Applied Veterinary Bacteriology and Mycology: Identification of anaerobic bacteria
Applied Veterinary Bacteriology and Mycology:
Identification of anaerobic bacteria
Author: Dr. J.A. Picard
Licensed under a Creative Commons Attribution license.
TABLE OF CONTENTS
INTRODUCTION ............................................................................................................................................................3
Methods of growing anaerobes ..................................................................................................................................3
The anaerobic jar......................................................................................................................................................3
Anaerobic cabinets and glove boxes ........................................................................................................................4
Reducing agents.......................................................................................................................................................4
Media ........................................................................................................................................................................4
Preparation of media ................................................................................................................................................5
The Non-Clostridial Anaerobes ...................................................................................................................................5
Genus: Bacteroides ..................................................................................................................................................6
Bacteroides fragilis ........................................................................................................................................7
Prevotella melaninogenica (formerly Bacteroides) ........................................................................................7
Bacteroides corrodens ..................................................................................................................................8
Bacteroides oralis ..........................................................................................................................................8
Bacteroides capillosus ...................................................................................................................................8
Bacteroides praeacutus .................................................................................................................................9
Genus: Fusobacterium .............................................................................................................................................9
Fusobacterium necrophorum ........................................................................................................................9
Fusobacterium nucleatum ...........................................................................................................................10
Fusobacterium varium .................................................................................................................................10
Fusobacterium mortiferum ..........................................................................................................................11
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Applied Veterinary Bacteriology and Mycology: Identification of anaerobic bacteria
Genus: Dichelobacter .............................................................................................................................................11
Genus: Actinomyces ...............................................................................................................................................12
Actinomyces bovis.......................................................................................................................................12
Genus: Eubacterium and Propionibacterium ..........................................................................................................13
The clostridia ..............................................................................................................................................................13
Habitat and pathogenicity .......................................................................................................................................14
Pathogenicity ..........................................................................................................................................................15
Specimens ..............................................................................................................................................................15
Direct microscopy ...................................................................................................................................................15
Isolation procedures ...............................................................................................................................................16
Biochemical identification .......................................................................................................................................16
Serological identification .........................................................................................................................................16
Individual clostridia .................................................................................................................................................19
Clostridium chauvoei ...................................................................................................................................19
Clostridium novyi .........................................................................................................................................19
Clostridium septicum ...................................................................................................................................19
C. sordellii ...................................................................................................................................................20
Clostridium tetani.........................................................................................................................................21
Clostridium perfringens ...............................................................................................................................22
Clostridium botulinum ..................................................................................................................................22
REFERENCES .............................................................................................................................................................25
APPENDIX: MEDIA AND REAGENTS FOR THE ISOLATION OF ANAEROBIC BACTERIA ..................................26
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Applied Veterinary Bacteriology and Mycology: Identification of anaerobic bacteria
INTRODUCTION
Anaerobic bacteria are those bacteria that grow only in the absence of free oxygen but fail to multiply in the
presence of oxygen on the surface of nutritionally adequate solid media incubated in air or an atmosphere
containing 5-10% CO2.
Anaerobes comprise a wide range of bacteria that are separated into Gram-positive and Gram-negative cocci
and bacilli, and two major additional groups, namely whether or not they produce spores.
METHODS OF GROWING ANAEROBES
A variety of methods are available for the culture of anaerobic bacteria. Exclusion of oxygen from part of the
medium is the simplest method, and it is affected by growing of the organism within the culture medium as a
shake or fluid culture. When an oxygen-free or anaerobic atmosphere is required for obtaining surface growth of
anaerobes, anaerobic jars provide the method of choice. More sophisticated methods for surface culture of
anaerobes are the pre-reduced anaerobically sterilized roll-tube technique, and use of the anaerobic cabinet or
glove box. These complex techniques provide the most meticulous anaerobic conditions and are appropriately
used for the isolation and study of anaerobic species that are highly sensitive to oxygen.
The anaerobic jar
Anaerobic jars are cylindrical vessels made of metal, glass or plastic, flanged at the top to carry an air-tight lid
which is held firmly in place by a clamp. The lid carries on it’s under surface the room-temperature catalyst
capsule. The difference between the BTL and the Gaspak jars is that in the former the lid is provided with two
valves through which air can be withdrawn and an anaerobic gas mixture introduced. The lid of the standard
Gaspak jar is not vented, because the jar is specifically designed for use with a disposable hydrogen-carbon
dioxide generator.
Anaerobic gas for use in the BTL jar is obtained from a cylinder of the compressed gas. As hydrogen is highly
explosive a mixture containing 5% hydrogen, 10% carbon dioxide and 85% nitrogen is used. Carbon dioxide in a
10% concentration improves the growth of many anaerobes and no anaerobes are adversely affected by this
CO2 concentration. Nitrogen is an inert gas without any risk of explosion, even with the addition of 5% hydrogen
which renders the mixture stable.
The use of a catalyst in the anaerobic jar speeds the development of anaerobiosis. Palladium (0,5%) is
contained in wire gauze attached to the underside of the lid of the jar. The sachet contains pellets of alumina
coated with finely divided palladium. Certain gases such as chlorine, sulphur dioxide, carbon monoxide and
hydrogen sulphide, oil, the vapour of some organic solvents and strong acids poisons the catalyst. Inactivation
by hydrogen sulphide is especially relevant since many anaerobes produce large amounts of this gas. The
catalyst is also inactivated by moisture, which is plentiful in the anaerobic jar, but it is readily reactivated in a hot
air oven at 160°C for 2 hours.
An anaerobic indicator is included to check on the development of anaerobiosis. As the jar becomes anaerobic,
the indicator dye (methylene blue) becomes colourless. The most commonly used chemical methods for
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Applied Veterinary Bacteriology and Mycology: Identification of anaerobic bacteria
indicating anaerobiosis depend on the fact that when methylene blue is placed in an anaerobic environment it is
reduced from its coloured oxidised form to a colourless reduced leuco-compound. The fact that indicators are
necessary in anaerobic work draws attention to the importance of examining the anaerobic apparatus before use
to ensure that it is in proper working order.
Anaerobic cabinets and glove boxes
An anaerobic cabinet is an air-tight cabinet in which conventional bacteriological techniques may be done in an
oxygen-free atmosphere. This technique has the advantage that all the operations of isolating and sub- culturing
anaerobes are conducted in the absence of oxygen. In addition to the roll tube method it enables the use of Petri
dish plate cultures.
An anaerobic cabinet consists of an air-tight chamber which is provided with glove ports. It is commonly fitted
with an air-lock through which materials are transferred into and out of the chamber. The anaerobic glove box is
constructed in such a way that it can be almost completely evacuated of air with a vacuum pump since it is
collapsible. Air in the interchange is removed by vacuum and replaced with a mixture of 85%N2, 5%H2 and 10%
CO2 (the same mixture that is present in the glove box.)
In some cases the temperature in the glove box is maintained at 37°C. Other models have built-in incubators
and in some instances the plates have to be removed from the glove box in an anaerobic jar and incubated
in a separate incubator.
Reducing agents
Although the addition of reducing agents to fluid and shake cultures is not necessary, it is essential for the
satisfactory culture of more exacting anaerobes. The following are some of the reducing agents used:

Thioglycollic acid; 0,01-0,2%

Glucose; 0,5-1%

Ascorbic acid; 0,1%

Sodium sulphide; 0,025%


Metallic iron (iron filings etc. in fluid media.)
Meat (as in cooked meat medium)
Media
The use of pre-reduced anaerobically sterilized media is not generally necessary in the routine laboratory, since
almost all of the anaerobes that are pathogenic can be isolated and identified by conventional anaerobic
techniques. It is however recommended for anaerobic blood culture.
Plate cultures are inoculated in the usual way. When dealing with very strict or otherwise demanding anaerobes,
it is desirable to use freshly prepared plates, since during storage the medium takes up oxygen from the
atmosphere in sufficient amounts to prevent the growth of these, even though complete anaerobiosis has
apparently been obtained in the jar. Alternatively, but less satisfactory, it may be convenient to store a set of
uninoculated plates under anaerobic conditions, the whole jar being kept in the refrigerator or the plates kept in
an anaerobic cabinet.
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Applied Veterinary Bacteriology and Mycology: Identification of anaerobic bacteria
Fluid media are inoculated after dissolved oxygen has been driven off by steaming or heating in a boiling water
bath. When inoculating with a Pasteur pipette, the inoculum should be pipetted gently into the depths of the
medium, care being taken not to introduce any air bubbles.
Preparation of media
The methods of preparation of media for the culture of anaerobes are the same as for aerobic bacteriological
media. Useful basic media are Columbia agar and Tryptose blood agar. Basic broth media may be solidified by
the addition of 1,5% Bacteriological agar.
To these basic media, whether fluid or solid, may be added any required enrichment, selective or indicator
substance that is compatible with bacterial growth (Table 1). It is convenient to select one or two basic media
from which most other media can then be prepared.
The following may be added to the medium for enhancement of growth:

Glucose 0,5 - 1% (for all anaerobes)



Sodium bicarbonate 0,1 %( for organisms whose growth is encouraged by carbon dioxide.)
Bile 20% (B. fragilis etc.)
Vitamin K and haemin (P. melaninogenica)
Table 1: Selective agents for anaerobic bacteria
Organisms Selected For
Anaerobic cocci
Bacteroides and Fusobacterium.
Bacteroides
Some Bacteroides
Clostridium
Cl. perfringens
Fusobacterium
Fusobacterium and Veillonella.
Gram-positive non-sporing anaerobes
Agents
Neomycin
Sodium azide/Brilliant green
Oleandomycin
Neomycin
Vancomycin
Sodium azide/Bile(ox gall)
Sodium azide
Sorbic acid/Polymyxin B
Phenyl ethyl alcohol
Kanamycin
Sulphadiazine
Neomycin
Crystal violet / Streptomycin
Kanamycin
Neomycin
Polymyxin
Vancomycin
Amounts
(Per 100ml of medium)
10 mg.
30,0 mg/1,8 mg
50 mg
10 mg
750 g
20 mg./1,7 g.
20 mg.
0,12 g/2,0 mg
0,25 g
10 mg
10 mg
10 mg
1,0 mg/1,0 mg
7,5 mg
10mg.
1000 units
750 g
THE NON-CLOSTRIDIAL ANAEROBES
The non-clostridial anaerobic bacteria may be divided into four groups for diagnostic identification purposes. An
anaerobe is classified as either Gram-positive or Gram-negative and either a coccus or a bacillus (Refer to Table
2). Diseases caused by the non-clostridial anaerobes are listed in Table 3.
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Applied Veterinary Bacteriology and Mycology: Identification of anaerobic bacteria
Table 2: Non-clostridial anaerobes
Gram’s stain
Bacilli
Gram-Positive
Gram-Negative
Cocci
Actinomyces
Bifidobacterium
Eubacterium
Lactobacillus
Propionibacterium
Peptococcus
Peptostreptococcus
Bacteroides
Fusobacterium
Dichelobacter
Prevotella
Veillonella
Genus: Bacteroides
Bacteroides are Gram-negative, non-sporing bacilli. They are strict anaerobes and many species are obligate
parasites, not occurring outside the body except perhaps in sewage. They differ from species of Fusobacterium
as they are commonly actively saccharolytic and do not produce indole or threonine deaminase.
Twenty-two species of Bacteroides have been described, of which five are of animal origin, two have been
isolated from termites, and the remaining 15 are associated with man. Bacteroides fragilis and Prevotella
melaninogenica are the species most commonly implicated as pathogens. Other commonly encountered
species are B. corrodens, B. oralis, B. capillosis and B. praeacutus.
Useful characteristics used for identification of certain species of Bacteroides are:
1. B. fragilis is by far the most commonly encountered pathogen in animal infections and unlike most other
species it is resistant to penicillin G.
2. Prevotella melaninogenica is also commonly encountered in infections. It is the only Bacteroides which is
proteolytic, which produces black pigmented colonies on horse blood agar, and whose colonies show red
fluorescence in ultraviolet light.
3. B. corrodens is the only species that cause pitting of agar media, and is the only oxidase-positive
Bacteroides spp.
Table 3: Diseases caused by the non-spore-forming anaerobes
Organism
Host(s)
Disease
Actinomyces bovis
Cattle
Horses
Bovine actinomycosis ("lumpy jaw")
Fistulous withers
A. suis
Pigs
Pyelonephritis
A. viscosus
Dogs
Canine actinomycosis: Localised abscesses
-pyothorax
A. hordeovulneris
Dogs
Localised abscesses, pleuritis, peritonitis, septic
arthritis, visceral abscesses
A. israelii
Humans and rarely pigs and cattle
Human actinomycosis
Bovine or porcine actinomycosis (rare)
Dichelobacter (Bacteroides)
nodosus
Sheep
Goats, cattle and pigs.
Calves, lambs, foals, piglets.
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Contagious (virulent) foot rot.
Occasional infections of interdigital skin.
Applied Veterinary Bacteriology and Mycology: Identification of anaerobic bacteria
B. fragilis
Cattle
Pigs
Diarrhoeal disease
Mastitis
Abscesses
Dogs, cats, horses, cattle.
B. asaccharolyticus
Osteomyelitis
Cattle
Prevotella melaninogenica
Fusobacterium necrophorum
Foot rot
Cattle, sheep, dogs and cats.
Cattle
Sheep
Pigs
Horses
Chickens
Rabbits
F. russii
Osteomyelitis
Calf diphtheria, liver abscesses, metritis, cellulitis,
mastitis.
Foot abscess, interdigital dermatitis, lip and leg ulcers
'Bull - nose', necrotic enteritis, liver abscess
Thrush involving the frog, necrobacillosis of lower
limbs
Avian diphtheria
Necrobacillosis of lips and mouth
Soft-tissue infections
Cats
F. nucleatum
Non-specific infections
Several species
Bacteroides fragilis
B. fragilis is a Gram-negative bacillus, about 0,4 x 3-5 μm in size, regularly shaped, with a straight or
slightly curved axis and rounded ends. Cells may contain one or more unstained vacuoles that distort the
bacillary body and may be mistaken for spores. It grows well on horse blood agar. After 24 - 48 hours of
incubation, colonies are low convex circular domes, 1-3 mm. in diameter, and semi-translucent or greyishwhite in colour. Most strains are non-haemolytic. B. fragilis is not inhibited by 20% bile, a feature which
distinguishes it from most other members of the genus. Growth of B fragilis is favoured by haemin, in the
absence of which atypical or negative biochemical reactions may be given.
Most strains of B. fragilis are resistant to benzyl penicillin (2 units), neomycin (1mg) and kanamycin (1mg).
Its resistance to penicillin distinguishes B. fragilis from most other Bacteroides spp. The organism is
sensitive to erythromycin (60g) and rifampicin (15g).
Prevotella melaninogenica (formerly Bacteroides)
Provetella melaninogenica is implicated in a variety of infections. Three subspecies are recognised on the
basis of saccharolytic and proteolytic activity- melaninogenica, intermedius and asaccharolyticus.
The cells from surface colonies are short Gram-negative rods, commonly more coccal than coccobacillary in appearance. It grows well but slowly on horse blood agar. After 2-3 days incubation colonies
are 0,5 - 3mm. in diameter, circular, convex or umbonate, opaque and grey, brown or black in colour.
Blackening of the colony, which commences at its centre on about the third day of incubation, extends
towards the periphery to produce a shiny jet-black colony after 5 - 6 days. The development of
pigmentation is often associated with death of the culture. Prevotella melaninogenica is often difficult to
grow and maintain in pure culture although it usually grows well in the presence of other organisms such
as E. coli and B. fragilis. Many strains of P. melaninogenica require vitamin K in addition to haemin for
growth.
No strains of P. melaninogenica reduce nitrates and none grows in the presence of 20% bile.
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Applied Veterinary Bacteriology and Mycology: Identification of anaerobic bacteria
All strains of P. melaninogenica are sensitive to penicillin (2 units), erythromycin (60mg) and rifampicin
(15g). The organism is resistant to neomycin (1g) and sometimes to kanamycin (1g).
Bacteroides corrodens
B. corrodens is a Gram-negative bacillus, 0,5 x 1-2 μm in size with an occasional tendency for chain
formation.
It grows well, but slowly, on horse blood agar and produces small (1 mm. diameter), non-haemolytic
colonies after 4 - 5 days incubation. Young (48h) colonies are barely discernible with the naked eye and
present the appearance of pin-point depressions in the agar (pitting of agar). Mature colonies are circular
with entire or slightly undulating margins, low convex or umbonate, and semi translucent and greyishwhite in colour. Colonies of fresh isolates cause pitting of the agar which is best developed after 7 days of
incubation. The ability to pit agar media is commonly lost in stock laboratory strains and is not always
associated with fresh isolates.
Bacteroides corrodens is non-saccharolytic and non-proteolytic. Nitrate is reduced to nitrite and urease
and oxidase are produced. Neither indole nor hydrogen sulphide is formed.
Bacteroides corrodens is sensitive to penicillin (2 units), neomycin (1mg), kanamycin (1mg), erythromycin
(60g) and rifampicin (15g).
Bacteroides oralis
B. oralis forms part of the normal flora in animals. Although it is encountered in infections, its pathogenic
significance is not known.
Cells of B. oralis are 0,5 x 1-2 m in size with a marked tendency to chain formation. It grows well on
horse blood agar producing discreet circular colonies which are 0,5 - 2 mm. in diameter after 48 hours
incubation. Colonies are entire, shiny, translucent and non-haemolytic.
B. oralis ferments glucose, maltose, lactose and sucrose. It is also positive for aesculin and starch
hydrolysis. Unlike B. fragilis it does not ferment arabinose or xylose. The organism is non-proteolytic, does
not reduce nitrates and produces neither indole nor hydrogen sulphide. It does not grow in the presence
of 20% bile, but its growth is slightly enhanced by haemin and Tween 80.
B. oralis is resistant to kanamycin (1mg), but sensitive to penicillin (2 units), erythromycin (60g),
neomycin (1g) and rifampicin (15g).
Bacteroides capillosus
B. capillosus is part of the normal faecal flora of animals but has been encountered in a variety of soft
tissue infections.
Cells vary considerably in size, from 0,5 - 1,5 m wide and 1,5 - 8,0 m in length. Pleomorphism is often
prominent with bent filaments, distorted bacilli and irregular staining.
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Applied Veterinary Bacteriology and Mycology: Identification of anaerobic bacteria
Surface colonies on blood agar are 0,5 -2 m. in diameter, circular, entire, low convex, greyish,
translucent and non-haemolytic.
Bacteroides capillosus is non-proteolytic and weakly saccharolytic, fermenting only glucose with formation
of acid. Aesculin is hydrolysed, nitrate is not reduced and neither indole or hydrogen sulphide is produced.
It does grow in the presence of 20% bile.
Bacteroides capillosus is resistant to penicillin (2 units), kanamycin (1 mg) and neomycin (1 mg) but
sensitive to erythromycin (60 g) and rifampicin (15 g).
Bacteroides praeacutus
B. praeacutus forms part of the normal flora of animals but has been implicated in a variety of soft tissue
infections.
Cells of B. praeacutus show considerable variation in size, ranging from 0,5 - 1,5 m wide to 1,5 - 12 m
long. Pleomorphism is common with short chains, filaments and swollen forms.
Colonies on horse blood agar are small (0,5 mm. diameter) after 48 hours incubation, circular and flat with
scalloped or diffuse margins, greyish, translucent and non-haemolytic.
Bacteroides praeacutus is non-saccharolytic. Gelatinase is produced, but more complex proteins are not
attacked. Nitrate is reduced and hydrogen sulphide is produced but indole is not formed. It grows in the
presence of 20% bile.
Genus: Fusobacterium
The fusobacteria are Gram-negative, non-sporing mainly spindle-shaped bacteria and like the bacteroides, many
species are obligate parasites of animals. They are distinguished from the bacteroides by their weakly
saccharolytic activity and by their production of threonine deaminase and indole. The species most commonly
implicated as animal pathogens are F. necrophorum subsp. necrophorum, F. necrophorum subsp. fundiliforme,
F. russi, F. nucleatum, F. necrogenes, F. equinum and F. simiae.
Fusobacterium necrophorum
Fusobacterium necrophorum is a well recognised pathogen of man and a variety of animals. It occurs
normally in the upper respiratory and intestinal tracts. The most important virulence factor is a leukotoxin
that is produced by virulent strains of F. necrophorum. The organism is a cause of infections, especially in
relation to, or derived from the upper respiratory tract.
Two subspecies are recognised, namely, F. necrophorum subsp. necrophorum (formerly F. necrophorum
biotype A) and F. necrophorum subsp. fundiliforme (formerly F. necrophorum biotype B). Of the two F. n.
necrophorum is considered to be the most pathogenic causing calf diphtheria, interdigital phlegmon, and
liver and lung abscessation in ruminants. Occasionally F. n fundiliforme has been isolated from abscesses
in ruminants. A non-pathogenic and non-haemolytic strain that was classified as F. necrophorum biotype
C is now called F. varium.
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Applied Veterinary Bacteriology and Mycology: Identification of anaerobic bacteria
It is a Gram-negative bacillus with a tendency to pleomorphism and irregular staining. Cells are 0,5 x 5-10
m in size, the larger ones commonly being curved. Filament formation is common. Fusiform swelling of
bacilli is sometimes seen but free spheroids are rarely developed. In pathological material pleomorphism
may be the predominant feature - coccal, bacillary and filamentous forms all being represented.
Fusobacterium necrophorum grows well on horse blood agar. Colonies are 1 - 2 mm in diameter after 48
hours incubation, circular with scalloped or diffuse margins, high convex or umbonate, with a ridged
surface and semi translucent or opaque, and yellowish in colour. Most strains of the organism are betahaemolytic, and many are lipolytic on egg yolk agar.
It is non-saccharolytic although some strains ferment glucose weakly. Some strains produce gelatinase,
but more complex proteins are not attacked. Nitrate is not reduced but indole and hydrogen sulphide are
produced. It does not grow in the presence of 20% bile.
The organism is sensitive to penicillin (2 units), kanamycin (1mg/), erythromycin (60g) and rifampicin
(15g). It is resistant to vancomycin (5g).
F. n. necrophorum haemagglutinates a 15% solution of washed chicken blood cells suspended in saline,
whereas F. n. fundiliforme does not.
A RAPD-PCR using the WIL2 primers will distinguish the subspecies.
Fusobacterium nucleatum
Fusobacterium nucleatum is involved in soft tissue infections in man and animals, especially in relation to
the respiratory tract.
Cells of F. nucleatum are bacilli of classical fusiform appearance, being spindle-shaped, sharply pointed
and occasionally with central or eccentrically placed swellings. Cells are 1 x 5-10 m in size and free
spheroids are commonly seen.
It grows well on horse blood agar. Colonies are 1 - 2 mm. in diameter after 48 hours incubation, irregularly
circular, low convex, greyish, glistening, translucent and non-haemolytic.
Fusobacterium nucleatum is biochemically fairly inactive. Most strains weakly ferment fructose and some
weakly ferment glucose. The organism is otherwise non-saccharolytic, and is entirely non-proteolytic.
Indole is produced, nitrate is not reduced and hydrogen sulphide is not formed. It does not grow in the
presence of 20% bile.
The organism is sensitive to penicillin (2 units), kanamycin (1 mg.), rifampicin (15g), neomycin (1 mg.)
and erythromycin (60 g.) It is resistant to vancomycin (5 g.).
Fusobacterium varium
Fusobacterium varium has been implicated in a variety of soft tissue infections. It is a small Gramnegative bacillus, 0,5 x 1-2 m in size. Coccobacillary forms are common.
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It grows satisfactorily on horse blood agar. Colonies may appear as mere pin-points or may measure up
to 1 mm. in diameter after 48 hours incubation. Colonies are flat circular disks, greyish, translucent and
non-haemolytic.
The organism grows well in 20% bile. Glucose, fructose and mannose are weakly fermented and some
strains weakly hydrolyse galactose. It is non-proteolytic, indole is produced, nitrate is not reduced and
hydrogen sulphide is not formed.
F. varium is sensitive to penicillin (2 units) and kanamycin (1 mg) but resistant to erythromycin (60 g),
rifampicin (15 g) and vancomycin (5 g).
Fusobacterium mortiferum
Fusobacterium mortiferum is implicated in a variety of soft tissue infections. It is a highly pleomorphic
bacillus. The cells are extremely variable in size, from 0,5 -2 m wide and 1 -10 m in length. Many cells
are grossly irregular in shape, and some are distorted with centrally placed swellings; large "ball" forms
are common.
It grows well on horse blood agar. Colonies are 1-2 mm. in diameter after 48 hours incubation, irregular,
low convex or slightly umbonate, greyish, semi translucent and non-haemolytic.
The organism is moderately saccharolytic, weakly fermenting glucose, lactose, mannose, salicin and
cellobiose. It is non-proteolytic, does not produce nitrate or produce indole or hydrogen sulphide. Aesculin
is hydrolysed and the organism grows well in the presence of 20% bile.
Fusobacterium mortiferum is sensitive to penicillin (2 units) and to kanamycin (1 mg), but it is resistant to
erythromycin (60 mg), rifampicin (15mg) and vancomycin (5 mg).
Genus: Dichelobacter
The only species of importance is Dichelobacter nodosus (formerly Bacteroides nodosus). Both virulent and
benign strains cause of foot rot in sheep where it is usually associated with other pathogens such as
Fusobacterium necrophorum, Trueperella (Arcanobacterium) pyogenes and Treponema penortha. Benign
strains are also known to cause occasional infections of the interdigital skin of goats, cattle and pigs.
Dichelobacter nodosus is a Gram-negative, fairly large (1,7 x 3-6mm), slightly curved, non-motile rod which is
often swollen at one or both ends. They occur singly or occasionally in pairs. Isolations of D. nodosus are most
likely to be made when Gram staining shows its presence in active foot rot lesions. The typical rods are in many
cases surrounded by radially disposed Gram-negative bacilli.
Samples for culture should be taken from necrotic material at the junction of healthy and separating tissue in the
feet of sheep, and transported in PRAS Cary-Blair or Stuart’s medium to the laboratory. Direct inoculation onto
hoof agar has proven to be a very sensitive method of isolation. Those portions of necrotic material showing
greatest numbers of D. nodosus on smears should be shaken vigorously for 30 seconds in 10ml. 0,25M sterile
sucrose and the suspension then seeded on anaerobic media, especially hoof agar.
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The ability to produce protease is associated with virulence. This is tested by the proteolytic index, gelatin-gel
protease thermostability test, the elastase test, the zymogram test and the protease ELISA.
Three basic colonial types are described for D. nodosus.

B-type: papillate or beaded (most pathogenic) from ovine footrot.

M-type: mucoid (less pathogenic) from non-invasive infections in sheep.

C-type: circular (non-pathogenic) and resulting from repeated passage in media.
The colonies, generally, are greyish-white and 0,5 - 3,0 mm. diameter in 3-7 days.
It is totally asaccharolytic and do not ferment any sugars. It is also negative for aesculin hydrolysis, catalase
production and urease and indole negative. It does not grow in 20% bile. It is however proteolytic and is
gelatinase and caseinase positive.
Genus: Actinomyces
Actinomyces occurs as part of the indigenous oral flora of mammals. A. israelii, A. eriksonii and A. odontolyticus
occur normally only in the human mouth while A. bovis appears to be normally confined to the bovine mouth.
Actinomyces species of veterinary importance A. bovis, A. suis, A. viscosus, A. hordeovulneris and A. israelii.
As a group the Actinomyces have been aptly described as "facultative but preferentially anaerobic organisms",
whose growth is usually enhanced by carbon dioxide.
Fluorescent antibody techniques provide the most rapid and specific diagnostic approach, on the basis of which
6 serological groups are recognised.
Actinomyces viscosus will not be dealt with in this section as they grow equally well in air + CO2 and are
therefore regarded as facultative anaerobes. Differential characteristics of the anaerobic animal pathogens and
other species encountered in clinical material are summarised in Table 4.
Actinomyces bovis
It is the cause of bovine actinomycosis (lumpy jaw), as well as poll evil and fistulous withers (supra-atlantal
and supraspinous bursitis, respectively) in horses.
“Sulphur” granules are the best specimens for direct examination in infections caused by A. bovis. If
smears are made from the granules and stained with the Gram stain, delicate, Gram-positive, branching
filaments can be observed. Occasionally short filaments or pleomorphic diphtheroidal forms may
predominate.
Actinomyces bovis is a carbon dioxide-dependent facultative organism. On brain-heart infusion agar micro
colonies are about 0,06 mm. in diameter after 24 hours incubation, circular, entire and flat, with a smooth
or granular appearance. Macro colonies are 0,5-1mm. in diameter after 7-14 days incubation. They are
circular, entire, low to high convex, opaque and white with a smooth or granular surface. The organism is
non-haemolytic on horse blood agar.
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Applied Veterinary Bacteriology and Mycology: Identification of anaerobic bacteria
A. hordeovulneris
pyogenes
Trueperella
(Arcanobacterium)
A. viscosus
A. naeslundi
A. howellii
A. odontolyticus
A. israelii
TESTS
Actinomyces bovis
Table 4: Actinomyces of animal importance and others isolated regularly from clinical specimens
Catalase
-
-
-
+
-
+
-
+
Urease
-
-
-
0
+
v
-
-
Gelatin hydrolysis
-
-
-
0
-
-
+
0
Nitrate reduction
-
V
+
0
+
V
-
-
Mannitol fermentation
-
V
-
-
-
-
-
-
Salicin fermentation
-
+
V
-
V
V
-
0
Raffinose fermentation
-
+
-
+
+
+
-
W
Xylose fermentation
-
+
V
+
V
+
V
+
Aero-tolerance
M or An
M or An
M or An
F
F
F
F
M An
V=variable W=weak positive
M=carboxyphylic
An=anaerobic
F=grows aerobically and anaerobically
Genus: Eubacterium and Propionibacterium
The only species of veterinary importance occurring within the genera Eubacterium and Propionibacterium is
Eubacterium suis which causes pyelonephritis in sows. Eubacterium suis is a pleomorphic Gram-positive rod in
palisade and Chinese letter formation when viewed microscopically and stained with Gram’s stain. Colonies on
horse blood agar are non-haemolytic, 2-3 mm. in diameter, grey, smooth, circular with a shiny centre and a dull
edge. Although, with the exception of Eubacterium suis, it is adequate to identify these bacteria to only genus
level, care must be taken not to confuse them with non-sporing Clostridia or Actinomyces.
THE CLOSTRIDIA
The Clostridium species are large (0,3-1,3 x 3-10 m), Gram-positive, anaerobic, endospore producing rods
which usually bulge the cell. All the pathogenic species are straight rods except C. spiriforme that is curved or
spiral. Cells from older cultures have a tendency to decolourise and stain Gram-negative. Most species are
motile by peritrichate flagella except C. perfringens that is non-motile and also produces a capsule in animal
tissues.
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Applied Veterinary Bacteriology and Mycology: Identification of anaerobic bacteria
The clostridia are fermentative, oxidase-negative and catalase-negative. The strictness of anaerobic
requirements varies among the species but they all prefer 10 per cent CO2. Most clostridia require enriched
media that include amino acids, carbohydrates, vitamins and blood or serum.
Optimum growth of most of the pathogenic species occurs at 37C. There are over 80 Clostridium species of
which about 11 are of veterinary importance. Most of the pathogenic species produce one or more exotoxins of
varying potency.
Habitat and pathogenicity
The clostridia have a wide distribution in soil, fresh water and in marine sediments throughout the world,
although some species or types are present only in localised geographical areas. Many of the pathogenic
clostridia are normal inhabitants of the intestinal tract of animals and man, and often cause endogenous
infections. Other clostridia are more commonly present in the soil and cause exogenous infections from wound
contamination or by ingestion.
Table 5: Diseases caused by the pathogenic clostridia
Clostridium species
Hosts
Diseases
NEUROTOXIC SPECIES
Clostridium tetani
Horses, ruminants, humans etc.
Tetanus
C. botulinum (types A-F)
Man and other animals
Botulism
C. argentinense
HISTOTOXIC SPECIES
C. chauvoei
Humans (Argentina)
Botulism
Cattle, sheep, pigs
Blackleg
C. septicum
Cattle, sheep, pigs
Sheep
Chickens
Malignant oedema
Braxy
Necrotic dermatitis
C. novyi
type A
Sheep
Cattle and sheep
Big-head of rams
Gas gangrene
type B
Sheep, (cattle, pigs)
Black disease
type C
Water buffalo
Osteomyelitis
C. haemolyticum
(C .novyi type D)
C. sordellii
Cattle, (Sheep)
Bacillary haemoglobinuria
Cattle, sheep, horses
Gas gangrene
C. colinum
Game birds, young chickens, turkey
poults
Quail disease
(ulcerative enteritis)
Humans
Lambs
Food poisoning, gas gangrene
Enterotoxaemic jaundice
type B
Lambs (< 3 wks)
Neonatal calves and foals
Lamb dysentery
Enterotoxaemia
type C
Piglets, lambs, calves, foals
Adult sheep
Chickens
Haemorrhagic enterotoxaemia
Struck
Necrotic enteritis
type D
Sheep (all ages except neonates)
(goats, calves)
Pulpy kidney disease, FSE
ENTEROTOXAEMIAS
C. perfringens
type A
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Applied Veterinary Bacteriology and Mycology: Identification of anaerobic bacteria
type E
Calves and lambs (rare)
Enterotoxaemia
Rabbits
Rabbits and guinea-pigs
Possible role in mucoid enteritis
Spontaneous and antibiotic-induced diarrhoea
Foals and pigs
Humans, hamsters, rabbits etc.
Dogs, foals, pigs, lab. animals
Enterocolitis (natural)
Antibiotic-induced enterocolitis
Naturally occurring diarrhoea
SPECIES ACTIVATED BY
ANTIMICROBIAL DRUGS
C. spiriforme
C. difficile
Pathogenicity
Clostridia mainly cause disease through the production of potent exotoxins. These toxins vary in potency and
effect. Diseases caused by clostridia are listed in Table 5. Pathogenic Clostridium species are divided into the
following groups based on the type of toxin produced:

Neurotropic clostridia (C. tetani and C. botulinum) that produce potent neurotoxins but are non-invasive

and colonise the host to a very limited extent.
Histotoxic clostridia (C. chauvoei, C. septicum, C. novyi, C. haemolyticum, C. sordellii, C. perfringens
type A and C. colinum.) that produce less potent toxins than the first group but are invasive. This

includes the gas-gangrene producing clostridia.
Clostridia that produce enterotoxaemias (C. perfringens type A-E). Enterotoxins are formed in the

intestines and absorbed into the bloodstream producing a generalised toxaemia.
Clostridia (C. difficile and C. spiriforme) producing enteric diseases that can be antibiotic-induced.
Specimens
Specimens should be taken from recently dead animals as bacteria such as C. perfringens, C .septicum and
enteric facultative anaerobes are rapid post-mortem invaders. For isolation, blocks of affected tissue (20 mm x
20 mm x 20 mm) or fluids in air-free containers should be collected when possible rather than swab-taken
samples that expose the clostridia to the lethal action of atmospheric oxygen. Commercial systems are
satisfactory and the swab may be placed in Cary-Blair transport medium. In some clostridial diseases, such as
the enterotoxaemias, the toxin is required for diagnosis. The contents of the small intestine is collected from a
recently dead animal and submitted to the laboratory as soon as possible, as the toxins are labile.
Direct microscopy
Gram-stained smears from specimens are used to observe the morphological types of organisms present. The
fluorescent antibody (FA) stain is useful for specific identification.
1.
Gram-stained smears from affected tissues may reveal large Gram-positive rods that tend to decolourise
easily when sporing C. spiriforme is an exception being curved or helical. The characteristic 'drumstick'
forms of C. tetani, due to the spherical forms being terminal and bulging the cell, may be seen in necrotic
material from wounds associated with tetanus. This is suggestive, but by no means conclusive, as other
clostridia, such as C. tetanoides and C. tetanomorphum have a similar morphology. In cases of suspected
enterotoxaemia, the presence of large numbers of fat Gram-positive rods in a smear of the small intestinal
mucosa, from a recently dead animal, is presumptive evidence of the condition.
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Applied Veterinary Bacteriology and Mycology: Identification of anaerobic bacteria
2.
The direct fluorescent antibody test is used routinely for disease associated with C. chauvoei, C. septicum,
C. novyi and C. sordellii as fluorescein-labelled antiserum can be obtained commercially. Affected tissue as
well as a piece of rib containing bone marrow (about 14cm long) is a useful specimen. A bacteraemia
usually occurs with these clostridial diseases so the bacteria would be expected to be present in the bone
marrow. This tissue has the added advantage of giving low background auto fluorescence and being one of
the last tissues to be invaded, post-mortem, by bacteria such as C. septicum.
Isolation procedures
Freshly prepared or pre-reduced blood agar is necessary for the isolation of clostridia, but special media are
required for isolation of more fastidious species such as C. chauvoei, C. haemolyticum and C. novyi types B and
C. A blood and MacConkey agar plate inoculated and incubated aerobically will detect any aerobic pathogens
that may be present and also indicate the degree of contamination of the specimen by facultative anaerobes.
Liquid and semi-solid media with a low REDOX potential such as cooked meat broth and thioglycollate medium
can be used to grow and maintain pure cultures of the clostridia. They are of limited use for primary inoculation
as any fast-growing anaerobes or facultative anaerobes will outgrow the Clostridium species of interest.
Immediately before inoculating broth media, boiling to expel absorbed oxygen should be undertaken, followed by
rapid cooling to 37C.
Most of the pathogenic species are strict anaerobes, the exception being C. perfringens that is relatively aero
tolerant. Isolation should however be performed under strict anaerobic conditions with the atmosphere
containing 2 - 10 % CO2 as this enhances the growth of clostridia.
Biochemical identification
Clostridia can be identified to species level by biochemical testing. Toxin types of, for example, C. perfringens or
C. botulinum can only be successfully identified by toxin-antitoxin neutralization tests (later in this section) or
PCR.
Lactose-egg-yolk-milk agar medium provides information not only on the production of lecithinase and lipase, but
also on lactose fermentation and proteolytic activity as evidenced by the digestion of milk. It also enables a
presumptive diagnosis of some anaerobes to be made from a single plate culture (Table 6).
Some differential characteristics of the most important species of Clostridium are shown in Table 7.
Serological identification
Neutralisation or protection tests are used to specifically identify the toxin(s) present and hence the clostridial
pathogen involved in the disease. These procedures are most commonly used in tetanus, botulism and in the
enterotoxaemias caused by C. perfringens. The methods used are described in the following section.
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C. perfringens
C. barati
C. bifermentans
C. sordellii
C. botulinum A
C. botulinum B
C. botulinum C
C. botulinum D
C. botulinum E
C. botulinum F
C. botulinum G
C. sporogenes
C. novyi type A
C. novyi type B
C. novyi type C
C. haemolyticum
C. cadaveris
C. cochlearium
C. tetani
C. tertium
C. fallax
C. chauvoei
C. septicum
C. sphenoides
C. butyricum
C. histolyticum
17 | P a g e
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
-
Proteolysis
Lactose
Lipase
Agent
Lecithinase
Table 6: Reactions of the most important clostridial species on lactose-egg-yolk-milk agar
+
+
+
+
+
+
+
+
-
+
+
+
+ or + or +
+
+
Applied Veterinary Bacteriology and Mycology: Identification of anaerobic bacteria
C. novyi C
C. histolyticum
C. butyricum
C. sphenoides
C. fallax
C. tertium
C .cochlearium
C. cadaveris
C .barati
C. colinum
C. sporogenes
C. tetani
C .botulinum
C. bifermentans
C. sordellii
C. haemolyticum
C. novyi B
C. novyi A
C .chauvoei
C. septicum
TEST
C.perfringens
Table 7: Differential biochemical characteristics of the most important clostridia
Nitrate
+
+
+
-
+
-
-
-
-
-
-
-
v
-
-
v
v
v
-
-
-
Indole
-
-
-
-
-
+
+
+
-
+
-
-
-
+
v
-
-
v
-
-
-
Glucose
+
+
+
+
+
+
+
+
+
-
+
+
+
+
-
+
+
+
+
-
+
Maltose
+
+
+
+
v
-
+
+
v
-
+
+
w
-
-
+
+
+
+
-
+
Lactose
+
+
+
-
-
-
-
-
-
-
-
-
+
-
-
+
+
+
+
-
-
Salicin
v
+
v
-
-
-
-
+
v
-
v
+
w
-
v
+
-
w
+
-
-
Sucrose
+
-
+
-
-
-
-
-
v
-
v
+
+
-
-
+
+
-
+
-
-
Urease
-
-
-
-
-
-
+
-
-
-
-
-
-
-
-
-
v
-
-
-
-
+
+
+
+
+
+
+
+
+
+
+
-
-
+
-
-
-
-
-
+
+
Gelatin
+ = positive
18 | P a g e
- = negative
w = weak positive
v = variable reactions
Applied Veterinary Bacteriology and Mycology: Identification of anaerobic bacteria
Individual clostridia
Clostridium chauvoei
C. chauvoei grows poorly on blood agar, but good growth may be obtained by supplementing the
medium with liver extract. Identification of the organism is greatly facilitated by incorporating sheep
blood into agar, resulting in colonies that are surrounded by a wide zone of haemolysis. It produces
oval subterminal spores together with numerous pleomorphic forms. Unlike C. septicum it ferments
sucrose but rarely salicin.
Clostridium novyi
C. novyi is divided into four types based on the production of the extracellular toxins. C. novyi type D
has recently been renamed C. haemolyticum. The distribution of the two main lethal toxins is shown in
Table 8. Unlike the other types of C. novyi, C. novyi type A can grow on normal blood agar and
produces large irregular colonies with a rhizoidal edge surrounded by a large zone of haemolysis. The
organisms are large Gram-positive rods with oval to cylindrical sub terminal spores not swelling the
sporangium.
Types B, C, and C. haemolyticum are extremely demanding, both in their nutritional requirements and
in their tolerance of oxygen and the medium of Moore should be used. The medium should be
prepared freshly and plates pre-incubated in an anaerobic gas mixture prior to inoculation. Provided
that such pre-incubated plates are quickly streaked and re-established under anaerobic conditions,
good growth ensues.
Table 8: Toxins produced by the different types of C. novyi
TYPE
C. novyi A
C. novyi B
C. novyi C
C. haemolyticum
Alpha toxin
+
+
-
Beta toxin
+
+++
Colonies are smaller than those of C. novyi type A and usually rhizoidal in nature. The success of this
medium is largely attributable to the presence of cysteine and dithiothreitol. Under the usual conditions
of medium preparation, pouring and inoculation, oxidation of the cysteine in the medium proceeds
rapidly so that cysteine is likely to be largely inactivated by the time the cultures are set up.
Dithiothreitol protects the free thiol group of cysteine from oxidation, thus preventing its reducing activity
Clostridium septicum
Clostridium septicum grows readily on blood agar and, because of its tendency to spread, the agar
concentration is increased in order to obtain isolated colonies. Colonies of C. septicum are usually
irregular with a rhizoidal edge, but smooth, round colonies are produced by some strains.
Morphologically, the organism has characteristic filamentous forms and produces oval subterminal
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spores. It can be differentiated from C. chauvoei on the basis of antiserum neutralization tests,
fluorescent staining, and fermentation of salicin but not sucrose.
C. sordellii
C. sordellii grows well on stiff agar and produces irregular colonies. Colonies lose their translucent
appearance and become white on aging due to the production of spores. The organisms are largely
Gram-positive rods with cylindrical spores not swelling the sporangium. The organism can be
differentiated from the closely related C. bifermentans by production of urease.
Identification of C. septicum, C. chauvoei, C. novyi and C. sordellii
1.
Make 5 smears of the affected muscles and subcutaneous tissues. Liver impression smears may
also be made. Samples should be taken soon after death as false positives can be obtained with
decomposed specimens, particularly C. septicum and C. novyi.
2.
3.
Stain one of the smears with Gram’s stain and if no clostridia are observed, discontinue the test.
If clostridia are found, stain with Clostridium FA conjugates (see below).
4.
Cultures can be undertaken in conjunction with the FA stains, particularly when it is important to
establish whether there has been a vaccine breakdown.
5.
Two Columbia blood agar plates are streaked, one incubated aerobically and the other
anaerobically, both at 37°C for 48 hours.
Fluorescent antibody test
Specific fluorescein-labeled antisera are used for identification of C. chauvoei, C .septicum, C. novyi
and C. sordellii as follows:
1.
Label two slides with a diamond pencil or wax pencil, pencil if frosted glass and circle two areas on
2.
each slide with a diamond pencil (one circle per conjugate).
Smear a portion of the suspected lesion onto a microscope slide and leave until air dry.
3.
Fix by immersing in cold 1:3 acetone-methanol solution (1 ml acetone plus 3 ml methanol) for 20
minutes.
4.
Allow to air-dry by placing the slides in an incubator at 37°C for 10 minutes.
5.
Place one drop of the appropriate conjugated antiserum on each circle, using a separate plastic
inoculation loop or pipette per conjugate, and spread gently.
6.
Leave in a covered moist chamber, in an incubator at 37°C for 45 minutes. A large Petri dish
containing moistened filter paper is quite adequate.
7.
Rinse off the excess of the reagents with 0,15M PBS, pH 9.
8.
Place in a glass beaker or Petri dish and cover the slide/s with PBS and stir gently for 10 minutes,
using a magnetic stirrer.
9.
Decant the PBS.
10. Allow the slides to air-dry. Cover the smear with a cover-slip using phosphate-buffered glycerol (9
parts glycerine to 1 part PBS, pH 8,4) as mounting fluid.
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11. The slide is then examined with a conventional fluorescence microscope employing the filters
routinely used in fluorescence microscopy with FITC as conjugate. Switch on the microscope 10
minutes before use. Fluorescence is specific and the species is positively identified if fluorescence
(apple-green rods) occurs with the specific antiserum.
12. Positive control smears should be prepared with each batch of slides.
Clostridium tetani
C. tetani grows readily on blood agar, produces haemolysis and tends to have a spreading, swarming
growth. If 3% agar is used, separate colonies with a rhizoidal edge can be obtained. The organism
produces spherical terminal spores and the typical drumstick appearance is seen on smears. There are
10 serological types of C. tetani, based on flagella antigens, but the neurotoxin is antigenically uniform.
Injection of culture or culture filtrate into mice produces a characteristic spasm.
Tetanus toxin test
Suitable samples are tissue, serum or culture supernatant.
Preparation of samples
Macerate tissues and add an equal amount of Normal saline. Leave to stand for at least 3 hours or
overnight in the refrigerator. Serum is used as is.
Test
Centrifuge the sample or culture until the supernatant is clear. Collect the supernatant and mix 0,5 ml
with 0,5 ml of antitoxin diluted to 20 IU/ml. For the control, mix 0,5 ml supernatant with 0,5 ml normal
serum. Allow both solutions to stand for 30 minutes at room temperature.
Inject each of 2 mice intramuscularly in the back leg with 0,5 ml of test mixture and each of 2 mice
with 0,2 ml of the control. Observe for 4 days.
Results
Positive: Mice that have received antiserum stay alive, and those that did not die after showing
spasms and paralysis, especially at the injection site.
Negative: All 4 mice survive and do not show signs of tetanus.
Notes and precautions

Only use mice as they are the most susceptible of all the laboratory animals

Overnight cultures usually contain enough toxin. Cultures that are weak toxin producers can be
incubated for up to I week before testing.

Do not inject the mice subcutaneously or intravenously as delayed or irregular results could occur.
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
If the toxin levels in the samples are very high, all the mice may die but the mice receiving
antiserum will live longer. Dilute the supernatant 1:10 and 1:100 and retest.
Clostridium perfringens
This organism grows well on blood agar, producing smooth, round, glistening colonies. Colonies are
surrounded by a double-zoned haemolysis on blood agar because of theta toxin which produces a
clear zone of haemolysis which is surrounded by a zone of partial haemolysis produced by alpha toxin.
The organism is divided into five types, A - E, based on the extracellular production of alpha, beta,
epsilon and iota toxins. The toxins, and thus the type of C. perfringens can be identified both in culture
and in intestinal contents.
Clostridium perfringens toxin identification tests
The PCR may be used to establish whether alpha, beta or epsilon toxins are present (Table 9). It takes
one to two days to do, is accurate and can be done on cultures or samples.
Table 9: Toxin production by C. perfringens
C. perfringens type
A
B
C
D
Toxin/s
Alpha
Alpha, beta, epsilon, iota
Alpha, beta
Alpha, epsilon
Should a PCR not be available, animal tests may be used.
Intestinal contents or culture supernatant are centrifuged to obtain a clear supernatant containing
the toxin. This is mixed with varying amounts of antitoxins to neutralize the toxins and either injected
into mice
(intravenously) or into guinea pigs (intradermally). Some of the test mixtures contain trypsin to
activate epsilon toxin. There are usually 8 mixtures in total.
The following day the mice are examined for deaths and/or the guinea pigs for skin lesions. From the
pattern of deaths or lesions, the toxin types can be determined.
Clostridium botulinum
C. botulinum is divided into types A - G, based on the production of specific lethal toxins. The name of
C. botulinum type G has been changed to C. argentinense. Intoxication results from the consumption of
foodstuffs containing toxin as a result of growth of the organism.
Types A, B, F and C. argentinense are responsible for classical botulism in humans, type E for human
botulism from marine sources, and type C and D for botulism in animals. Types A, B, F and C.
argetinense are all, except for a few strains of type B, strongly proteolytic. C. botulinum types C, D and
E resemble each other morphologically and culturally. They consist of large rods with oval to cylindrical
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terminal spores not swelling the sporangium, and they produce irregular colonies with a granular
surface and rhizoidal margin. All are non-proteolytic.
Recovery of C. botulinum from food is not sufficient to establish a diagnosis of botulism. Food can be
contaminated without toxin being produced. A diagnosis is based upon the demonstration of a sufficient
amount of toxin.
Isolation
Great care should be taken when working with food or cultures that may contain botulinum toxin. To
culture C. botulinum from the suspected food, several samples are suspended in a small amount of
saline and heated at 65 - 80C for 30 minutes to eliminate non-sporing bacteria. Blood agar plates are
inoculated and incubated anaerobically. If the organism is identified biochemically as C. botulinum,
tests are conducted for toxigenicity. Filtrates are prepared from 5 - 10 day old cultures incubated at
30C in cooked meat medium.
Clostridium botulinum toxin test
Suitable samples are rumen and caecum contents and feed, but other samples such as intestinal
contents from any part of the intestine, liver or serum may be used. Toxin levels in the serum are
usually low.
Sample preparation
Organs such as liver should be minced or finely chopped. All samples such as intestinal contents are
placed in an equal amount of brain-heart infusion broth (BHI) or saline. Feed should be left in the broth
for at least 3 hours or preferably overnight to extract the toxin. 6 - 10ml of the supernatant is then used
for the test.
Serum is used without diluting it. At least 12 ml of serum is required for the complete test.
Screening test
1.
Centrifuge the samples at 3 000 g for 15 minutes.
2.
Collect the clear supernatant fluid.
3.
Thoroughly mix 2 ml of supernatant fluid with 0,4 ml of penicillin/streptomycin and let stand at
room temperature for 30 minutes.
4.
Store the rest of the supernatant fluid in the refrigerator.
5.
Inject 0,5 ml of the supernatant/antibiotic mixture intraperitoneally into 2 adult mice. Store the rest
of the mixture in the refrigerator.
6.
Serum is treated with pen/strep similar to supernatant and injected into mice twice a day at a dose
of 0,5 ml for a maximum of 4 days, until clinical signs or death occurs.
7.
Examine the mice daily for 4 days for typical clinical signs. If the mice show a wasp waist or die
showing a hollow abdomen and enteritis, proceed to the definitive test. If the mice are negative on
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the second day, they should again be injected with the mixture. If the mice remain negative for 4
days, the test is reported as negative.
Definitive test
Use the stored supernatant, and prepare the following suspensions:
1. 1 ml supernatant + 0,2 ml pen/strep + 0,2 ml D antiserum
2. 1 ml supernatant + 0,2 ml pen/strep + 0,2 ml C antiserum
3. 1 ml supernatant + 0,2 ml pen/strep + 0,2 ml normal serum
4. 1 ml supernatant heated at 100°C for 10 minutes.
5. Allow the suspensions to stand at room temperature for 30 minutes and inject mice in the same way
as above. If the mice remain negative they may be injected twice more on 2 consecutive days.
Serum samples are treated in the same fashion.
Possible results
Type C: Groups 1 and 3 are dead or show typical clinical signs and 2 and 4 are healthy.
Type D: Groups 2 and 3 are dead or show typical clinical signs and 1 and 4 are healthy.
If the results differ from this, the following may apply.

Group 3 is dead or shows typical clinical sings and groups 1, 2 and 4 are healthy. There may be
too little toxin present. Dilute the C and D antisera 1:10 and 1:100 with normal serum and repeat
the definitive test.

Groups 1, 2 and 3 are dead or show typical clinical signs and group 4 are healthy or dead. There
may be too much toxin or there may be a different toxin such as ionophore present. Dilute the
supernatant 1:10 and 1:100 and repeat the definitive test. Examine the mice more frequently so
that clinical signs as well as deaths are noted. If the results remain the same, there is probably
another toxin present.

If all the mice merely die without showing clinical signs, even if group 4 (botulinum toxin destroyed
by heat) dies, there is probably another toxin present.
Notes and precautions
The toxin may be present in the material in a protoxin form that needs activation by trypsin to become
toxic. This is more important if types B or E are suspected. It is not necessary to add trypsin to intestinal
contents. As farm animals usually are affected by toxins C or D and the toxin is usually present in large
amounts, veterinary samples are not usually treated with trypsin. Trypsin may be added to the
suspension at the same time as the broth or saline is added.
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Applied Veterinary Bacteriology and Mycology: Identification of anaerobic bacteria
REFERENCES
1. Carter,G.R and Cole,J.R. Jr. Diagnostic Procedures in Veterinary Bacteriology and Mycology,
1990. Academic Press. ISBN 0-12-161775-0.
2. Donahue,J.M. Non-spore-forming Anaerobic Bacteria, In: Diagnostic Procedures in Veterinary
Bacteriology and Mycology (fifth ed.) 1990, edited by G.R.Carter and J.R.Cole, Jr. Academic Press.
ISBN 0-12-161775-0.
3. Quinn,P.J; Carter,M.E; Markey,B & Carter, G.R. Non-spore-forming Anaerobic Bacteria, In Clinical
Veterinary Microbiology,1994.Wolfe Publishing.ISBN 0 7234 1711 3.
4. Willis, A.T. Anaerobic Bacteriology: Clinical and Laboratory Practice (third edition),1977; Butterworths
and Co.; ISBN0-407-00081-X.
5. Palmer MA (1993) A gelatin test to detect activity and stability of proteases produced by
Dichelobacter (bacteroides) nodosus Veterinary Microbiology 36:113-122
6. Stewart DJ, Peterson JE, Vaughan JA, Clark BL, Emery DL, Caldwell JB and Kort AA (1986b)
The pathogenicity and cultural characteristics of virulent, intermediate and benign strains of
Bacteroides nodosus causing ovine footrot. Australian Veterinary Journal 63:317-326
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Applied Veterinary Bacteriology and Mycology: Identification of anaerobic bacteria
APPENDIX: MEDIA AND REAGENTS FOR THE ISOLATION OF
ANAEROBIC BACTERIA
1.
VL (Viande-Levure) broth
Tryptone
NaCl
Beef extract
Yeast extract
Cysteine HCl
Agar
Distilled water.
10g.
5g.
2g.
5g.
0,4g
0,6g
1000ml
capped bottles and autoclaved at 121°C for 20
minutes.
This medium is used for the detection of
proteinase activity.
4.
Gelatin agar.
Heat to dissolve, adjust pH to 7,2-7,4, dispense,
sterilise by autoclaving at 115°C for 10 minutes.
Prepare the required amount of VL agar and add
0,4% gelatin.
This is a most useful basal broth from which an
Sterilise by autoclaving at 115°C for 10 minutes.
almost complete range of fluid and solid media for
the identification of anaerobes can be prepared.
This medium is used for the detection of
gelatinase activity.
Sugars (filter sterilised 10% solutions of the
various sugars) are added after the broth has
been autoclaved.
5.
Bile broth.
This is VL broth containing 1% glucose and 20%
Agar can be added at 15g /liter for plate media.
ox gall (Oxoid), dispensed in capped test tubes. It
2.
is used to determine if growth is inhibited or
stimulated by bile.
Egg yolk agar.
Prepare an egg yolk emulsion by mixing egg yolk
6.
Indole production.
with an equal volume (about 20 ml. /egg yolk) of
sterile saline. Add the emulsion to sterile molten
Indole is detected in cultures growing in trypticase
VL agar base (at 50 - 55°C) in the proportion of
10%. Pour plates immediately.
nitrate broth. On the addition of Kovac's reagent to
3.
Milk agar.
an aliquot of the culture, a dark red colour in the
amyl alcohol surface layer constitutes a positive
indole test.
Melt the required amount of VL agar base and
7.
cool to 55°C. Add skimmed milk to a concentration
of 25%. Pour plates immediately.
Nitrate reduction is tested for in cultures growing in
Nitrate reduction.
trypticase nitrate broth. On addition of the 2 nitrate
Skimmed milk is prepared from fresh cow's milk by
reagents to an aliquot of the culture, a pink colour
centrifugation to separate the cream. The milk is
developing in the culture indicates the presence of
nitrite.
pipetted and distributed in 20ml volumes in screw-
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Applied Veterinary Bacteriology and Mycology: Identification of anaerobic bacteria
8.
Urea medium.
To 100ml. of sterile VL broth base add aseptically
Tryptose
15 g
Yeast extract
5g
Soytone
5g
Ferric ammonium citrate
1g
Sodium metabisulfite
1g
Agar
20g
Distilled water
900ml
5 ml. of a 40% filter sterilised urea solution. Mix
well and distribute in sterile capped test tubes. On
the addition of phenol red (yellow at pH 6,8 and
red at pH 8,4) to the culture, a pink colour
indicates hydrolysis of urea.
9.
Hydrogen sulphide production.
A lead acetate strip is wedged in the top of a
culture tube of VL broth, VL glucose broth or
cooked meat medium. Blackening of the strip
indicates a positive reaction.
Heat with agitation to dissolve. Adjust pH to
7.6±0.2. Autoclave 15 minutes at 121°C.
10. Aesculin hydrolysis.
Cool in a water bath to 50°C.
For the aesculin hydrolysis test either VL broth or
agar medium may be used with the addition of
0,01% aesculin and 1% ferric ammonium citrate.
A positive reaction in the broth is indicated by
blackening of the medium and on the agar
medium by blackening of the colonies.
Add 80 ml of D-cycloserine solution (1 g in 200
ml distilled water).
Add 80 ml of 50% egg yolk emulsion.
Pour plates.
Positive: Black colonies surrounded by an
11. Tryptose-sulfite-cycloserine (TSC) agar
For the selective isolation of Cl. perfringens
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opaque white zone are indicative of lecithinase
activity.