeria selective agar

Blood agar
containing Aesculin

Non-pigmented translucent haemolytic colonies Black colonies with a black /brown zone
(Non-haemolytic strains of L. monocytogenes occur occasionally) around colonies (Listeria species)

Colonial morphology and haemolysis varies with other

Gram positive rods (see 3.3)

Gram stain on pure culture

Gram positive rods

If there is a different Gram stain appearance refer to the appropriate UK SMI

Catalase Motility test

Tumbling motility Other motility eg

Brownian motion
Positive Negative

Positive Negative
Further identification if
Arcanobacterium haemolyticum clinically needed
All Listeria sp Trueperella pyogenes
Propionibacterium sp Some Listeria stains* Aureobacterium sp*
Trueperella bernardiae All Listeria sp Propionibacteriumsp Microbacterium sp*
Aureobacterium sp Bifidobacterium species Brevibacterium sp Arcanobacteriumsp*
Brevibacterium sp Erysipelothrix rhusiopathiae Rhodococcus sp Send isolate to
Cellulomonas sp Gardnerella vaginalis Rothia sp Reference laboratory
Dermabacter sp Lactobacillus sp Turicella sp
Oerskovia sp Propionibacterium propionicum Cellulomonas sp*
Rothia sp Microbacterium sp*
Turicella sp Trueperella pyogenes
Microbacterium sp Trueperella bernardiae Commercial
Oerskovia sp* identification system
Arcanobacterium sp*
Bifidobacteriumsp
Dermobacter hominis
Erysipelothrix rhusiopathiae
Gardnerella vaginalis
Lactobacillus sp

*Denotes that these species give variable reactions.

The flowchart is for guidance only

Presumptive Identification of Anaerobic Cocci
Clinical
Clinical specimens
specimens-- Primary
Primary isolations
isolations plate
plate

Fastidious
Fastidious anaerobe
anaerobe agar
agar or
or blood
blood agar
agar with
/without
with/without neomycin
neomycin incubated
incubated at
at35-37°C
35-37°C for
for48hr
48hr

Colony
Colony morphology
morphology and
andhaemolytic
haemolytic varies
varies(see 3.3))
(see 3.3

Gram
Gram stains
stains on
on pure
pure culture
culture

Gram
Gram postive
postive Gram
Gram negative
negative
cocci
cocci cocci
cocci

Peptococcus
Peptococcusspecies
species Veillonella
Veillonellaspecies
species
Peptostreptococcus
Peptostreptococcusspecies
species Acidaminococcus
Acidaminococcusspecies
species
Peptoniphilus
Peptoniphilusspecies
species Megasphaera
Megasphaeraspecies
species
Anaerococcus species
Anaerococcus species
Finegoldia
Finegoldia magna
magna
Parvimonas
Parvimonasmicra
micra
Atopobium
Atopobiumspecies
species
Blautia species
Blautia species
Coprococcus
Coprococcusspecies
species
Sarcina
Sarcinaspecies
species
Ruminococcus
Ruminococcusspecies
species
Murdochiella
Murdochiellaspecies
species

Metronidazole
Metronidazole Urease
Urease test
test Indole
Indole test
test Carbohydrate
Carbohydrate
sensitivity
sensitivity (TP36)
(TP36) (TP13)
(TP13) fermentation
fermentation tests
tests

Sensitive
Sensitive Resistant
Resistant Positive
Positive Negative
Negative Positive
Positive Negative
Negative Positive
Positive Negative
Negative

All
All Non-anerobes
Non-anerobes and
and Blautia
Blautia Peptococcus
Peptococcussp sp Peptoniphilus
Peptoniphilussp*
sp* Peptococcus
Peptococcussp sp Coprococcus
Coprococcussp sp Veillonella
Veillonellaspecies
species
anaerobes aa few
few GPAC
GPAC strains species Peptostreptococcus
Peptostreptococcussp sp Anaerococcus
Anaerococcussp*
sp* Peptoniphilus
Peptoniphilussp*
sp* Peptostreptococcus
Peptostreptococcussp sp Peptococcus
anaerobes strains species 11 Peptococcussp sp
Peptoniphilus sp
Peptoniphilus sp Murdochiella Peptostreptococcus
Peptostreptococcussp sp Sarcina sp
Sarcina sp Parvimonas
Parvimonasmicra
Murdochiellasp
sp micra
Parvimonas
Parvimonasmicra
micra Veillonella
Veillonellaspecies
species Megasphaera
Megasphaerasp sp Murdochiella sp
Murdochiella sp
Finegoldia
Finegoldiamagna
magna Finegoldia
Finegoldiamagna
magna Finegoldia
Finegoldiamagna
magna
Anaerococcus
Anaerococcussp sp Anaerococcus
Anaerococcussp* sp* Anaerococcus
Anaerococcussp sp
Atopobium
Atopobiumsp sp Atopobium
Atopobiumsp sp Atopobium
Atopobiumsp sp
Ruminococcus
Ruminococcussp sp Ruminococcus
Ruminococcussp sp Ruminococcus
Ruminococcussp sp
Murdochiella
Murdochiellaspsp Blautia
Blautia species
species Blautia
Blautiasp
sp
Veillonella
Veillonellaspecies
species

Additional
Additional tests:: catalase
tests catalase and
and or
or nitrate
nitrate reduction
reduction tests
tests could
could be
be done
done

Further
Further identification
identification ifif clinically
clinically indicated
indicated send
send to
to the
the reference
reference laboratory
..
laboratory
*These
*These give
give variable
variable test
test resutls..
resutls
 Identification of Bordetella species
Clinical specimens
Primary isolation plate

Charcoal blood agar with cefalexin

Butyrous glistening greyish-white colonies at 3-7
days (may be 2 days for B. parapertussis)

Gram stain of pure culture

Gram negative thin coccobacilli

Oxidase test

Positive Negative
B. pertussis B. parapertussis
B. bronchiseptica B. ansorpii
B. hinzii B.trematum
B. petrii B.holmesii
B. avium* B. avium*
other Gram negatives

Agglutination with specific antiserum on

pure culture, and organisms giving the
biochemical profile of Bordetella species

Positive Autoagglutination Negative

agglutination agglutination
Strongly suspected to beBordetella
species based on colonial morphology
or clinical details

Strongly suspected to be Not strongly

Bordetella species based on
biochemical or clinical
details.
Carry out additional tests
suspected to be
Bordetella species based
on colonial morphology or
clinical details
Report as Bordetella
species not isolated

Further identification if clinically indicated

Refer to the Reference Laboratory
If required, save the pure isolate on to a charcoal blood agar slope

Identification of Yersinia species

 Clinical
Clinical specimens
specimens
Primary
Primary isolate
isolate plate
plate

CIN
CIN agar
agar incubated
incubated in
in air
air at
at Blood
Blood agar
agar incubated
incubated in
in air
air at
at
28-30°C
28-30°C for 24-48hr
24-48hr 28-30°C for 24-48hr
28-30°C for 24-48hr

Suspect
Suspect colonies
colonies
Y.
Y. enterocolitica
enterocolitica –– typical
typical “bull’s
“bull’s eye”
eye” colonies
colonies Suspect
Suspect colonies
colonies
with a deep red centre surrounded by a Y.
Y. pestis
pestis –– small
small grey-white
grey-white translucent
translucent
transparent
transparent border
border colonies
colonies and
and 1-2mm
1-2mm in diameter
Y. pseudotuberculosis
pseudotuberculosis –– smaller
smaller deep
deep red
red
colonies
colonies with
with aa sharp
sharp border
border

Gram
Gram stains
stains ofof pure
pure culture
culture
Gram
Gram negative rods that
negative rods that may
may show
show
bipolar staining
bipolar staining

Further
Further identification
identification ifif clinically
clinically indicated
indicated
Commercial
Commercial identification
identification systems
systems or or other
other biochemical
biochemical identification;
identification; send
send to
to the
the
Reference
Reference Laboratory
Laboratory ifif required
required

The flowchart is for guidance only.

Identification of Vero cytotoxin-producing
Escherichia coli including E. coli O157
Clinical specimen
Primary isolation plate

CT-SMAC agar
incubated in air at
35-37°C for 16-
24hr

Sorbitol fermenting
Non sorbitol colony (pink)
fermenting colony Some rare strains of
(colourless) VTEC O157 ferment
sorbitol

Agglutination with
Consider clinical
VTEC O157
details
antiserum reagent

Bloody
Positive Negative diarrhoea or Diarrhoea only
HUS

Agglutination
with VTEC O157
antiserum
reagent

Consider
non-O157 Negative Positive
VTEC

Identify as E. coli by
commercial identification system or other biochemical
method. Save isolate on slope. Report presumptive
Discard
(locally-confirmed) E. coli O157.
Send to Reference Laboratory to confirm ID , VTgene
type, phage type

The flowchart is for guidance only.

Note: Refer to clinical details: in cases and particularly clusters of cases where isolation or identification
fails, but the symptoms are consistent with VTEC infection, the following actions are recommended:
 send a faecal sample to the Reference Laboratory
 send a serum sample to the Reference Laboratory for the testing for the presence of antibodies
to E. coli O157 lipopolysaccharide
Identification of Campylobacter species

Clinical
Clinical specimens
specimens
Primary
Primary isolation
isolation plate
plate

CCDA
CCDA oror
Campylobacter
Campylobacter Blood
Blood agar
agar or
or Fastidious
Fastidious anaerobe
anaerobe
selective
selective media
media agar
agar
incubated
incubated incubated
incubated microaerobically
microaerobically oror
microaerobically
microaerobically at
at anaerobically
anaerobically at
at 42°C
42°C for
for 40-48hr
40-48hr
42°C
42°C for
for 40-48hr
40-48hr

Moist
Moist grey/white
grey/white moist
moist colonies
colonies Translucent
Translucent moist
moist colonies
colonies

Gram
Gram stain
stain of
of pure
pure culture
culture
Gram
Gram negative
negative typically
typically curved
curved or
or “S”
“S” shaped
shaped rods
rods

Suspect
Suspect
Campylobacter
Campylobacter
species
species

Oxidase
Oxidase test
test
(TP
(TP 26)
26)

Positive
Positive Not Campylobacter
Negative
Negative species

Campylobacter
Campylobacter species
species

Further
Further identification
identification ifif clinically
clinically indicated.
indicated. IfIf aa colony
colony morphologically
morphologically resembling
resembling
Commercial
Commercial identification
identification system
system or or other
other Campylobacter
Campylobacter species
species is
is oxidase
oxidase
biochemical
biochemical identification
identification or or send
send toto the
the negative
negative subculture
subculture onto
onto blood
blood agar
agar and
and
Reference
Reference Laboratory.
Laboratory. retest
retest after
after 24hr
24hr incubation.
incubation.

The flowchart is for guidance only.

Identification of Salmonella species
Clinical specimens
Primary isolation plate

Selective/Non Selective agar

Suspect colonies

Oxidase test Urease test Indole test

(TP 26) (TP 36) (TP 19) - optional

Positive Negative Positive Negative Positive Negative

Discard Discard Discard

Agglutination with polyvalent

* Follow manufacturer’s instructions for
O and Vi antiserum
agglutination tests. (TP 3)*

Positive

Negative
(Not all O antigens are
included in the Polyvalent
Consider Possible O antisera)
S.typhi if Vi Salmonella
positive species

Further biochemical
tests using
Agglutination tests
commercial
identification systems

If required, save the pure isolate on a

One or all nutrient agar slope for referral to the
All negative
positive Reference Laboratory for confirmation,
phagetyping and serotyping.

Submit to Reference
Laboratory for confirmation

Consider clinical details.

Repeat agglutinations from fresh
subculture on non selective agar if required.
Identification of Helicobacter species
Clinical specimen
Primary isolation plates

Blood agarincubated at 5% O2 with 5-10% CO2

at 35-37°C for up to 7 days

Small translucent grey colonies

, may be slightly haemolytic

Gram stain on pure culture

Gram negative straight, curved or comma-shaped rods.
Spiral or helical shapes are less evident
.

Oxidase Catalase
(TP 26) (TP 8)

Positive Negative Positive

Negative
All Helicobacter Not Helicobacter All Helicobacter
H.canis
species species species

Nitrate reduction
Urease test
test (where
(TP 36)
appropriate)

Positive Negative Positive Negative

H. cineadi
H. pullorum
H. pylori H. canadensis
H. bizzozeroni
H. bizzozeroni H. ganmani H. pylori
H. cynogastricus
H. cynogastricus H. pullorum H. fennelliae
H. salomonis
H. salomonis H. felis H. suis
H. bilis
H. suis H. cinaedi H. canis
H. canadensis*
H. bilis H. fenelliae H. canadensis*
H. ganmani
H. heilmannii H. canis
H. heilmannii
H. felis

* H. canadensis gives variable results

Where clinically indicated refer isolates of suspected Helicobacter species to the Reference Laboratory
for identification and typing.
If required, contact the Reference Laboratory to obtain suitable transport medium for referral of biopsies
and isolates.
The flowchart is for guidance only.
 Presumptive identification of anaerobic gram negative rods

Clinical
Clinical specimens
specimens
Primary
Primary isolation
isolation plate
plate

Fastidious
Fastidious anaerobe
anaerobe agar
agar or
or equivalent
equivalent with
with or
or without
without neomycin
neomycin
incubated
incubated anaerobically
anaerobically at
at 35
35-37°C
-37°C for
for 40 -48hr
40-48 hr

Gram
Gram stain
stain of
of pure
pure culture
culture
Gram
Gram negative
negative rods
rods of
of variable
variable lengths
lengths (may
(may bebe filaments
filaments or
or
pleomorphic).
pleomorphic). For
For other
other gram
gram stain
stain results,
results, refer
refer to
to the
the appropriate
appropriate
SMI.
SMI.

Susceptibility
Susceptibility to
to 5µg
5µg metronidazole
metronidazole discdisc
Metronidazole
Metronidazole sensitive
sensitive
(although
(although a
a few
few metronidazole
metronidazole resistant
resistant B.
B. fragilis
fragilis have
have been
been reported)
reported)

May
May report
report as
as “Gram
“Gram negative
negative anaerobe
anaerobe isolated”
isolated”

Further
Further identification
identification if
if clinically
clinically indicated.
indicated. Commercial
Commercial identification
identification kit
kit or
or other
other
biochemical
biochemical identification
identification or or GLC.
GLC. If If required,
required, save
save thethe pure
pure isolate
isolate in
in fastidious
fastidious
anaerobe
anaerobe broth
broth with
with cooked
cooked meat
meat forfor referral
referral to
to the
the Reference
Reference Laboratory
Laboratory..

Genus Characteristics of growth on fastidious anaerobe agar after anaerobic

incubation at 35-37°C

Bacteroides Colonies are 1-3mm diameter, circular, low convex, smooth, semi-opaque grey
and are often moist or even mucoid. Mostly non-haemolytic and resistant to an
ox-bile disc.

Fusobacterium Colonial appearance is variable, but most are 1-3mm diameter, with an
irregular or dentate edge. They vary from translucent to granular and opaque;
F. necrophorum may be beta-haemolytic. Indole positive, fluorescent yellow-
green under long wave UV light.

Porphyromonas Colonies are <1.0mm diameter after 48hr incubation, smooth, shiny and grey.
Dark brown or black pigment develops after 3-7 days. Growth may be
enhanced by “satellitism” around colonies of other organisms eg staphylococci.

Prevotella Colonies are similar to those of Bacteroides species, except some species are
pigmented (may be pale brown to black). Most pigmented species are
haemolytic.

Identification of Enterobacteriaceae
 Clinical Specimens
Primary isolation plate

BA
CLED B or CLED A, MAC
DCA, XLD, CT-SMAC, TCBS, CIN agar
Incubation temperature and conditions vary
. See 3.2

Gram stain on pure culture.

Colonial appearance and Gram negative rods– some
morphology varies. See 3.3 may show bipolar staining
(Yersinia species)

Carbohydrate Carbohydrate
(lactose) (lactose) non
fermenting fermenting

Oxidase test (TP 26)

Performed from non selective
medium

Positive Negative

All Enterobacteriaceae (except Plesiomonas

Plesiomonas shigelloides
shigelloides)
Possible Pseudomonas species
or
Other identification tests (optional) that could be
Pasteurella species
done are indole test, serology, commercial
(see ID 17 and 13)
identification system

For further identification if clinically indicated, send

to the Reference Laboratory

The flowchart is for guidance only.

Identification of Pseudomonas species and other
Non-Glucose Fermenters

Clinical
Clinical specimens
specimens
Primary
Primary isolation
isolation plate
plate

Pseudomonas
Pseudomonas selective
selective agar
agar containing
containing cetrimide
cetrimide incubated
incubated inin air
air at
at 35-37°C
35-37°C
for
for 16–48hr,
16–48hr, thenthen at
at 30°C
30°C forfor up
up to
to 55 days.
days.
Burkholderia
Burkholderia cepacia
cepacia selective
selective agar
agar incubated
incubated in in air
air at
at 35-37°C
35-37°C for
for 48-72hr,
48-72hr,
then
then for
for up
up to
to 55 days
days ifif necessary.
necessary.
Blood or chocolate agar incubated in 5-10% CO at 35-37°C
Blood or chocolate agar incubated in 5-10% CO2 at 35-37°C for 16-48hr.
2 for 16-48hr.
CLED/
CLED/ MacConkey agar incubated in air at 35-37°C 35-37°C forfor 16-48hr.
16-48hr.

See
See typical
typical colony
colony morphology
morphology and
and pigment
pigment production
production of
of Pseudomonas
Pseudomonas
species
species and
and morphologically
morphologically simlar
simlar organisms
organisms in
in section
section 3.3
3.3

Gram
Gram stain
stain of
of pure
pure culture
culture
Pseudomonas
Pseudomonas species –– Gram
Gram negative
negative rods
rods which
which are
are straight
straight or
or slightly
slightly
curved
curved on
on some
some strains
strains of
of P. putida
putida can
can appear
appear elongated.
elongated.
See
See section
section 3.1
3.1 for
for Gram
Gram stains
stains of
of other
other morphologically
morphologically similar
similar organisms.
organisms.

Oxidase
Oxidase test
test (TP
(TP 26)
26)

Positive
Positive Negative
Negative

Pseudomonas
Pseudomonas species
species
Burkholderia
Burkholderia species
species
Ralstonia
Ralstonia species
species
Brevundimonas
Brevundimonas species
species Pseudomonas
Pseudomonas luteola
luteola
Comamonas
Comamonas species
species Pseudomonas
Pseudomonas oryzihabitans
oryzihabitans
Achromobacter
Achromobacter species
species Some
Some Burkholderia
Burkholderia species*
species*
Alcaligenes
Alcaligenes species
species Stenotrophomonas
Stenotrophomonas
Delftia
Delftia species
species Maltophilia
Maltophilia
Methylobacterium
Methylobacterium species
species Acinetobacter
Acinetobacter species
species
Oligella
Oligella species
species Roseomonas
Roseomonas species
species
Shewanella
Shewanella species
species Pandoraea
Pandoraea species
species
Sphingobacterium
Sphingobacterium species
species
Elizabethkingia
Elizabethkingia meningoseptica
meningoseptica
Pandoraea
Pandoraea species
species
Acidovorax
Acidovorax species
species

Further
Further identification
identification ifif clinically
clinically indicated,
indicated, commercial
commercial identification
identification systems
systems or
or other
other
biochemical
biochemical indetification
indetification could
could be
be done
done or
or sent
sent to
to the
the Reference
Reference Laboratory.
Laboratory.

*Some Burkholderia species give variable reactions on oxidase test (B. contaminans, B. lata, B. pyrrocinia, B. gladioli, B. mallei)

The flowchart is for guidance only.

Members of the genus demonstrate considerable variation in cell and colony

morphologies and this may lead to difficulties in recognition of putative Actinomyces
 on primary isolation media.
Species Colonies Comments

A. israelii White to cream, breadcrumb or molar Slow growing

tooth, gritty, pitting
Old colonies may become pink

A. gerensceriae Bright white, breadcrumb or molar Slow growing

tooth, pitting and softer than A. israelii
A. naeslundii White, cream or pinkish, smooth, Occasional rough forms occur
convex, entire edged
A. odontolyticus Cream to red, smooth, convex, entire Old colonies may be dark brown
edged
A. meyeri Small, white, smooth, convex, entire Slow growing
edged
A. georgiae White or cream, smooth, convex,
entire edged
A. neuii sub sp. neuii White or cream, smooth, convex,
and anitratius entire edged
A. radingae Grey to white, semi-translucent,
smooth, low convex, entire edge
A. turicensis Grey, semi translucent, smooth, low
convex, entire edged
A. europaeus Whitish, semi translucent, smooth, low
convex, entire edged
A. graevenitzii* White pronounced molar tooth or Red fluorescence. Rough and smooth
smooth, convex forms occur together. Old colonies
may become dark brown
A. radicidentis Cream to pink, smooth, convex, entire Old colonies may become red
edged
A. urogenitalis Cream to-pink, with darker rings, Old colonies may become red
smooth
A. funkei Grey, semi translucent, opaque centre
(fried egg), low convex, entire edged

A. cardiffensis Cream to pink, smooth, convex, entire

edged
A. nasicola White or grey, smooth, convex, entire
edged
A. oricola White, breadcrumb, pitting on the agar

A. viscosus The two types of colonies: large and

smooth colonies with V, Y and T
configurations or small and rough
colonies with short branching
filaments.
Colonies are similar to A. naeslundii
A. johnsonii

A. oris Colonies are similar to A. naeslundii

A. massiliensis white, pinpoint, circular and shiny with

entire edges
A. dentalis tiny, white and breadcrumb-like and
pitting on the agar
A. hongkongensis non-haemolytic, pinpoint colonies

A. hominis white–greyish, convex, entire edges

A. timonensis α-haemolytic, pin-point, circular,

white, dry and embedded in the agar
P. propionicum* Off white to buff, breadcrumb, gritty, Red fluorescence, rough and smooth
pitting, or smooth, convex, entire forms occur together
edged
*Colonies of A. graevenitzii and Propionibacterium propionicum on blood containing media fluoresce red
under long-wave (366 nm) UV illumination.

Identification of Neisseria species

 Clinical specimens
Primary isolation plates

Whole Blood/Heated Blood

GC selective agar
(chocolate) agar

N. gonorrhoeae and N. meningitidis are

Colonial appearance varies according to
smooth round; moist, uniform grey/brown
Species N. gonorrhoeae grows poorly on
colonies with a greenish colour in the agar
underneath at 48hr whole blood agar in some cases

Oxidase test (TP 26)

Positive Negative

Neisseria species,
Not Neisseria species
M. catarrhalis
Consider Kingella species (catalase negative)
Oligella species

Gram stain (TP 39)

Gram -ve rods Not N. gonorrhoeae

Gram -ve cocci
arranged in short chains or
arranged in pairs
or as diplococci N. meningitidis

If further identification is
Perform confirmatory tests*
required, refer to the
*Please refer to the rest of the document for more
appropriate reference
detailed instructions regarding tests to use
.
laboratory

Identification of Staphylococcus Species,

Micrococcus species and Rothia species
 Clinical specimens
Primary isolation plate
Opaque, white, cream, yellow or orange colonies on blood agar

Gram stain
Gram positive cocci in clusters Suspected
If there is a different Gram stain S. aureus
appearance refer to the appropriate UKSMI

Catalase

Negative Positive

Catalase negative
S. aureus subsp anaerobius S.capitis Catalase positive
S. saccharolyticus All Staphylococcus,
(grows anaerobically) Micrococcus and Rothia
Rothia dentocariosa* species
Consider other organisms

Novobiocin
sensitivity test

Resistant
S. saprophyticus,
S. sciuri and
Sensitive
S. cohnii groups
All Staphylococcusspecies
S. hominis subsp.
novobiosepticus
S. cohnii group

Coagulase (slide or tube)

DNAse test, Commercial
identification system

Negative Positive

S. aureus
Coagulase-negative S. hyicus*
Staphylococcusspecies S. intermedius*
S. schleiferi subsp coagulans*

Confirm with tube

coagulase if required

Further identification if clinically indicated

Refer to the Reference Laboratory
If required, save the pure isolate
onto a nutrient agar slope

* Strains may be positive to certain tests

Identification of Moraxella species and
Morphologically Similar Organisms

Clinical specimens
Primary isolation plate

Blood agar or chocolate agar

incubated in 5-10% CO 2 at 35-37°C
for 16-48hr

Moraxella species are white or buff, convex colonies on blood agar

Neisseria species form smooth, round, moist, uniform large grey/brown colonies with a
glistening surface and entire edges
Kingella species are smooth entire convex or spreading colonies 0.5–1mm in diameter
K. kingae is β haemolytic
Oligella species are small, white, opaque and non - haemolytic
Psychrobacter species are non-pigmented, smooth, opaque colonies

Gram stain on pure culture

Gram negative large rods or coccobacilli

Oxidase test

Positive
Possible Moraxella species Negative
or Psychrobacter species or Possible Acinetobacter species
Kingella species or Oligella species or (ID 17)
Neisseria species

Positive
Tributyrin
Moraxella species
(presumptive)

Negative DNAse Test Positive

Neisseria species (supplementary ) M. catarrhalis
(ID 6)

Negative

Further identification if clinically indicated

Commercial identification systems or other identification
(Commercial kits may misidentify Brucella species as P. phenylpyruvicus,
Acinetobacter species)
If required, save pure isolate on a blood agar slope

This flowchart is for guidance only.

 Summary of the biochemical tests:

Organism Catalase Oxidase Urease

H. influenzae + + (+)

H. aegyptius + + +

H. ducreyi - + Unknown

H. haemolyticus + + +

H. parainfluenzae d + d

H. pittmaniae d d -

H. parahaemolyticus d + +

H. paraphrohaemolyticus + + +

H. sputorum V + +

A. actinomycetemcomitans + + -

A. aphrophilus - - -

C. hominis - + -

C. valvarum - + -

E. corrodens - + -

K. kingae - + -

K. denitrificans - + -

K. oralis - + -

K. potus - + -
+ = positive, - = Negative, (+) = 80-89% positive, d= 11-89% positive, V= variable result
Growth requirement for X and V f Summary of X and V test results

Organism X factor V factor X + V factor Porphyrin

H. influenzaea No growth No growth Growth Negative

H. haemolyticusb No growth No growth Growth Negative

H. parainfluenzae No growth Growth Growth Positive

H. pittmaniae No growth Growth Growth Positive

H. parahaemolyticus No growth Growth Growth Positive

H. paraphrohaemolyticus No growth Growth Growth Positive

Haemophilus ducreyi Growth No growth Growth Positive

Haemophilus sputorum No growth Growth Growth Positive

aH. aegyptius is indistinguishable from H. influenzae biotype III in normal laboratory tests.
-haemolytic on horse blood agar.
b
 Identification of Vibrio species
Clinical specimen
Primary isolation plate

Blood agar TCBS

incubated at 35-37°C for 18-24hr agar incubated at 35-37°C for 18-24hr

Colonies are 2-3mm in diameter Yellow or green colonies and are

Some colonies may be haemolytic 2-3mm in diameter

Gram stain on pure culture

Gram negative rods characteristically curved or
comma -shaped but can also be straight

Oxidase test (TP 26)

Positive Negative

All Vibrio species

Possible V. metschnikovii
Check biochemistry before discarding
(V. metschnikovii produces yellow colonies on
Pteridine 0129 sensitivity TCBS. Oxidase negative isolates
(optional ) with a different morphological
( 10µg and 150µg discs ) appearance may be discarded )

Sensitive 10µg Resistant 10µg Resistant 10µg

Sensitive 150 µg Resistant 150 µg Sensitive 150µg

Possible
Aeromonas species V. parahaemolyticus
or V. cholerae O1 V. alginolyticus
or V. cholerae O139 V. fluvialis
V. furnissii

Agglutinations with
O1 or O139 antisera

Positive Negative

V. cholerae (non O1, O139)

V. cholerae V. vulnificus
Vibrio species

Further identification if clinically indicated

Commercial identification system (these may require
supplementation with NaCl ) or send to the Reference Laboratory

The flowchart is for guidance only

Identification of Shigella species

Clinical specimen
Primary isolation plate

XLD and / or DCA agar incubated at35-37°C for 18-24hr in

air.
Colony appearance and morphology varies . See 3.3.

Purity plate on a non-selective plate

Gram stain on pure culture

Gram negative rods arranged singlyin pairs and in chains

Oxidase test Urease test

(TP 26) (TP 36)

Positive Negative Negative Positive

Non-Shigella Shigella Shigella Non-Shigella

species species species species

Carbohydrate fermentation
test (Mannitol)

Positive Negative

Shigella flexneri
Shigella boydii Shigella dysenteriae
Shigella sonnei

Agglutination with polyvalent Agglutination with polyvalent

antiserum for each species antiserum S. dysenteriae

Positive Negative Positive Negative

Shigella sonnei(1)
Shigella flexneri (1-6, x,y) Pure culture
Not
Shigella boydii(1-6, 7-11, 12-15) specific agglutination with
S. dysenteriae
Note: Not all serotypes are monovalent antisera
contained in polyvalent antisera

Further identification, if
clinically indicated, send to
Reference Laboratory

4b Identification of Aeromonas species

 Clinical specimen
Primary isolation plate

Blood agar incubated at35-37°C for CIN agar/or MacConkey agar

18-24hr incubated at 37-37°C for 24-48hr

On CIN agar – colonies appear as

pink bulls eye due to fermentation of
D-mannitol.
Colonies are distinctively circular
,
On MacConkey agar– colonies are
large, raised and are 1-3mm in
typically non-lactose fermenting
diameter
although some lactose-fermenting
Aeromonas species have been
observed

Gram stain on pure culture

Gram negative rods characteristically straight
, coccobacillary to bacillary with rounded ends
.
They appear singly or in pairs

Oxidase test (TP 26)

Positive Negative

Not Aeromonas
All Aeromonas/
species. Possible
Vibrio species
V. Metschnikovii

Pteridine O129
sensitivity
(optional) (10µg x
150µg discs)

Resistant 10µg Resistant 10µg Sensitive 10µg

Resistant 150µg Sensitive 150µg Sensitive 150µg

All Aeromonas species

or Vibrio O1 or Vibrio
O139. Other
identification tests such
Not Aeromonas species Not Aeromonas species
as ornithine
decarboxylase hydrolysis
or Voges–Proskauer
tests could be done

Further identification if clinically

indicated. Commercial identification
system or send to the Reference
Laboratory

The flowchart is for guidance only

4 Identification of Streptococcus species, Enterococcus species and
Morphologically Similar Organisms
Clinical specimen
Primary isolation plate

(Blood, CLED, Staph/Strep medium or fastidious anaerobe agar)

Gram stain
Gram positive cocci in pairs and/or short chains
Positive
(Probable Staphylococcus)
Catalase A weak catalase or pseudocatalase reaction may be
produced by some strains of Aerococcus &
Enterococcus species

Negative

a - haemolytic non-haemolytic
(Consider Leuconostoc , b- haemolysis
(Consider Gemella) Gemella , Helcococcus )

Suspected Enterococcus
(1-2mm may be a, β or non -haemolytic .
Consider clinical details )
Optochin
Lancefield Group
Rapid Aesculin hydrolysis

Sensitive Resistant A,B,C,D,F,G

Non groupable
(A ,C,G, consider
(repeat , consider Listeria ,
S.anginosus
check previous
Positive Negative group )
tests)

S. pneumoniae: “viridans”
Some S. pneumoniae may be Streptococci:
resistant to optochin : if there is a Occasional strains of S. oralis may Enterococcus sp
clinical suspicion of pneumococcal be optochin sensitive : Lancefield group D Consider
infection, confirm by performing S. pseudopneumoniae optochin Lactococcus sp Pediococcus sp
bile solubility resistant when incubated in 5 % Lancefield Group B
Some strains of Aerococcus and
S.pseudomoniae when incubated CO 2 S. anginosus group
at ambient air is positive .
Leuconostoc sp
Pediococcus sp Aerococcus urinae
Globicatella sp

Further identification if clinically indicated

Commercial identification system or other biochemical identification or send to the
Reference Laboratory

The flowchart is for guidance only.

Identification of Clostridium species
Clinical specimens
Primary isolation plate

Blood containing agar incubated anaerobically at

35-37°C for 40-48hr

β or non haemolytic colonies which may spread all round the agar plate
Optional
(swarming)

Gram stain on pure culture

Gram positive rods which may possess a single endospore. Some species
may be gram variable.
Spore stain/phase contrast microscopy
used to determine the shape and position of the spore

Subculture to egg yolk agar and incubate anaerobically at

35-37°C for 16-24hr

Nagler Test Indole Test Urease Test

Lipase Test Swarming (TP 19) (TP 36)
(TP 22)

Positive Negative Positive Negative Yes No Positive Negative Positive Negative

C. tetani*
C. difficile
C. botulinum* C. novyi Type B C. perfringens
C. perfringens C. difficile C. novyi Type C C. novyi Type A C. bifermentans
C. sordellii C. histolyticum C. perfringens C. botulinum C. septicum C. novyi Type B*
C. novyi Type A C. novyi Type C C. novyi Type A C. cadaveris C. sporogenes C. novyi Type A* C. sporogenes C. novyi Type D*
C. novyi Type B C. cadaveris C. sporogenes C. histolyticum C. novyi C. sordellii C. histolyticum C. novyi Type A
C. perfringens C. sordellii
C. botulinum* C. septicum C. botulinum C. baratii C. septicum C. tetani* C. tetani* C. perfringens
C. difficile
C. baratii C. tetani C. tetani* C. haemolyticum C. tetani C. bifermentans C. difficile C. septicum
C. bifermentans C. butyricum C. ghonii C. bifermentans C. sordeilii C. botulinum C. botulinum
C. haemolyticum C. tertium C. butyricum C. bifermentans C. butyricum C. tetani
C. ghonii C. sporogenes C. sordellii C. baratii
C. innocuum C. septicum
C. tertium
C. innocuum

Further identification if clinically indicated

, send to the appropriate reference laboratory

* These give variable test results

Identification of Bacillus species

 Clinical specimen
Primary isolation plate

Blood agar incubated in air/ PEMBA incubated in air/CO2

CO2 at 35-37C for 24-48hr at 35-37C for 24-48hr

B. cereus group - flat, grey/green/cream haemolytic B. cereus, B. thuringiensis - 5mm diameter,

colonies, ground glass appearance, 2-5mm diameter. turquoise to peacock blue, with zone of precipitation.
B. anthracis - grey/white non-haemolytic flat, colonies with B. subtilis group - colonies are cream to light yellow
irregular edges and resembles “medusa head”. with no zone of precipitation.
B. mycoides - rhyzoid/hairy/spreading colony.
Oher Bacillus sp. 2-5mm diameter, smooth/mucoid/
crenated–generally β haemolytic.

β haemolytic colonies, B. cereus group, B. subtilis group

Non haemolytic
colonies (B. anthracis) Gram stain of pure culture
Gram-positive rods (may have single endospore)
Some species may be Gram variable

Lecithinase Motility Penicillin susceptibility

Negative Positive Postitive Negative Resistant Sensitive

Presumptive Presumptive Presumptive

B. cereus, B. cereus group B. anthracis
B. thuringiensis

For further identification if clinically indicated, refer to a Reference Laboratory

Genus
 Characteristics of growth on fastidious anaerobe agar after incubation at
35 - 37°C for 40 – 48hr

Nocardia species Wrinkled often dry, crumbly, chalky-white appearance to orange or tan pigment

Streptomyces species Waxy heaped colonies with variable morphology

Oerskovia species Yellow pigmented, extensive branching that grows on the surface and in to the
agar

Gordonia, Rhodococcus, Non-haemolytic, round, often mucoid with salmon-pink/red colonies developing
and Tsukamurella within 4 to 7 days
species

Dermatophilus Round adherent grey-white colonies, that later develop orange pigments; often
congolensis beta-haemolytic. Colonies may be adherent to the agar.

Actinomadura species White to pink colour. Colonies are usually mucoid, wrinkled and have a molar
tooth appearance

Rothia species Small, smooth to rough colonies and dry in appearance

Nocardiopsis species Coarsely wrinkled and folded with well- developed aerial mycelium

Identification of Haemophilus species

 Clinical Specimens
Primary isolations plate

Blood
Blood or
or chocolate
chocolate agar
agar
incubated
incubated in
in 5-10%
5-10% CO2
CO2 at
at
35-37°C
35-37°C for
for 24-48hr
24-48hr

Haemophilus
Haemophilus species
species are
are small,
small, round,
round, convex,
convex,
colourless
colourless to
to grey
grey colonies
colonies and
and may
may bebe iridescent .
iridescent.
H.
H. haemolyticus
haemolyticus have
have β-haemolytic
β-haemolytic colonies.
colonies.

Gram’s
Gram’s stain
stain on
on pure
pure culture
culture
Gram-
Gram- negative
negative spherical,
spherical, oval
oval or
or rod
rod shaped
shaped cells
cells
with
with marked
marked pleomorphisim
pleomorphisim oror filament
filament formation
formation

Oxidase
Oxidase Catalase
Catalase Urease
Urease Growth
Growth for
for X
X and
and VV factors
factors
(TP
(TP 26)
26) (TP
(TP 8)
8) (TP
(TP 36)
36) (TP
(TP 29
29 or
or TP
TP 38)
38)

Positive
Positive Negative Positive
Positive Negative
Negative Positive
Positive Negative
Negative Positive
Positive Negative
Negative
Negative

H.
H. influenzae
influenzae H.
H. pittmaniae*
pittmaniae* H.
H. influenzae
influenzae H.
H. ducreyi
ducreyi H.
H. influenzae
influenzae H.
H. pittmaniae
pittmaniae H.
H. parainfluenzae
parainfluenzae H.
H. influenzae
influenzae
H.
H. aegyptius
aegyptius H.
H. aegyptius
aegyptius H.
H. sputorum#
sputorum# H.
H. aegyptius
aegyptius H.
H. parainfluenzae*
parainfluenzae* H.
H. pittmaniae
pittmaniae H.
H. aegyptius
aegyptius
H.
H. ducreyi
ducreyi H.
H. haemolyticus
haemolyticus H.
H. parainfluenzae*
parainfluenzae* H.
H. haemolyticus
haemolyticus H.
H. ducreyi
ducreyi H.
H. parahaemolyticus
parahaemolyticus H.
H. haemolyticus
haemolyticus
H.
H. haemolyticus
haemolyticus H.
H. parainfluenzae*
parainfluenzae* H.
H. pittmaniae*
pittmaniae* H.
H. parainfluenzae*
parainfluenzae* H.
H. paraphrohaemolyticus
paraphrohaemolyticus H.
H. ducreyi
ducreyi
H.
H. parainfluenzae
parainfluenzae H.
H. pittmaniae*
pittmaniae* H.
H. parahaemolyticus*
parahaemolyticus* H.
H. parahaemolyticus
parahaemolyticus H.
H. sputorum
sputorum
H.
H. pittmaniae*
pittmaniae* H.
H. parahaemolyticus*
parahaemolyticus* H.
H. paraphrohaemolyticus
paraphrohaemolyticus
H.
H. parahaemolyticus
parahaemolyticus H.
H. paraphrohaemolyticus
paraphrohaemolyticus H.
H. sputorum
sputorum
H.
H. paraphrohaemolyticus
paraphrohaemolyticus H.
H. sputorum#
sputorum#
H.
H. sputorum
sputorum

** shows
shows 11-89%
11-89% of
of strains
strains are
are postitive
postitive
## shows
shows H.
H. sputorum
sputorum gives
gives variable
variable results
results Further
Further ID
ID ifif clinically
clinically indicated,
indicated, Refer
Refer to
to the
the appropriate
appropriate Reference
Reference laboratory
laboratory

This flowchart is for guidance only.

4b Identification of HACEK group

 Clinical specimens
Primary isolation plate

Blood agar

Incubate aerobically at
35-37°C for
24-48hr

Growth may require CO 2 Requires 5-10% CO2 CO2 not required

Grows in air, may require CO2
Grows in air + CO2 addition Small moist colonies with clear Either spreading corroding
Star shaped non-haemolytic
Yellowish non-haemolytic Smooth, convex and opaque centres surrounded by flat colony or smooth convex
colonies with rough surface
,
colonies colonies growth colony, often produces mucoid
may produce pitting of agar
1.5mm at 24hr Slight a -haemolysis Non-haemolytic colonies with a small zone of
Colonies 1mm at 48hr
Colonies 1-2mm at 48hr 0.5 - 1mm after 48hr β-haemolysis

Catalase positive Catalase negative Catalase negative Catalase negative Catalase negative
Oxidase positive Oxidase negative Oxidase positive Oxidase positive Oxidase positive
Urease negative Urease negative Urease negative Urease negative Urease negative

C. hominis
A. actinomycetemcomitans A. aphrophilus E. corrodens All Kingae species
C.valvarum

This flowchart is for guidance onl

Identification of Pasteurella species and Morphologically Similar Organisms
Clinical specimens
Primary isolation plate

Blood Agar incubated

at 5-10% CO 2 at 35-
37°C for 16-48hr

Avibacterium species - Actinobacillus species - The

Pasteurella species are grey,
viscous, non-haemolytic
colonies on blood agar . Rough,
irregular colonies may also
occur. M. haemolytica and B .
trehalosi are β-haemolytic on
blood agar .
Bibersteinia species - Mannheimia species -
Colonies on sheep -blood agar colonies are non -haemolytic ,
Colonies on blood agar are Colonies are smooth and
are non-haemolytic , greyish, translucent and 1-2mm in
round, regular , greyish or greyish on blood agar and are
opaque , but eventually diameter on blood agar . Most
yellowish , semi-transparent at 1-2mm in diameter after 24hr
translucent at the periphery , strains will grow on MacConkey
the periphery and are about incubation . Most strains show a
with a butyrous consistency , agar apart from A.
2mm in diameter . Some strains characteristic β-haemolysis on
smooth and shiny , circular and pleuropneumoniae and some
are haemolytic . bovine blood agar .
raised with an entire margin . strains of A. suis.

Gram stain on pure culture

Gram negative rods or cocco -bacilli occurring singly , in pairs or short chains
If there is a different Gram stain appearance refer to the appropriate UK SMI

Catalase Oxidase test

(TP 8) (TP 26)

Negative
P. bettyae Positive Positive
Negative
P. caballi Possible Pasteurella species Possible Pasteurella species
P. bettyae
Bibersteinia trehalosi * Avibacterium sp* Bibersteinia sp*
Bibersteinia sp*
Avibacterium sp* Bibersteinia trehalosi * Avibacterium sp*
Mannheimia sp*
Actinobacillus sp* Mannheima sp Mannheima sp*
Actinobacillus sp*
(Some strains of Actinobacillus sp* Actinobacillus sp*
A. hominis )

A zone of inhibition to
Penicillin

Sensitive Resistant
Possible Pasteurella Not Pasteurella
species species

Further identification if clinically indicated . Commercial identification systems or other identification .

If required , save pure isolate on a blood agar slope

* These species give variable results in these tests