chapter 4: the identification of species with special ... · bacillus anthracis is generally...

Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria

Chapter 4: The identification of Bacillus species with special reference to Bacillus anthracis

1 | P a g e



Author: Dr. Valerius de Vos

Licensed under a Creative Commons Attribution license.

TABLE OF CONTENTS

INTRODUCTION ........................................................................................................................................... 2

IDENTIFICATION .......................................................................................................................................... 2

Table 4.1: Identification of Bacillus species ................................................................................... 4

Table 4.2: Secondary characteristics in the identification of Bacillus spp. in Morphological

Group 1 .......................................................................................................................................... 7

Table 4.3: Biochemical characteristics of Morphological Group 1 ................................................. 7

Table 4.4: Identification of Bacillus species ................................................................................... 8

Table 4.5: Suggested tests to differentiate B. anthracis from B. cereus (numbers positive to

numbers tested from Brown et al, 1958) ........................................................................................ 9

APPENDIX 1 ............................................................................................................................................... 16

http://creativecommons.org/licenses/by/3.0/



2 | P a g e

INTRODUCTION

Most species in the genus Bacillus are large, aerobic of facultatively anaerobic, Gram-positive,

endospore-producing rods. Spore-producing bacteria embrace a large number of bacterial species

with a great diversity of properties. Most of them are contaminants or saprophytes with the ability to

adapt to a wide range of environmental conditions. In spite of the fact that some have attained

pathogenic status for humans and animals, it remains the general rule in diagnostic laboratories to

dismiss aerobic spore-bearers as contaminants. There is evidence that while Bacillus anthracis, B.

cereus, B.licheniformis and B. subtilis have attained the status of potential pathogens, other species

within this genus may also be incriminated if studied more closely (Gilbert et al, 1983; Gordon, 1981;

Parry et a1, 1983; Tuazon et al, 1979). Especially B. cereus, but also B. licheniformis and B. subtilis,

have been associated with a wide range of infections, such as bacteraemia and septicaemia, wound

and respiratory infections, ophthalmia, peritonitis, gastroenteritis, kidney and urinary tract infections,

endocarditis, meningitis and bovine rnastitis. B. anthracis however, stands out in this group of bacteria,

causing anthrax in humans and animals. Anthrax is a peracute, acute or subacute, highly contagious

disease of domestic and wild animals and humans caused by the bacterium Bacillus anthracis. In

most species of animals it is characterized terminally by the development of a rapidly fatal

septicaemia, resulting, in sudden death. The principal lesions are those of widespread oedema,

haemorrhage and necrosis.

IDENTIFICATION

Smith et al (1946, 1952) found that the genus Bacillus, or aerobic spore-bearers, can be divided into 3

groups on the basis of the shape of the spore and swelling or absence of swelling of the sporangium

by the spore. Morphological Group 1 is defined by the absence of sporangial swelling and possession

of ellipsoidal spores and includes all of the known pathogens of this genus. Morphological Group 2

includes species whose sporangia are swollen by oval spores, and Morphological Group 3 includes

species that produce round spores. (Figure 1)



3 | P a g e

Fig. 1: A schematic representation of the different morphological groups. (From Parry et al, 1983)

In the majority of cases the clinical microbiologist will be faced with the identification of the principal

Bacillus species in Morphological Group 1. Figure 2 depicts a flow chart, giving suggested primary

steps in the identification of those species. Table 4.1 lists secondary identification characteristics and

the final biochemical identification tests are given in Table 4.2.

Bacillus anthracis characteristics

Actively growing (vegetative) B. anthracis organisms are typically rod-shaped, measure 1,0-1,5 by 3,0-

10,0 m. In stained smears of blood or tissue fluid obtained from infected animals, the organisms

appear truncated, commonly occur singly or in short chains, and are surrounded by a well-developed

capsule, (Figures 2 and 3). Capsules are not formed in cultures unless special conditions for their

development are provided. In unstained preparations, the organisms are robust, transparent rods and,

in contrast to those in animal tissues, have rounded ends. In vitro B. anthracis grows in long, undulant

chains composed of many cells which resemble the segments of a bamboo pole.

The spores, which are never found in the living animal, are ellipsoidal or oval, and are formed

equatorially without causing a swelling of the sporangium. Spores develop under suitable,

environmental conditions and are liberated by lysis of the bacilli. They germinate by polar rupture.



4 | P a g e

Sporulation in cultures on the surface of solid media, commences at about the end of logarithmic

growth, is far advanced by 24 hours, and is usually complete by 48 hours. The shape, wall thickness

and size of the spores relative to the sporangium, are important criteria in the taxonomy of the genus

Bacillus and are of considerable assistance in distinguishing B. anthracis from other members of the

genus.

Bacillus anthracis belongs to Morphological Group 1 (absence of sporangial swelling, and ellipsoidal

spores). Other Bacillus spp., such as B. megaterium, B. cereus, B. cereus var. mycoides, B.

thuringiensis, B. licheniformis, B. subtilis however, also possess these characteristics; consequently

other methods must be used to differentiate them (Table 4.3. Figure 2)

Table 4.1: Identification of Bacillus species

Species

Mo

tility

Cata

lase

Para

sp

ora

l b

od

ies

Lip

id g

lob

ule

s i

n p

roto

pla

sm

Le

cth

ov

itellin

reacti

on

Cit

rate

Uti

lizati

on

An

aero

bic

Gro

wth

V-P

Reacti

on

PH

in

V-P

med

ium

<6

Gro

wth

at

50°C

Gro

wth

at

60°C

Gro

wth

in

7%

NaC

l

Acid

fro

m A

S g

luc

ose

Acid

an

d g

as f

rom

AS

g

luc

ose

Nit

rate

red

uc

tio

n

Casein

Hyd

roly

sis

Sta

rch

Hyd

roly

sis

Pro

pio

na

te U

tilizati

on

Morphological Group 1

B. megaterium V + - + - + - - V - - + + - V + + N

B. cereus + + - + + + + + + - - + + - + + + N

B. cereus mycoides - + - + + + + + + - - + + - + + + N

B. anthracis - + - + + V + + + - - + + - + + + N

B. thuringiensis + + + + + + + + + - - + + - + + + N

B. licheniformis + + - - - + + + + + - + + - + + + +

B. subtilis + + - - - + - + V V - + + - + + + -

B. pumulis + + - - - + - + + V - + + - - + - -

B. firmus v + - - - - - - - - - + + - + + + -

B. coagulans + + - - - V + + + + V - + - V V + -


B. polymyxa + + - - - - + + V - - - + + + + + N

B. mascerans + + - - - V + - + + - - + + + - + N

B. circulans V + - - - V V - + V - V + - V V + N

B. stearothermophilus

+ V - - - - - - + + + - + - V V + N

B. alvei + + - - - - - + + - - - + - - + + N

B. laterosporus* + + - - (+) - + - + - - - + - + + - N

B. brevis + + - - - V + - - V V - + - V + - N

V-P: Voges-Proskrauer; AS: Ammonium salt; N: Not applicable.

Morphological Group 1: Sporangium not swollen by the spore; spore is ellipsoid or cylindrical, central or terminal

Morphological Group 2: Sporangium swollen by an ellipsoid spore, spore central or terminal

Morphological Group 3: Sporangium swollen by spherical spore; spore sub terminal or terminal

* Spore and sporangium have a characteristic canoe shape



5 | P a g e

Fig. 2: Primary steps in the identification of principal Bacillus species of Morphological groups 1, 2, and 3



6 | P a g e

Figure 3: Flow diagram of suggested procedures for isolation and identification of B. anthracis and

confirmation of diagnosis



7 | P a g e

Table 4.2: Secondary characteristics in the identification of Bacillus spp. in


Set A: LV positive Characteristics

B. anthracis B. cereus B. thuringiensis B. cereus var. mycoides B. laterosporus

Non-motile, non-haemolytic, Morphological Group 1. Marked haemolysis, motile, Morphological Group 1. Identical to B. cereus, contains parasporal bodies in young culture. Variant of B. cereus, colonies rhizoid and spreading, usually non-motile. Typical canoe-shaped cells containing Morphological Group 2 spores.

Table 4.3: Biochemical characteristics of Morphological Group 1

B. m

eg

ate

riu

m

B.

cere

us

B.

cere

us

var

.myco

ide

s

B.

an

thra

cis

B.

thu

rin

gie

ns

is

B.

lich

en

ifo

rmis

B.

su

bti

lis

B.

pu

mil

us

B.

firm

us

B.

co

ag

ula

ns

LV (egg yolk) reaction - + + + + - - - - -

Citrate utilization + + + v + + + + + v

Anaerobic growth - + + + + + - - - +

V-P reaction - + + + + + + + - v

Nitrate reduction v + + + + + + - + v

Indole production - - - - - - - - - -

Growth in 7% NaCl + + + + + + + + + -

Starch hydrolysis + + + + + + + - + +

Casein hydrolysis + + + + + + + + + v

Gelatine hydrolysis + + + + + + + + + -

Urease activity v v v - v V v - - -

*Acid from: Glucose + + + + + + + + v +

Mannitol v - - - - + + + + v

Xylose v - - - - + + + v v

Arabinose v - - - - + + + v v

Haemolysis (blood agar) + + - +

Motility + - - +

Propionate utilization + -

Parasporal bodies - - - +

Tyrosine hydrolysis +/- + +/- - + - - - -/+ -



8 | P a g e

Growth in 0,001% lysozyme - + + + + - -/+ +/- - -

V =variable; +/- = more often +; -/+ = more often -;

Use ammonium salt sugars as base

Table 4.4: Identification of Bacillus species

Species

Mo

tility

Cata

lase

Para

sp

ora

l b

od

ies

Lip

id g

lob

ule

s i

n p

roto

pla

sm

Le

cth

ov

itellin

reacti

on

Cit

rate

Uti

lizati

on

An

aero

bic

Gro

wth

V-P

Reacti

on

PH

in

V-P

med

ium

<6

Gro

wth

at

50°C

Gro

wth

at

60°C

Gro

wth

in

7%

NaC

l

Acid

fro

m A

S g

luc

ose

Acid

an

d g

as f

rom

AS

glu

co

se

Nit

rate

red

uc

tio

n

Casein

Hyd

roly

sis

Sta

rch

Hyd

roly

sis

Pro

pio

na

te U

tilizati

on


B. megaterium V + - + - + - - V - - + + - V + + N

B. cereus + + - + + + + + + - - + + - + + + N

B. cereus mycoides - + - + + + + + + - - + + - + + + N

B. anthracis - + - + + V + + + - - + + - + + + N

B. thuringiensis + + + + + + + + + - - + + - + + + N

B. licheniformis + + - - - + + + + + - + + - + + + +

B. subtilis + + - - - + - + V V - + + - + + + -

B. pumulis + + - - - + - + + V - + + - - + - -

B. firmus v + - - - - - - - - - + + - + + + -

B. coagulans + + - - - V + + + + V - + - V V + -


B. polymyxa + + - - - - + + V - - - + + + + + N

B. mascerans + + - - - V + - + + - - + + + - + N

B. circulans V + - - - V V - + V - V + - V V + N

B. stearothermophilus + V - - - - - - + + + - + - V V + N

B. alvei + + - - - - - + + - - - + - - + + N

B. laterosporus* + + - - (+) - + - + - - - + - + + - N

B. brevis + + - - - V + - - V V - + - V + - N


B. sphearicus + + - - - V - - - - - V - - - V - N

V-P: Voges-Proskauer; AS: Ammonium salt; N: Not applicable.

Morphological Group 1: Sporangium not swollen by the spore; spore is ellipsoid or cylindrical, central or terminal

Morphological Group 2: Sporangium swollen by an ellipsoid spore, spore central or terminal

Morphological Group 3: Sporangium swollen by spherical spore; spore sub terminal or terminal

* Spore and sporangium have a characteristic canoe shape

Bacillus anthracis is generally Gram-positive, but this attribute is often lost with age. Gram's staining of

smears of the organism grown in culture should therefore be carried out as soon as possible, usually

within 24 hours of the commencement of growth. The capsule, although also Gram-positive, is more

easily decolourized than is the body of the young bacilli, with the result that individual Gram-positive

bacilli may be enveloped by a Gram-negative capsule. Several Bacillus spp., including B. anthracis, B.

subtilis, B. licheniformis and B. megaterium under appropriate growth conditions, produce

carbohydrate capsules containing varying amounts of the polypeptide, poly-D-glutamic acid. The

organisms are readily stained by the usual stains (Parry et al, 1983). In contrast to the other

polysaccharide capsule-producing bacilli, the capsule of B. anthracis consists predominantly of poly-D-

glutamate and only shows up well with Wright's and Giemsa stains, polychrome methylene blue

(M'Fayden reaction stain), and 0,1% toluidine blue in a 1% aqueous solution of alcohol, or by the

application of immunofluorescence techniques. Of the techniques described, the McFayden and



9 | P a g e

Giemsa staining methods are preferred by most laboratories. Demonstration of the capsule by staining

blood smears helps with the confirmation of the diagnosis of anthrax. Other Bacillus spp., such as B.

subtilis, B, licheniformis and B. megaterium also have capsules that contain polypeptides which impart

similar staining characteristics. These species are however, unlikely to be encountered in specimens

of blood or tissues from animals or humans with suspected anthrax. Although the spores of B.

anthracis can be stained by the usual spore stains, Schaeffer and Fulton's malachite green technique

is recommended (Parry et al, 1983). The use of phase contrast microscopy is also helpful in the

examination of spores.

Table 4.5: Suggested tests to differentiate B. anthracis from B. cereus (numbers

positive to numbers tested from Brown et al, 1958)

B. anthracis B. cereus/

B. thuringiensis B. cereus var. mycoides

No. +/Total tested

No. +/Total

tested

No. +/Total tested

Motile - 0/122 + (feeble) 115/115 Slight/- 30/38 all

slight

‘Cotton wool’ growth in broth culture

+/- 78/122 - 16/115 - 4/38

Haemolysis on 5% blood agar1

- 45/122 + 89/115 + 38/38

LV reaction -/+ 19/89 + 89/97 + 15/15

Sensitive to penicillin (10 units)1

+ n - n - n

Susceptible to gamma phage + 122/122 - 0/115 - 0/38

‘Inverted pine tree’ growth in gelatine stab

-/+ 21/122 -/+ 41/115 -/+ 13/38

Capsule formation: fresh animal isolates

1 + n - n - n

Reduction of methylene blue (48 hours)

- 0/122 -/+ 36/115 -/+ 3/27

Pathogenecity2: mice

guinea pigs rabbits

+ + +

107/120 41/47 34/42

-/+ -/+ -

26/63 7/26 0/24

-/+ -/+ -/+

7/70 3/27 0/27

Lethal toxin3,4

- n - n n n

Requiring thiamine5

+ n + n - n

Growth at 45°C6

- n + n - n

Tyrosine decomposition7

- n + n +/- n

N = not done, not recorded, not applicable or only a few strains tested;

1. In general these together with colony tenacity and Gram staining appearance constitute the basis

and most practical set of tests for differentiating B. anthracis from other members of the B. cereus

group.

2. 0.2 ml of an 18 hour broth administered subcutaneously

3. 0.5 ml cell-free culture filtrate intravenously in mice

4. Bonventre and Johnson (1970)

5. Proom and Knight (1955)

6. Burdon (1956)

7. Gordon et al (1973)

Wild, virulent types of B. anthracis are aerobic, but do grow in partial anaerobic atmospheres. The

optimum temperature for growth is between 35 and 37°C. Growth is slower at lower temperatures.

Bacillus anthracis utilizes simple sources of nitrogen and carbon for energy and growth, and grows on

most of the ordinary culture media, but the provision of suitable concentrations of thiamine and



10 | P a g e

metallic ions, particularly potassium, calcium, iron and manganese is important. After 24 hours growth

on nutrient agar, the colonies are 3-5 mm in diameter and have a grey, frosted appearance, especially

when viewed with transmitted, oblique light from above. The margins of colonies are very irregular

because of tangled outgrowths of bacterial filaments from the edges of the colonies which impart the

so-called "Medusa head" appearance. Typically these outgrowths taper and re-curve in the same

direction so that the colony as a whole almost appears to be spinning. This feature is best

demonstrated by placing a cover slip on top of a young colony growing on an agar surface and

examining the colony edges under a microscope using a XIO objective. This appearance is also seen

in colonies of many strains of B. cereus, B. thuringiensis, B. cereus var. mycoides and B, megaterium,

although the re-curvature of the outgrowths of B. cereus colonies is less noticeable than that which

can be seen in the other Bacillus spp.

Tenacity is another characteristic feature of cultures of B. anthracis. Typical colonies are very viscid

and have a marked tenacity. The effect produced by drawing a bacteriological loop across them, has

been described as "strings" or "tacky resembling drying glue". The “strings" can be made to stand up

perpendicular to the agar surface without support. This form of growth is probably responsible, for

"spiking" or "tailing" along the inoculation line which, in turn, is associated with virulence; avirulent

strains tend to lack these outgrowths.

After 24 hours, growth is less characteristic and colonies become whitish-opaque, and contain

scattered, darker lacunae. Colonies are then also typically butyrous, lack the tenacity of young growth

and are easily emulsifiable.

Bacillus anthracis bacteria produce capsules after inoculation into suitable hosts, but not when grown

on or in ordinary culture media. Capsule formation can however, be induced in vitro by special

conditions such as a high partial pressure of CO2 and media containing serum (Sterne, 1937),

bicarbonate (Gladstone, 1946; Thorne et al, 1952), and activated charcoal with or without serum

(Meynell & Meynell, 1964) or milk (Weaver et al, 1970). Capsule formation in bicarbonate agar is an

effective way of differentiating between B. cereus and B. anthracis (Parry et al, 1983). Capsulated B.

anthracis cultures on solid media are thick, smooth and very slimy and have no resemblance to the

usual dry, flat, tough "Medusa head" type of growth.

Nutrient broth, when inoculated with B. anthracis, becomes turbid and as the floccules which develop

become more dense they sediment to the bottom. Freshly isolated strains usually form a deposit in

broth.

In gelatine stab-cultures, delicate lateral projections grow out from the needle tract, with the longest

projections at the upper part of the culture and the others decreasing in length progressively

downwards, giving the growths an inverted fir tree-effect. Liquefaction is slow and crateriform. This has

some diagnostic application as B. cereus has a more pronounced arborescent, filamentous growth

and causes saccate liquefaction.

Biochemically, B. anthracis is much less active than other morphologically similar, but non-pathogenic

Bacillus spp. These biochemical characteristics are not specific enough to distinguish it from other

Bacillus spp.



11 | P a g e

Isolation and diagnosis of Bacillus anthracis

In animals it is generally very difficult to demonstrate the presence of B. anthracis in the blood during

the early stages of the disease, but later on the organisms may be cultured from blood.

The organisms are seldom present in sufficient numbers in the blood to be demonstrable in blood

smears of live animals, unless these are made when the disease is terminal, but they may be found,

sometimes with difficulty, in smears made from local swellings if these are present.

In order to prevent or minimize the contamination of the immediate surroundings of the carcass with B.

anthracis spores, and also to avoid possible infection of the prosector, a necropsy should not be

performed if the history and clinical signs indicate anthrax. An appropriately stained blood smear using

blood obtained from a small wound is made by puncturing the skin of the lip, ear or hoof coronet with a

sharp instrument should be examined microscopically. It must be borne in mind that as soon as an

animal dies from the anthrax bacillus (in the unopened carcass), it undergoes changes in its

morphology. The capsule commences to disintegrate, and the protoplasm to degenerate, absorbing

the stain more and more faintly until only ghost-like bacilli are seen. The capsular material is the last to

disappear. At death only a few, if any, B. anthracis rods are present in the peripheral blood of horses,

pigs, carnivores and some wild animal species. A diagnosis may be made by preparing smears from

the oedematous fluid that surrounds localized lesions, such as that which occurs in the region of the

throat and neck of many pigs which die from the disease.

When after the examination of a blood smear, anthrax is still suspected but unconfirmed, suitable

samples should be collected and submitted to a laboratory for bacteriological examination. The

isolation and identification of B. anthracis from specimens originating from a relatively fresh carcass

are not particularly difficult, but attempts to do these procedures using material obtained from severely

decomposed carcasses are often unsuccessful. Usually by 48 hours after death, organisms can only

be isolated with difficulty, but in cool environments it may be able to isolate them for as long as four

weeks after death. The preferred specimens for diagnostic purposes depend on the state of the

carcass and the length of time that has elapsed between death and the collection of the specimens.

In fresh, unopened carcasses, blood collected from peripheral blood vessels or the jugular vein and

kept at 4°C should be submitted for bacteriological examination. In carcasses in which decomposition

is well advanced, blood should be obtained from the extremities furthest away from the gastrointestinal

tract. Specimens obtained from the coronets of the hooves offer the best chances to detect the

organism in smears or by isolation.

If a suitable blood sample is unobtainable, the tip of the tongue or a superficial lymph node such as

the prescapular, are the specimens of choice. If the carcass has been opened for necropsy, a pooled

specimen taken from the spleen and several lymph nodes is preferred. When carcasses are

dehydrated and putrefaction is advanced, samples should be collected from areas which might have

been contaminated with blood at an earlier stage where sporulation of B. anthracis could have taken

place, such as at natural body openings or parts of the body mutilated by scavengers. Even when

bones are all that remains of a carcass, bacteria may be more readily isolated from specimens of the



12 | P a g e

bones forming the eye sockets, mandible and ischium than from those taken randomly. Soil from

below the carcass may also be submitted for culturing,

Bacillus anthracis grows readily on artificial culture, and when isolation is attempted from

uncontaminated fresh specimens, nutrient agar can be used for this purpose, but best results are, in

general, obtained on media containing serum or blood. Severely contaminated material, such as

samples of soil or bone-meal, should be cultured on selective media of which there are several

available. PLFT medium is suitable for the purpose of isolating B. anthracis spores in soil, even when

they are present in concentrations as low as 3 spores per gram. Seeded plates should be incubated

for 12 to 24 hours at 37°C and thereafter examined under a stereo microscope with transmitted light

from the side and above. Colonies suspected of being B. anthracis on the grounds of colonial

morphology and tenacity, are lifted with a bacteriological needle and inoculated on a 5% blood agar

plate. If no haemolysis occurs within 24 hours at 37°C and the organisms conform to Morphological

Group 1 bacilli, further tests are required to differentiate B. anthracis from the other members of this

group.

Additional tests in laboratory animals are considered essential if B. anthracis is to be conclusively

identified. In this respect, no universal standardized procedures have been formulated. It is generally

recommended that 0,2 ml of a broth suspension should be inoculated subcutaneously into mice or

guinea pigs, intramuscularly into guinea pigs, or intraperitoneally into mice. The intraperitoneal

injection in mice of 1 to 5 x 105

B. anthracis spores is favoured.

An API Bacillus system (API Laboratory Products, Ltd) has been developed to aid in the identification

of Bacillus spp. and to facilitate in the identification of both typical and atypical strains of B. anthracis.

Separation of slightly virulent and avirulent strains of B. anthracis from closely related Bacillus spp. is

based on API and phage-sensitivity tests.

Lysis by bacteriophage (gamma) is a highly specific differential test for B. anthracis and is popular as

a diagnostic aid in laboratories dealing with anthrax.

When B. anthracis is grown in the presence of low concentrations of penicillin, the bacilli swell and

filaments of them appear as chains of spores or of round cellular forms referred to as "strings of

pearls". This phenomenon, which was first described by Jensen and Kleemeyer, is specific for B.

anthracis, although exceptions do occur as some strains of B. anthracis do not grow under these

circumstances. Because of the specificity and relative ease at which it can be performed, this is a

useful diagnostic test for the identification of B. anthracis.

Generally, serological and immunofluorescent diagnostic methods are unreliable for the diagnosis of

anthrax. Direct and indirect immunofluorescence assays, immunoradiometric assay and the enzyme-

linked immunosorbent assay may be developed in future for determining the serological relatedness of

B. anthracis and other Bacillus spp. An enzyme immunoassay and the production of monoclonal

antibodies against the protective antigen of B. anthracis have also been considered for diagnostic

purposes, and specialist laboratories use the PCR technique with great success.

For practical purposes a battery of tests may be required to confirm a diagnosis of anthrax.



13 | P a g e

Specialized media, reagents and procedures

Only specialized media and reagents needed for isolation, identification, diagnosis and confirmation of

anthrax are provided.

Staining

1. Giemsa

Mature, at least two weeks on shelf.

Dilute the stain 1:10 and insert the slide for 30 minutes to 1 hour. Wash with distilled water, dry and

examine. This stain is the best stain to be used when blood films are made from carcasses that have

been for dead a while.

2. Polychrome methylene blue (McFayden stain reaction)

Prepare a saturated solution of methylene blue in 95% ethanol by mixing ± 0.5g of the dye in 50 ml of

the alcohol. Add 30ml of this to 100ml of a 0.01% KOH solution in distilled water. Add K2CO3 to a final

concentration of I%.

Allow to stand in half-filled bottles stoppered with cotton-wool plugs. The bottles should be shaken

periodically for fuller aeration. Oxidation ("ripening") takes several months.

Make thin smears and air dry. Fix by dipping in absolute or 95% methanol or ethanol for 30-60

seconds and re-dry. Put a large drop of polychrome rnethylene blue on the smear and spread to cover

all parts of the smear. Leave for 30-60 seconds. Wash with water, blot and dry. Under oil immersion

(100X) the capsule is seen clearly (pink) surrounding the blue-black, often square-ended bacilli.

Although the McFayden stain reaction gives the best results in fresh cases, it was found that during

putrefaction the capsule loses its affinity for methyIene blue

3. CAM's Quick stain

It can be used with great success, but mainly on fresh cases.

"PLET" Selective medium

PLET (Knisely, 1966) is the best selective agar currently available for isolation of B. anthracis from

animal or environmental specimens contaminated with other organisms, including other Bacillus

species.

"Difco" heart infusion agar (or Difco heart infusion broth with agar base) is made up according to the

manufacturer's instructions. EDTA (0.3g/1) and thallium acetate (0.04g/1) are added before

autoclaving. After autoclaving, the agar is cooled to 50°C and polymyxin (30 000 units/1) and

lysozyme (300 000 units/1) are added. After swirling the, agar is poured into Petri dishes.



14 | P a g e

Polymixin blood agar

This is useful for testing an unheated suspension of old, decomposed or processed animal or

environmental specimens and reduces or prevents growth of many Gram-negative species.

Polymixin.B sulphate should be added to a level of 100 000 units/litre of medium to the cooled blood

agar base at the same time as adding the blood.

Blood culture

Capsule formation can be demonstrated by transferring a pin-head quantity of growth from a suspect

colony to approximately 2,5ml defibrinated blood in a sterile test tube or small bottle and incubating 5

hours to overnight. A smear is made from the blood, stained and examined microscopically.

Defibrinated horse blood seems to be the best.

Laboratory animal tests

In view of concerns about animal welfare, and of the increasing reliability and sophistication of

alternative in vitro methods, the use of animals for isolation or confirmation of identity of B. anthracis

can and should generally be avoided. There are however, still occasions where it is used, such as

animals that were treated before the specimen was taken and where environmental samples contain

sporostatic substances.

Confirmation of identity or of virulence can be done by injecting light suspensions (approximately

10 000 colony-forming units/ml) into mice (0.05-0,1 ml subcutaneously) or guinea pigs (0,1-0.2 ml

intramuscularly). Virulent B. anthracis will kill the animals in about 42 - 48 hours. Blood smear

examination should reveal large numbers of capsulated bacilli.

If soil or environmental samples are used the animals should be inoculated the day before with

subcutaneous doses of mixed gas gangrene antisera and anti-tetanus sera. If unavailable, the

material to be exacmined should first be heated at 62.5°C for 15 min.

"String of Pearls’ test

Prepare a solution of sodium benzyl penicillin in sterile distilled water to contain 50 units/ml. Add 1 ml

of this to 100 ml of melted blood agar base and pour 25 ml into Petri dishes. Allow to set. Using a

scalpel blade, cut a block approximately 1.5 cm square from the penicillin agar plate and place it on a

microscope slide. Put the slide in a Petri dish together with a small piece of moistened cotton wool.

Make a line with a suitable marker along the length of a clean cover slip and about 5 mm from one

edge. Touch a loop to the edge of a young vigorous growing colony of the suspect culture and streak it

along the centre of the agar block on the slide. Place the cover slip so that the line (see above) is face

down along the streak of the culture. The line then acts as focusing and location guides. Put the lid on

the Petri dish and place in a 37°C incubator. After 2 hours, place the slide on a microscope stage.

Focus on the line with the X10 objective and bring the high dry or oil immersion lens into use to look

for the ‘string of pearls’



15 | P a g e

Gamma phage

Inoculate a blood agar plate or other suitable medium evenly over its entire surface with a loopful of

the test culture. If necessary, allow the plate to dry for a few minutes. Place a loopful or small drop of

phage suspension in the centre of the plate and incubate at 37°C. The phage inhibition should be

readily apparent at 6-8 hours of incubation, although it can be read overnight.

Motility

Various tests for motility are available. The two most reliable methods are the hanging drop method

and growth in semi-solid agar (in a Craigie tube).



16 | P a g e

APPENDIX 1

Propagation and concentration of the gamma phage

Please note that a few isolates will be gamma-phage negative and a few B. cereus isolates with be

gamma-phage positive. Thus it should be used in a panel of tests.

i) Spread a blood agar plate with the Sterne’s vaccine strain of B. anthracis.

ii) Inoculate 10ml of nutrient broth with growth from the blood agar plate and incubate for 4 hours

(until just cloudy), then refrigerate.

iii) Spread 100µl of the culture from ii) and spread onto three blood agar plates and incubate at 37°C

for 30 to 60 minutes.

iv) Spread 100µl of the gamma phage suspension over the plates and incubate overnight at 37°C.

v) Harvest the phage-lyzed growth on the blood agar plate into 5 ml nutrient broth followed by a

second wash in 5 ml nutrient broth. Incubate overnight at 37°C.

vi) Filter using a 0.45µm filter and retain the filtrate.

vii) Repeat steps iii) to, vi) once more to increase the concentration of gamma phage.

viii) Inoculate 100 ml of brain heart infusion broth with 2.5 ml of the culture from ii) and incubate on a

rotary shaker at 37°C until just turbid.

ix) Add 20 ml of the filtrate from vii) and incubate overnight at 37°C.

x) Filter. The resultant filtrate should be checked for sterility and titrated in ten-fold dilutions to

determine the concentration of the phage. Running the test in triplicate, 20µl of diluted filtrate is

placed on lawns of the B. anthracis culture. For the best results 108 – 10

9 plaque-forming units

per ml should be obtained.

chapter 4: the identification of species with special ... · bacillus anthracis is generally...

Documents