Applied Veterinary Bacteriology and Mycology: Introduction  Chapter 5: Identification systems used in diagnostic
bacteriology
Applied Veterinary Bacteriology and Mycology:
Bacteriological techniques
Chapter 5: Identification systems used in diagnostic
bacteriology
Author: Dr. J.A. Picard
Licensed under a Creative Commons Attribution license.
TABLE OF CONTENTS
BACTERIOLOGICAL IDENTIFICATION FOR THE CLINICAL LABORATORY ........................................ 2
INTERPRETATION OF GROWTH/COLONY MORPHOLOGY ................................................................... 2
SECONDARY CULTURES........................................................................................................................... 4
IDENTIFICATION OF BACTERIA ................................................................................................................ 4
TYPES OF IDENTIFICATION SYSTEMS .................................................................................................. 17
Biochemical profiles ............................................................................................................................... 17
Gas-liquid chromatography .................................................................................................................... 18
THE NORMAL FLORA ............................................................................................................................... 21
Mouth, nasopharynx .............................................................................................................................. 21
OCCURRENCE OF PATHOGENS IN ANIMAL SPECIES ........................................................................ 22
Laboratory animal infections .................................................................................................................. 22
Rats and Mice ........................................................................................................................................ 22
Guinea Pigs ............................................................................................................................................ 22
Rabbits ................................................................................................................................................... 23
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Applied Veterinary Bacteriology and Mycology: Introduction  Chapter 5: Identification systems used in diagnostic
bacteriology
BACTERIOLOGICAL IDENTIFICATION FOR THE CLINICAL
LABORATORY
Although new technologies such as the polymerase chain reaction (PCR), DNA hybridization and the
ELISA test are available for the identification of various bacteria, they are often too expensive for routine
laboratory use. Thus clinical microbiologists continue to depend on morphological and biochemical criteria
e.g. substrate utilization systems for the identification of micro-organisms isolated from clinical specimens.
Identification of bacteria follows primary isolation and colony purification. This is normally based on the
following tests:


Colony morphology
Gram’s stain (in some cases specialized stains e.g. for mycobacteria).

Catalase reaction

Oxidase reaction

Growth on MacConkey agar

Oxidation-fermentation test

Motility test

Secondary identification tests
INTERPRETATION OF GROWTH/COLONY MORPHOLOGY
Interpretation of bacterial growth requires considerable experience. In short, it hinges on the ability of the
microbiologist to distinguish between what is significant (abnormal) and what is to be expected from
normal tissue. The clinical history, necropsy findings and smear examination must be borne in mind when
evaluating the growth.
Examine all the cultures of each specific case. The stereo-microscope can be invaluable at this stage. If a
stereo-microscope is not available, a hand lens can be used. This helps to distinguish between different
bacterial colonies. A good light source is required to examine the cultures. Circle suspect colonies on the
under-surface of the plate. Colonies should be fully described according to shape, size, colony elevation,
opacity, the presence of pigments, consistency and odour. Each circled colony should then be subcultured to obtain a pure culture for identification.
Below are a few tips:
1. Examples of micro-organisms exhibiting distinctive odours include:

Pseudomonas aeruginosa: grape juice odour

Proteus spp.: burnt chocolate

Pasteurella spp.: mouse urine
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Applied Veterinary Bacteriology and Mycology: Introduction  Chapter 5: Identification systems used in diagnostic
bacteriology

Nocardia & Streptomyces spp: musty basement


Clostridium spp.: faecal, putrid
Prevotella melaninogenica: acrid
2.
It is often preferable to re-incubate very small colonies so as to be able to assess purity.
3.
Although growth rate is not an indication of pathogenicity, slow growing bacteria are often more
pathogenic than those which grow fast on culture.
4.
The growth of the suspected pathogen on the various media must be closely compared before a
5.
decision is made as to which colonies should be isolated.
Some bacterial species, such as some streptococci, clostridia and Bacillus, do not retain their Gram-
6.
positive properties very well and consequently often stain Gram-negative.
Clostridium perfringens seldom forms spores on artificial media.
Figure 1: Terms used to describe gross colony morphology
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Applied Veterinary Bacteriology and Mycology: Introduction  Chapter 5: Identification systems used in diagnostic
bacteriology
SECONDARY CULTURES
After 24 hours incubation (or as long as it requires to distinguish the individual colonies clearly), make a
subculture from a single colony of the suspect pathogen by touching the colony with a sterile (flamed and
cooled) inoculation needle and then inoculating it onto a non-selective agar medium.
When the bacterial growth is very heavy and the colonies too close together to distinguish from each
other, a stereo-microscope may be used to advantage. A straight sharp needle may facilitate the isolation
of a particular colony.
Only select colonies from selective media, should there be no alternative i.e. the colony is not
represented on non-selective media. Inoculate a non-selective agar medium. Isolations from selective
solid media into nutritious fluid media should not be attempted, even if the culture appears “pure”.
Selective media often inhibits, but may not kill, bacteria.
Make a smear of the colony and stain it using Gram’s method, and any other stains that may be deemed
necessary, from the remainder of the same colony.
Incubate agar for at least 18 hours before carrying out the appropriate identification tests. Should there be
enough isolated colonies on the initial non-selective agar medium, identification and an antimicrobial
sensitivity test can be done from the primary plates. Please note that all tests should be done using a
pure culture as using mixed cultures can lead to spurious results.
IDENTIFICATION OF BACTERIA
Primary identification of the majority of bacteria is based on the Gram’s stain. Bacteria are divided into
Gram-positive and Gram-negative bacteria. Thereafter they are divided according to colony and
microscopic morphology as well as catalase, oxidase and oxidation-fermentation tests.
Based upon these results secondary identification tests are done.
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Applied Veterinary Bacteriology and Mycology: Introduction  Chapter 5: Identification systems used in diagnostic
bacteriology
Figure 2: General procedures for the isolation of bacteria and fungi from clinical specimens
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Applied Veterinary Bacteriology and Mycology: Introduction  Chapter 5: Identification systems used in diagnostic
bacteriology
Figure 3: Primary identification of some Gram-positive bacteria of veterinary importance
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Applied Veterinary Bacteriology and Mycology: Introduction  Chapter 5: Identification systems used in diagnostic
bacteriology
Figure 4: Primary identification of some Gram-negative bacteria of veterinary importance
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Applied Veterinary Bacteriology and Mycology: Introduction  Chapter 5: Identification systems used in diagnostic
bacteriology
Table 1: Selection of commercial biochemical tests for the Gram-negative bacteria, according to microscopic
morphology, colony appearance, catalase and oxidase tests
Shape
Short & long rods
Colony
Colonies
Morphology
Large on BTA
Cat.
+
Oxi.
-
Biochemical tests
Enterobacteriaceae, API
20E
API 10S
Enterobacteriaceae
Short & long rods
Yellow on MacConkey
+
-
API 20E
API 10S
Enterobacteriaceae
Short & long rods
Black on XLD
+
-
Salmonella
API 20E
API 10S
Pseudomonas
Short & long rods
(beaded)
Large or fine,
MacConkey positive.
+
Diplococci
Large or fine
+
D
API 20NE
Microbact 12A & 12B
Pseudomonas
D
API 20NE
Microbact 12A & 12B
Pseudomonas
Diplococcobacilli
Large or fine
+
D
API 20NE
Microbact 12A & 12B
Pleomorphic
Fine
D
D
Haemophilus, Pasteurella,
Actinobacillus
API 20NE
Microbact 12A & 12B
Fowl Pasteurella
Pleomorphic
from chickens or
ostriches
D
D
Pleomorphic
Large and sometimes
beta-haemolytic
D
+
Fish bacteria (with salt
broth)
Pleomorphic
Large or fine, growth at
25°C. Found in fish,
tortoises & snakes.
D
D
Fish bacteria (with salt
broth)
Rods
Flat & spreading at
25°C. Found in fish
D
D
Myxobacteria
Pleomorphic, bent
or spiral-shaped
Fine. Growth better with
added CO2
D
D
Campylobacter
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API20NE (identification is
not listed, you must use
own tables).
Applied Veterinary Bacteriology and Mycology: Introduction  Chapter 5: Identification systems used in diagnostic
bacteriology
Table 2: Selection of biochemical tests for the Gram-positive bacteria, according to microscopic morphology,
colony appearance, catalase and oxidase tests
Shape
Colony
Colonies
Morphology
Cat.
Oxi.
Biochemical tests
Short & long rods,
sometimes with spores
Usually large
+
D
Short & long rods
Fine
D
D
Short & long rods,
sometimes with spores
AnO2,
-
D
Fine rods
Fine alpha-haemolytic
and from ostrich
stomach.
-
D
Pleomorphic
Large or fine
+
D
Pleomorphic and
sometimes branched
Fine white betahaemolytic
-
D
CAMP test
Cocci or tetrads
Large, usually white,
sometimes yellow and
beta-haemolytic.
+
D
Staphylococcus,
Micrococcus
Cocci or coccobacilli in
broth can occur as
chains)
Fine alpha- or beta-, or
gamma-haemolytic
-
-
Streptococcus
Large oval to round cells
with budding.
Large dull, sticky
colonies. Rarely slimy
+
D
Yeasts
Branched large hyphae,
sometimes with fruiting
bodies
Large, chalky, fluffy or
leathery.
D
D
Fungus
Branched filamentous
bacteria, sometimes
breaks up into cocci.
Usually grows into the
agar.
+
D
Nocardia,
Streptomyces,
Actinomyces
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Beta-haemolytic
Bacillus
Gram-positive
CoryneAPI
Clostridium
API32A
Ostrich bacillus
Gram-positive
CoryneAPI
Applied Veterinary Bacteriology and Mycology: Introduction  Chapter 5: Identification systems used in diagnostic
bacteriology
Table 3: Recognition of the different bacterial genera by their Gram’s stain and colony morphology
Blood agar
Bacterium
Colony
Salmonella spp.
Greyish,round
and shiny
Gram’s stain
MacConkey agar
Haem
olysis
-
Growth
+
LF/NLF
NLF
Reactio
n
-
General comment
Shape
R
Pale colonies on MAC
agar. No smell (unlike
most other members of
the Enterobacteriaceae
Non-haemolytic but
otherwise similar to the
haemolytic strains.
Characteristic “coliform”
smell.
2-3 mm
Non haemolytic E.
coli
Yersinia spp.
Greyish, round
and shiny
-
+
LF
-
R
-
+
NLF
-
R
2-3 mm
Greyish, round
and shiny
2-3 mm
Serratia
marcenscens and
S. rubidea
Klebsiella spp.
Red/orange
convex, round
and shiny.
-
+
NLF
-
R
Produces red pigment
(prodigosin). Some
strains are white at 37°C
(no pigment)
-
+
LF
-
R
Colonies tend to be large,
mucoid and light pink on
MAC agar. Non-motile.
-
+
LF
-
R
Very similar to colonies of
Klebsiella spp. Motile.
2-3mm
Grey, mucoid
colonies that
coalesce.
2-4mm
Enterobacter spp.
Grey, mucoid
colonies that
coalesce.
2-4mm
Proteus spp.
Grey, swarming
growth over the
agar.
Pseudomonas
spp. other than
Grey or
yellowish-green,
flat and
spreading.
P. aeruginosa
-
+
NLF
-
R
Characteristic swarming
on non-selective agar
which can be in waves.
Turns BTA brown. Very
foul smell. Colonies pale
and discreet on MAC, but
edges may be irregular.
-
+
NLF
-
R
Some may produce the
yellowish-green pigment,
pyoverdin.
-
-
+
R
2.5-4mm
Corynebacterium
renale
Brucella spp.
Grey-white,
round and moist
0.5-1 mm
Urease positive
Translucent,
convex and
round
-
-
-
R(C)
Some brucellae require
10% CO2 for growth.
Colonies not visible until 23 days after incubation.
MZN positive.
-
-
-
R
Requires 2-3 days for
growth under reduced
0.5 mm
Campylobacter
10 | P a g e
Colonies older than 48
hours become drier.
Small, “dewdrop”, round and
Applied Veterinary Bacteriology and Mycology: Introduction  Chapter 5: Identification systems used in diagnostic
bacteriology
fetus
opaque
curved
oxygen tension. Curved
rods, if in pairs they have
a "seagull" appearance
-
R
Usually urease positive
-
R
Colonies appear pinkish
on BTA. Characteristic
sweetish smell. Indole
positive.
-
R
Colonies very small at 24
hours but become much
larger later. Unreactive
bacterium.
+
R(C)
The colour becomes more
definite with time. Mucoid
colonies tend to merge.
+
C
Colonies are similar to
coagulase +
staphylococci, but always
white and non-haemolytic.
+
C
Colonies are usually
pigmented.
C
Human and bovine strains
are bright yellow. Hold
plate to bright light to see
characteristic double
haemolysis. Are catalase
positive.
+
R
Aerobic with double or
target haemolysis.
Colonies tend to have
irregular edges.
0.5 mm
Actinobacillus spp.
Grey to
translucent,
round and shiny
-
V
-
-
V
0.5-1mm
Pasteurella
multocida
Translucent,
smooth, round
and shiny
1-2 mm
Bordetella
bronchiseptica
Rhodococcus equi
Staphylococcus
epidermidis
(coagulase
negative)
Micrococcus spp.
Small geyishwhite and round
-
+
-
-
NLF
0.5-2 mm
Salmon pink and
mucoid.
Colonies
coalesce
White, shiny,
round and
convex
-
+
-
-
LF
Most
2-3 mm
White, yellow,
tan or pink.
Round, convex
and shiny.
2-3 mm
S. aureus or
S.
pseudintermedius
Clostridium
perfringens
White or yellow,
smooth rounsd
and shiny
+
target
+
LF
+
2-3 mm
Grey, flat and
often irregular
edge
+
target
-
2-3 mm
Grey, smooth
and round
+
+
LF
-
R
Characteristic “coliform”
smell.
+
+
v
-
R
2-3 mm
Foul smell, different to that
of E. coli. Good growth on
MAC. Oxidase positive.
P. aeruginosa
Blue-green, flat,
round. Some
have a metallic
sheen.
2.5-4
mm
+
+
NLF
-
R
Amount of pyocyanin
(blue-green pigment)
varies between strains.
Characteristic fruity-musty
smell.
Bacillus spp.
Grey, dry,
granular with
irregular edges
±
v
v
+
Haemolytic E. coli
2-3 mm
Aeromonas
hydrophila
11 | P a g e
Grey, flat, round
and shiny
R
spores
Usually large, dry rhizoid
colonies. Motile except for
B. anthracis. B. anthracis
Applied Veterinary Bacteriology and Mycology: Introduction  Chapter 5: Identification systems used in diagnostic
bacteriology
3-5 mm
C.
pseudotuberculosi
s
Trueperella
pyogenes
Beta-haemolytic
streptococci
Opaque, dry,
crumbling
non-haemolytic.
V
R
The cells tend to be less
pleomorphic than A.
pyogenes.
R(C)
Hazy haemolysis along
streak lines, even before
colonies are seen. Very
pleomorphic in the Gram
stained smear. Catalase
negative.
C
The size of the clear zone
of beta-haemolysis varies
with species. Catalase
negative.
R(C)
Colonies resemble that of
beta-haemolytic
streptococci. Young
colonies may yield coccoid
cells. Catalase positive.
-
R(C)
Colonies very similar to
the above two bacteria.
Gram-negative and cells in
pairs as rods or fat cocci.
LF
-
R
Some strains are
haemolytic only under the
colonies.
LF
+
C
Red pin-point colonies on
MAC.
+
C
-
+
0.5-1 mm
Translucent, pinpoint
0.5 mm
Translucent,
glistening and
round
+
hazy
+
-
+
-
+
0.5-1 mm
Listeria
monocytogenes
Moraxella bovis
White, smooth
and round
White, smooth
and round
0.5-1 mm
Pasteurella
haemolytica
+
-
+
0.5-1 mm
White/grey,
smooth and
round
+
(-)
+
-
+
pin-point
0.5-1.5 mm
Enterococcus
faecalis
Alpha-haemolytic
streptococci
Erysipelothrix
rhusiopathiae
White, smooth
and round
0.5-1 mm
White, smooth
and round
0.5-1 mm
White, smooth
and round.
Some strains
rough
0.5-1.5 mm
+
+
alpha
pin-point
+
alpha
-
+
alpha
48
hours
-
+
R
Alpha-haemolysis under
the colonies only at 48
hours. Rough, dry colonies
especially from chronic
forms of the disease.
Catalase negative.
+ = positive; - = negative; v= variable reaction; LF = lactose fermenter; NLF = non-lactose fermenter; MAC = MacConkey agar; BTA
= blood agar; MZN = modified Ziel-Neelsen; C = cocci, R = rods
Size of colonies can be variable. Size is given and described after 48 hours of incubation, as even with fast-growing bacteria the
colonies are more characteristic at this stage.
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Applied Veterinary Bacteriology and Mycology: Introduction  Chapter 5: Identification systems used in diagnostic
bacteriology
Table 4: Biochemical tests recommended for the identification of Gram-negative bacteria
Standard tests
Fowl Pasteurellas
Haemophylis, Pasteurella,
Actinobacillus
Pseudomonas
Lactose
3 ml MRP broth
3 ml MRP broth
42°C BTA
Dulcitol
Urea
Urea
Nitrate
Sucrose
Glucose
Glucose
Urea
Maltose
Mannose
Mannose
O/F without oil
Inositol
Trehalose
Sucrose
Glucose
Phenylalanine
Mannitol
Lactose
Citrate
Urea
Inositol
Maltose
Aesculin
Malonate
Aesculin
Trehalose
Gershman’s
Mannitol
3 ml MRP broth
Inositol
Brucella glucose with H2S
Sorbitol
Horse serum
Citrate
TSI
3 ml MRP broth
BTA with staph streak (CAMP
test)
Gershman’s
If Salmonella suspected
add:
Salicin
If it cannot be identified with
these tests add:
Aesculin
BTA for ONPG & Porphyrin
Ornithine
Salicin
Arabinose
Sorbitol
Xylose
Lysine & control
Rhamnose
Galactose
Sucrose
Lactose
Maltose
Adonitol
Dulcitol
Sorbitol
Salicin
Table 5: Identification of some Gram-negative bacteria
Campylobacter
Fish bacteria
Myxobacteria
AnO2 BTA
Gershman’s
Maltose
BTA in Campylobacter gas
0% Sout broth
Sucrose
At 25°C:
0% Salt broth
BTA in candlejar @ 42°C
5% Salt broth
Mannitol
2% Salt broth
BTA in candle jar at 25°C.
7% Salt broth
Trehalose
Gelatin
TSI
3 ml MRP broth
Glycerol
Aesculin
Sodium hippurate
Methylred
Sorbitol
Glucose
Nitrate
Citrate
Salicin
O & F without oil
Bruc. glucose & H2S
Arginine
5% BTA
Casein agar
Selenite
Lysine
37°C BTA-ONPG
DNAse agar
Nutrient broth
Ornithine
0129 BTA disc
Nitrate
25°C for motility
Urea
Starch
Bruc. gluc. & H2S
Citrate
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Applied Veterinary Bacteriology and Mycology: Introduction  Chapter 5: Identification systems used in diagnostic
bacteriology
Gelatine
Vibrio agar
Gershman’s
Glucose
Starch
Arabinose
AnO2 @ 25°C
AnO2 Cytophaga agar
Eggyolk
Phosphatase agar
Table 6: Biochemical tests recommended for the isolation of Gram-positive bacteria
Gram-positive
Staphylococcus
Streptococcus
Bacillus
Actinomyces/
Corynebacterium
O & F (x2)
Nitrate
Lactose
Egg yolk
Xylose
Nitrate
Glucose
Mannitol
Anaerobic BTA
Galactose
Glucose
Maltose
Raffinose
Citrate
Mannose
Urea
O&F
Salicin
3ml MRP broth
Lactose
Aesculin
DNAse
Sorbitol
Nitrate
Sucrose
Trehalose
Glucose
Trehalose
Inulin
Horse serum
Raffinose
Horse serum
Brucella glucose &
H2S
Sodium Hippurate
For identification of
coagulase negative
staphs:
Anaerobic BTA
Thioglycollate
Starch
Methylred
DNAse
Xylose
Casein
Arabinose
If identification not
possible with these
tests, then add:
Raffinose
Ornithine
Maltose
If identification not
possible with these
tests, then add:
Lysine
Mannitol
Arabinose
Arginine
Mannose
Fructose
Mannitol
Trehalose
Galactose
Salicin
lactose
Xylose
Adonitol
Galactose
Arginine
Arabinose
Nitrate
Glycerol
Raffinose
Arginine
Dolcitol
Rhamnose
Urea
Lactose milk
Xylose
45°C BTA
Bruc. glucose & H2S
Trehalose
Phosphatase agar
Adonitol
Aesculin
DNAse agar
Starch agar
Sorbitol
BTA & nalidixic acid
disk
Phosphatase agar
Salicin
Sucrose
anaerobic BTA
14 | P a g e
Aesculin
Salicin
Methylene blue
Phosphatase agar
Sodium hippurate
Inositol
45°C BTA
CAMP test
Streptex
Applied Veterinary Bacteriology and Mycology: Introduction  Chapter 5: Identification systems used in diagnostic
bacteriology
Table 7: Identification of some Gram-positive bacteria
Micrococcus
Listeria
Anthrax
Nocardia
Megabacteria
Anaerobic BTA
Galactose
Salicin
45°C BTA
Glucose
Lactose
Methylin blue milk
Casein agar
Glycerol
Mannitol
Gelatin
Galactose
Aesculin
Rhamnose
Lactose milk
Rhamnose
Nitrate
Sucrose
Penicillin disk
Lactose
Urea
Xylose
3 ml MRP broth
Sucrose
Phosphatase agar
Gershman’s
Raffinose
CAMP test with
Rhodococcus equi & S.
aureus.
Gamma-phage
Salicin
Brucella glucose
slant & H2S
Mannitol
Aesculin
Anaerobic BTA
MAC
Table 8: Summary of selected, commonly used biochemical tests for the identification of bacteria
Test
Citrate
utilization
Decarboxyla
se (lysine
and
ornithine)
and
dehydrolase
(arginine
tests)
Gelatin
liquefaction
Hippurate
15 | P a g e
Medium
Simmons citrate.
Inoculate solid agar
slant.
Incubation
Product tested for and
reagent used
Up to 7 days
at 37°C
Ability to use citrate as the
sole carbon source
Up to 4 days
at 37°C
Arginine and ornithine to
putrescine. Lysine to
cadaverine. Products are
alkaline. Bromocresol
purple used as the
indicator.
Result
Negative
Positive
Green pH 6.9
Blue
(E. coli)
(Salmonella)
Yellow (acid),
glucose only
attacked.
Purple (alkaline)
Broth base & 0,1%
glucose with:
0.5% L-arginine or
0.5% L-lysine or
0.5% L-ornithine
OR
Lysine iron agar
(Salmonella)
(Proteus)
MIO medium
(ornithine)
Stab inoculation of
nutrient gelatin.
22°C for 30
days or 37°C
for 14 days
Proteolytic activity
(gelatinases) and gelatin
liquefied.
No liquefaction
Liquefaction (not
solid at 4°C)
Charcoal gelatin
discs, placed in a
broth
37°C for 14
days
As above. Charcoal
particles are released
when the gelatin is
liquefied
No change
Free charcoal
particles.
X-ray film method.
Small strip in heavy
inoculum of bacteria
in trypticase soy
broth
37°C for 48
hours
Sodium hippurate
24 hours at
As above.
Gelatin layer on X-ray film
strip
Centrifuge test. Add 0.2ml
No change
(Enterobacter
spp.)
No precipitate
Removal of
gelatin layer
leaving blue
plastic film.
(Serratia
marcescens)
Permanent
Applied Veterinary Bacteriology and Mycology: Introduction  Chapter 5: Identification systems used in diagnostic
bacteriology
hydrolysis
37°C with an
uninoculated
control
ferric chloride reagent to
0.8ml of the supernatant
Iron salts in media
e.g. TSI and SIM
(least sensitive
method)
16 hours at
37°C
Hydrogen sulphide gas
production
Lead acetate paper
strip (most sensitive
method). Strip
suspended over
trypticase soy broth
or serum glucose
agar slants
(Brucella)
35°C for up to
7 days.
Change lead
acetate strip
daily.
Hydrogen sulphide gas
production
Tryptone water
1-2 days at
30°C
Trytophan splits to Indole.
Add 0,5ml Kovac’s
reagent to medium and
shake. Read in 1 minute.
Reagent layer:
yellow
Reagent layer:
deep red
(Salmonella)
(E. coli)
Kovac’s reagent (0,2ml) to
tube. Stand for 10 minutes
No change in
reagent colour
Oxalic acid test paper
suspended over medium
No change in
test strip
Spot test for indole
Use bacterial
colonies on
either blood
or nutrient
agar incubated
24-48 hours at
37°C
Filter paper saturated with
Kovac’s reagent. Rub
colony over filter paper
with a glass rod.
No reaction (P.
haemolytica)
Malonate
utilization
Malonate broth
(0,3% sodium
malonate)
24 hours at
37°C
Utilization of malonate as
a sole carbon source.
Bromothymol blue
indicator.
No change
(most
Salmonella spp.)
Growth and a
deep blue colour
Methyl red
(MR) test
Glucose phosphate
peptone water (5 ml)
MR-VP broth
2 days at 37°C
or 3-5 days at
30°C
Yellowish (E.
cloacae)
Red (acid)
Colourless. Add
a pinch of zinc
dust.
Red (NO3 to
NO2)
Hydrogen
sulphide
Indole test
10g in 1 litre of brain
heart infusion broth.
SIM medium in
tubes
Nitrate broth (0,1%
KNO3: 5ml)
16 | P a g e
(A. ligniersii,
No change
(E. coli)
No change to
lead acetate
strip
Blackening of
medium
(Salmonella)
Blackening of
the lead acetate
strip.
(Brucella spp.)
Reagent dark
red
As above
Small molecular weight
acids such as formic and
acetic.
Add 5 drops of MR
reagent to medium
24 hours at
37°C (rarely
up to 5 days)
Nitrite (NO2)
37°C and
examine at 4
Pink colour at
lower end of
paper
Blue colour on
streak within 30
seconds (P.
multocida)
(S. arizonae)
(E. coli)
Nitrogen gas (N2)
Add 5 drops of reagents A*
and B#. Shake and wait 12 minutes.
KNO3 (40%) on filter
paper. Place on
blood agar and stab
precipitate
S. agalactiae)
Nitrate (NO3)
Nitrate
reduction
(A. equuli, S.
pyogenes)
Nitrate reduction
NO3 converted
to NO2
(negative: red)
Colourless
(positive)
No reaction or
very narrow
zone of
Wide zone of
browning of
medium
Applied Veterinary Bacteriology and Mycology: Introduction  Chapter 5: Identification systems used in diagnostic
bacteriology
inoculate test
bacterium 20mm
from paper strip.
Use a heavy
inoculum and E. coli
as a positive control
and 24 hours
ONPG test
Peptone water &
0,15% onitrophenoly-beta-Dgalactopyranoside
24 hours at
37°C
Phenylalanin
e deaminase
test
Phenylalanine
medium (BBL) 0,2%
DL-phenylalanine
slant agar)
Inoculate
heavily. 35°C
for 4 or 18-24
hours
Phosphatas
e test
Nutrient agar &
0,01%
phenolpthalein
diphosphate
18-24 hours at
37°C
Urease tests
Christensen media:
either an agar slant
or broth base
containing 2% urea
Up to 24 hours
at 37°C
5ml glucose
phosphate peptone
water. (MR-VP
broth)
between colony
and strip (E. coli)
Colourless
(Salmonella
spp.)
Yellow
Add 4-5 drops of aqueous
ferric chloride. Rotate and
read in 1-5 minutes.
Sufficient phosphatase to
split to phenol-phtalehin
diphosphate.
Hold colonies on the agar
over an open bottle of
ammonia
Urease: splits urea with
formation of ammonia
(alkaline).
As above
Yellowish
Unchanged
colonies
(coagulase
negative
staphylococci)
Colonies bright
pink (coagulase
positive
staphylococci)
Yellow
(Salmonella
spp.)
No change
Acetoin derived from
glucose.
3-5 days at
30°C
3ml of 5 & alphanaphthol
in absolute ethyl alcohol
and then 1ml of 40% KOH.
Shake and leave for 5
minutes.
(S. arizonae)
Green reaction
in slant.
(Proteus,
Morganella and
Providencia
spp.)
Phenyl-pyruvic acid
formed.
Phenol red indicator
Spot test. Moisten
filter paper with a
few drops of 10%
urea agar base
concentrate and rub
some culture onto
the filter paper with
a glass rod.
Voges
Proskauer
(VP) test
The enzyme betagalactosidase. Identifies
potential lactose
fermenters
browning around
medium
Red
(Proteus spp.)
Pink or red
streak within 2
minutes
Red
Colourless (E.
coli)
(most
Enterobacter
spp.)
TYPES OF IDENTIFICATION SYSTEMS
Biochemical profiles
In the diagnostic laboratory, biochemical profiles are most commonly used to identify a micro-organism,
either by using a manual or automated system. Biochemical profiles are determined by the reactions of
individual organisms with each of the substrates in the system. Most bacteria can be identified to species
17 | P a g e
Applied Veterinary Bacteriology and Mycology: Introduction  Chapter 5: Identification systems used in diagnostic
bacteriology
level by these tests, allowing a small laboratory to identify a wide range of bacteria. Systems used rely on
the following indicators:

pH-based reactions (many 15- to 24-hour identification systems). As a general rule, carbohydrate
utilization by micro-organisms results in acid production, while protein utilization or the release of
nitrogen-containing products results in an alkaline pH.

Enzyme profiles (many 4-hour systems). Enzyme profile tests are usually based on pre-formed
enzymes and require minimal microbial growth. When a colourless chromogen (colour) or fluorogen
(fluoresces in U-V light) is hydrolyzed by an appropriate enzyme, the chromogen or fluorogen is
released resulting in a visible reaction.

Carbon-source utilization. This method measures metabolic activity. Tetrazolium-labelled carbon
sources are colourless until electrons activated by metabolic activity are transferred to the dye,
creating a purple colour.

Visual detection of growth (yeast identification systems). Assimilation assays depend on the ability of
micro-organisms to grow in the presence of a substrate. Visual detection of growth is a positive
result.
Gas-liquid chromatography
Chromatography has been used to separate various bacterial components such as cellular fatty acids, in
order to isolate, characterize and identify bacteria. As this is a technically difficult technique, requiring
specialized instrumentation, and limited to the identification of a small group of bacteria, it has only been
used in reference laboratories and for research purposes. This technique has been most commonly used
to identify obligate anaerobes, by identifying short chain fatty acids that are produced by glucose
fermentation. The mycolic acids of Nocardia spp., Rhodococcus spp., Corynebacterium spp. and
mycobacteria can be analysed in this fashion.
Recently, these methods have been standardized and automated (Microbial identification systems,
Newark, Del.), making them more easily available. They allow objective analysis, less labour input and
relatively low cost per sample. They are also able to identify both asaccharolytic organisms and those
with enzymic profiles that are not distinctive. Disadvantages are the large capital expense required for
initial equipment purchase, non-acceptance of alternative identification strategies, and the size of the
library of known organisms that is required. As these tests are developed further, they may in the future
become more accessible to diagnostic laboratories.
Immunoassays
Immunoassays are commonly used in diagnostic laboratories to both identify the micro-organism
and to detect antibodies. Tests that are most commonly used to identify micro-organisms include
agglutination tests e.g. Streptex, serotyping of E. coli and Salmonella sp., fluorescent antibody
tests i.e. for identification of histiotoxic clostridia and precipitation tests e.g. identification of
galactan produced by Mycoplasma mycoides subsp. mycoides.
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Applied Veterinary Bacteriology and Mycology: Introduction  Chapter 5: Identification systems used in diagnostic
bacteriology
Molecular methods
At present, DNA probes and nucleic acid amplification techniques are most useful for the
characterisation of micro-organisms for which culture and serologic methods are difficult, expensive
or unavailable. Amplified and non-amplified DNA probes are used for the identification of microorganisms in samples (in situ hybridization); for culture confirmation of slow-growing bacteria such
as the mycobacteria and pathogenic dimorphic fungi; and the identification of enterotoxin producing
E. coli. Amplification of microbial DNA is the most sensitive technique, making it suitable for the
identification of micro-organisms in samples.
As these techniques are expensive, detect both
viable and non-viable micro-organisms and are not available for all micro-organisms, they should
only replace conventional techniques where they would lead to a quicker patient isolation, a better
choice of patient therapy, and a decrease in time when the patient is infectious.
Rapid manual and automated methods
Commercial systems both automated and in kit form, are available for the identification of commonly
isolated species. These tests have enabled laboratories to more rapidly report results which are
often more accurate. They also are less labour-intensive as no complex media preparation is
required. These kits/systems, however, do not replace a good microbiologist, who is able to
determine the possibility of a false result e.g. Bacillus species often resembles Gram-negative
bacteria and may be misidentified by an automated system as members of the family
Enterobacteriacae. Errors in commercial systems include:

Human error: wrong isolate, misidentified module.

System inadequacies: insufficient data in database, inability to interpret atypical reactions.


Incorrect identifications.
Results for a given test may not be equally valid among all identification systems e.g.
Christensen’s urea agar is more sensitive in the detection of urease than that of the API-system.


Batch variation.
Laboratory practice variation.
Table 9: Summary of selected identification systems
System
Manufacturer
Organisms identified
Storage
temp. (°)
No. of
tests
Incubation
Automa
ted
ANI
BioMérieux
Vitek
Anaerobes
2-8
28
4 h: aerobic
Yes
API 20A
BioMérieux
Vitek
Anaerobes
2-8
21
24 h: anaerobic
No
API 20C
BioMérieux
Vitek
Yeasts
2-8
20
24 – 48h
No
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Applied Veterinary Bacteriology and Mycology: Introduction  Chapter 5: Identification systems used in diagnostic
bacteriology
API 20E
BioMérieux
Vitek
Enterobacterieae &
nonfermenting Gramnegative bacteria
2-8
21
4 – 24h
No
API 20 Strep
BioMérieux
Vitek
Streptococci & enterococci
2-8
20
4h: aerobic
No
API An-IDENT
BioMérieux
Vitek
Anaerobes
2-8
21
24 h
No
API Corne
BioMérieux
Vitek
Corynebacteria
2-8
20
24 – 48h
No
API NFT
(Rapid NFT)
BioMérieux
Vitek
Gram-negative nonEnterobacterieae
2-8
20
2–4h
No
API Rapid 20E
BioMérieux
Vitek
Enterobacterieae
2-8
21
4h
No
API StaphIDENT
BioMérieux
Vitek
Staphylococci & micrococci
2-8
10
API Staph
(STAPH-Trac)
BioMérieux
Vitek
Staphylococci & micrococci
2-8
20
24 h
No
Bacterial
Identification
Panel
Alamar
Enterobacterieae &
nonfermenting Gramnegative bacteria
RT
26
18 – 20h
Reader
only
Crystal E/NF
BDMS
Enterobacterieae &
nonfermenting Gramnegative bacteria
2-8
30
18 – 20 h
No
Crystal Rapid
Stool/Enteric
BDMS
Gram-negative stool
pathogens
2-8
30
18 – 20 h
No
Enterotube II
BDMS
Enterobacteriea
2-8
15
18 – 24 h
No
EPS (Enteric
Pathogen
Screen)
BioMérieux
Vitek
Edwardsiella, Salmonella,
Shigella & Yersinia
2-8
10
4 – 8h
No
ES MicroPlate
Biolog
Aerobic gram-negative
bacteria
2-8
95
4 – 24 h
No
Fox Dual GNI
Micro-Media
Systems
Enterobacterieae &
nonfermenting Gramnegative bacteria
-20 - 40
33
4 – 24 h
Yes
GM Microplate
Biolog
Aerobic Gram-negative
bacteria
2-8
95
4 – 13 h
Reader
only
GNI
BioMérieux
Vitek
Enterobacterieae &
nonfermenting Gramnegative bacteria
2-8
29
4 – 24 h
Yes
GP Microplate
Biolog
Most Gram-positive cocci &
bacilli
2-8
95
4 – 15 h
Reader
only
GPI
BioMérieux
Vitek
Gram-positive cocci & bacilli
2 – 8 - 20
29
16 – 20 h
Yes
ID Tri-Panel
Difco/Pasco
Gram-negative & Grampositive bacteria
MicroID
Organon
Teknika
Enterobacteriea
Minitek
BDMS
Anaerobes,
Enterobacterieae, Gram-
20 | P a g e
No
30
Reader
only
2-8
15
No
2 - 25
4 – 21
(dependi
Enterobacteriacc
eae & Neisseria
No
Applied Veterinary Bacteriology and Mycology: Introduction  Chapter 5: Identification systems used in diagnostic
bacteriology
positive bacteria, Neisseria
spp. nonfermenters &
yeasts.
ng on
need)
spp. For up to
72 h for yeasts
THE NORMAL FLORA
It is important that the veterinary microbiologist is familiar with the kinds of organisms encountered
normally in and on animals.
Such knowledge is necessary in the interpretation of the results of
microbiological examinations. The so-called normal flora consists of the wide variety of bacteria and fungi
that live in or on the normal animal without producing disease. Included in this flora are many potential
pathogens and opportunistic organisms. The term normal flora is a convenient concept, but it should be
kept in mind that the kinds and numbers of bacteria present vary greatly under different circumstances.
The intestinal flora of the young animal differs markedly from that of the older animal. The flora is also
influenced by geographic location, nutrition, and climate. The technical procedures are biased to recover
pathogenic organisms and thus frequently give a distorted idea of the kinds of numbers of bacteria
present. The normal flora of the domestic animals has not been studied in as detailed a fashion as that of
human beings.
What little information that is available, and firsthand experience in the diagnostic
laboratory, indicate a considerable similarity between the normal flora of humans and that of domestic
animals. Some of the bacteria that can be expected to occur normally in and on domestic animals are
tabulated below.
Mouth, nasopharynx
Micrococci (aerobic and anaerobic, pigmented and nonpigmented); Staphylococcus spp.; haemolytic and
non-haemolytic streptococci; Veillonella and other Gram-negative cocci; coliforms and Pasteurella spp.;
diphtheroids; pneumococci; yeasts, including Candida albicans; Haemophilus spp.
Jejunum, ileum
Only a small number of bacteria are present in this portion of the intestinal tract of animals.
Large intestine
Enteroccus spp.; E. coli; Klebsiella; Enterobacter; Pseudomonas spp.; Proteus spp.; staphylococci;
Cl. perfringens, Cl. septicum, and other clostridia; Gram-negative anaerobes; spirochetes;
lactobacilli.
Trachea, bronchi, lungs
Few, if any, bacteria and fungi reside in these structures; possibly very low numbers of Pasteurella
spp. may be present.
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Applied Veterinary Bacteriology and Mycology: Introduction  Chapter 5: Identification systems used in diagnostic
bacteriology
Vulva, prepuce
Vulva: diptheroids; micrococci; coliforms and Proteus spp.; enterococci; yeasts; Gram-negative
anaerobes.
Prepuce: the same kinds of organisms. A bull may be a carrier of C. foetus venerealis and a
stallion of certain serovars of Klebsiella pneumoniae and Taylorella equigenitalium.
Vagina
The numbers and kinds of bacteria vary with the reproductive cycle and age. The cervix and
anterior vagina of the healthy mare possesses few bacteria. Some of the organisms recovered
from the vagina are coliforms and Proteus spp.; certain salmonellas and klebsiellas; diphtheroids
and lactobacilli; mycoplasmas; yeasts and fungi.
Skin
Animals, by virtue of their habits and environment, frequently possess a large and varied bacterial
and fungal flora on their hair and skin. Stapylococcus epidermidis and S. aureus occur commonly,
as do other micrococci. Of the many other organisms isolated, it is not known which make up the
resident flora and which are “transients”.
Milk
Micrococci, staphylococci, non-haemolytic streptococci, mycoplasmas and diphtheroids including
Corynebacterium bovis are frequently shed from the apparently normal mammary gland.
OCCURRENCE OF PATHOGENS IN ANIMAL SPECIES
In Table 20, the organisms most frequently associated with infection in various organs and systems of the
more important animal species are listed.
Laboratory animal infections
Rats and Mice
Salmonella spp.; pyogenic streptococci; Bacillus piliformis; Pasteurella pneumotropica, P.
multocida; Corynebacterium kutscheri; Bordetella bronchiseptica; Streptobacillus moniliformis;
Streptococcus pneumoniae; mycoplasmas; Yersinia pseudotuberculosis.
Guinea Pigs
22 | P a g e
Applied Veterinary Bacteriology and Mycology: Introduction  Chapter 5: Identification systems used in diagnostic
bacteriology
Salmonella spp.; Bordetella bronchiseptica; Streptococcus pneumoniae, pyogenic streptococci;
Klebsiella pneumoniae; Yersinia pseudotuberculosis; Streptobacillus moniliformis.
Rabbits
Salmonella spp.; Pasteurella multocida; Bordetella bronchiseptica; Yersinia pseudotuberculosis, Y.
enterocolitica; pyogenic streptococci; Haemophilus spp.; Clostridium piliforme, Fusobacterium
necrophorum; Treponema cuniculi.
Table 10: Potentially pathogenic bacteria of different organ systems in different animals
Bovine
P. multocida,
The respiratory
system
Ovine
Porcine
M. haemolytica,
P. multocida,
P. trehalosi
M. haemolytica;
M. haemolytica; A. pyogenes;
P. multocida;
T (A). pyogenes;
B. bronchiseptica; H. somni; Actino.
actinoides (rare); mycoplasmas.,
including M. mycoides mycoides SC.
C. pseudotuberculosis
T (A). pyogenes; mycoplasmas;
H. parasuis, A.suis,
A. pleuropneumoniae
M. hypneumonia
chlamydia
M. hyorhinis
Streptococci;
Mastitis
S. aureus, S. epidermidis; Micrococcus
spp.; Strep. qalactiae, Strep.
dysqalactiae, Strep. uberis; C. bovis,
A.pyogenes; E. coli; Ps.aeruginosa;
M. haemolytica, P. multocida; Klebsiella
spp.; other Gram-negative organisms;
Mycoplasma bovis, yeasts
S. aureus; M. haemolytica, P.
multocida; S. agalactiae, S.
dysgalactiae, Strep. uberis; A.
pyogenes;
S. aureus;
C. pseudotuberculosis;
Histophilus ovis, mycobacteria;
mycoplasmas.
A. lignieressii;
F. necrophorum;
Actinomyces bovis;
A. pyogenes,
mycobacteria;
coliforms.
E. coli;
Escherichia coli; Salmonellas;
Cl. perfringens types B and C;
The gastrointestinal tract
M. paratuberculosis.
E. coli; Cl. perfringens type A
(red gut), type B (lamb
dysentery), type C (struck) type
D (pulpy kidney), type E;
M. paratuberculosis; Salmonella
Salmonella (especially
S. choleraesuis);
B. hyodysenteriae;
B. pilisicoli
Lawsonia
intracellularis;
Cl. perfringens type C.
Genital infections.
Streptococci,
staphylococci,
enteric bacteria
and Pseudomonas
aeruginosa are
commonly
associated with
genital infection in
all species
23 | P a g e
Campylobacter fetus subspp. fetus and
intestinalis; Brucella abortus; chlamydia;
Brucella ovis,
M. bovigenitalium;
C. fetus subsp. intestinalis;
L. monocytogenes;
L. monocytogenes; Histophilus
ovis (Haemophilus somnus),
Pasteurella spp., A. seminis;
T. pyogenes.
B. melitensis;
C. pseudotuberculosis;
Chlamydophila. abortus
Brucella suis;
Escherichia coli,
mycobacteria;
Ps. aeruginosa;
T. pyogenes,
multocida;
pyogenic streptococci
Applied Veterinary Bacteriology and Mycology: Introduction  Chapter 5: Identification systems used in diagnostic
bacteriology
Abscesses, ulcers
and infections of
the skin
Streptococci and
staphylococci are
the most common
causes.
A. pyogenes, A. bovis;
D. congolensis;
Actinobacillus lignieresii,
E. rhusiopathiae; S.
hyicus;
C. pseudotubercolosis,
Corynebacterium (?) sp. (Bolo
disease); Pseudomonas spp.
(fleece rot).
Group E streptococci
(jowl abscesses);
S. porcinus;
Sporothrix schenckii;
D. congolensis.
T. verrucosum
T. pyogenes. M. avium
E. coli;
The central
nervous system
The eyes
Joints
E. coli; Listeria monocytogenes;
Histophilus somni; streptococci;
P. multocida, Pasteurella strains EF4
group; C. psittaci; Staphylococcus
aureus.
E. coli; pyogenic and fecal
streptococci; E.rhusiopathiae;
Haemophilus agni; chlamydia;
Actinomyces pyogenes; Staphylococcus
aureus; chlamydia;
Strep. dysgalactiae Mycoplasma
agalactiae;
mycoplasmas; Haemophilus somnus.
A. pyogenes;F. necrophorum;
Histophilus ovis.
Str. canis; Nocardia
asteroides;
mycoplasmas;
Cryptococcus
neoformans;
Blastomyces
dermatitidis;
Actinomyces
viscosus
Staph. intermedius
(puppies); Borrelia
canis; Spirillum spp.;
Campylobacter.
P. multocida;
Nocardia
asteroides;
Bord.
bronchiseptica;
chlamydophila;
Cryptococcus
neoformans.
24 | P a g e
A. pyogenes; various
pyogenic streptococci;
mycoplasmas; Staph.
aureus; Haemophilus
parasuis; Brucella
suis; E. coli;
Actinobacillus suis.
Str. zooepidemicus
A. paragallinarum
Strep. pneumoniae
P. multocida,
R. equi (foals);
G. anatis, P.
gallinarum;
A. equuli (foals);
P. multocida; Ps. mallei;
Klebsiella;
B. bronchiseptica;
Mycoplasma felis
C. neoformans; Asperqillus.
Salmonella,
Candida
albicans
(kittens).
Brucella canis, other
brucella species
(rare); Klebsiella;
Enterobacter;
Proteus spp.;
Candida albicans;
S. intermedius;
E. rhusiopathiae;
Strep. equi, Str. equisimilis;
Salmonella
Clostridium perfringens
Clostridium sordelli
Salmonella; R. equi (foals);
A. equuli (foals).
The mare’s cervix
S. aureus; various streptococci;
Klebsiella; P. aeruginosa;
various fungi; R. equi; Candida
albicans; Enterobacter; E. coli;
A. equuli; T. equigenitalis
P. aeruginosa;
mycoplasmas.
The skin
P. multocida;
streptococci
E. coli; pyogenic streptococci;
Salmonella;
Salmonella; possibly
other enteric
bacteria;
Genital tract
L. monocytogenes;
P. haemolytica.
Moraxella ovis; Moraxella spp.;
mycoplasma; chlamydia.
P. multocida;
Klebsiella,
GIT
E. coli;
Staph. aureus;
Moraxella bovis; Branhamella ovis;
chlamydia.
Bordetella
bronchiseptica;
Respiratory
system
L. monocytogenes;
Pasteurella
Actinobacillus equuli; Klebsiella;
O. rhinotracheale ;
various mycoplasmas;
A. fumigatus.
Applied Veterinary Bacteriology and Mycology: Introduction  Chapter 5: Identification systems used in diagnostic
bacteriology
pyogenic
streptococci;
Nocardia asteroides;
Blastomyces
dermatitidis;
S. schenkii;
Actinomyces
viscosus; fungal
dermatophytes.
multocida,
N. asteroides;
fungal
dermatophytes.
Rhodococcus equi; Salmonella
abortus-equi; Candida albicans;
pyogenic streptococci; E. coli;
P. aeruginosa
P. aeruginosa;
Candida albicans
S. aureus;
P. aeruginosa;
various streptococci;
Canine otitis
externa
C. albicans; Proteus
spp.
Malassezia
pachydermatis;
C. pseudotuberculosis (chest
abscesses); Histoplasma
farciminosum; Sporothrix
schenckii; fungal dermatophytes
C. perfringens.
L. monocytogenes;
Strep. equi;
CNS
C. neoformans
Staph. aureus; Staph. epidermidis;
The eyes
P. aeruginosa; Cl. perfringens; Candida
albicans; Cryptococcus neoformans,
chlamydia; mycoplasmas; Moraxella
spp.,
C. neoformans
Staph. aureus; Staph.
epidermidis; Pseudomonas
aeruginosa; Clostridium
perfringens; Candida albicans;
chlamydia; mycoplasmas;
Moraxella spp., and Neisseria
flavus.
Staph. aureus.
Clostridium tetani
Strep. equi;
Strep. equisimilis;
Staph. aureus.
A. equuli;
Staphylococcus aureus; Pseudomonas
aeruginosa; various streptococci;
Joints
Staph. aureus;
pyogenic and faecal
streptococci; E. coli;
Rhodococcus equi;
Klebsiella; Salmonella
Candida albicans;
Malassezia pachydermatis; Proteus
spp.; Clostridium perfringens.
Proteus (usually mirabilis);
Urinary infections
25 | P a g e
P. aeruginosa; enterococci; E. coli;
Enterobacter; S. aureus; pyogenic and
fecal streptococci; Corynebacterium
pilosum (rare), C. cystitidis.
C. pilosum,
E. coli
C. cystitidis.