11 Bacillus cereus
11.1 Introduction 2012): B. anthracis is nonmotile and usually nonhemo-
Bacillus cereus is a pathogenic bacterium, which causes
foodborne diseases classified by the International Com-
mission on Microbiological Specifications for Foods
(ICMSF, 2002) in Risk Group III: “diseases of mod-
erate hazard usually not life threatening, normally of
short duration without substantial sequelae, causing
symptoms that are self-limiting but can cause severe
discomfort”.
11.1.1 B. cereus Group
Bacillus cereus, Bacillus anthracis, Bacillus thuringiensis,
Bacillus mycoides, Bacillus pseudomycoides and Bacillus
weihenstephanensis constitute a group of Bacillus species
(B. cereus Group) very closely related and, from a prac-
tical point of view, difficult to distinguish from each
other. They are Gram-positive rods, sporeforming,
facultative anaerobic and each species is differentiated
from B. cereus by, basically, for one single characteristic
(Bennett and Belay, 2001, Euzéby, 2003). The main
characteristics of B. cereus Group are summarized in
Table 11.1.
B. anthracis is pathogenic to man as well as B. cereus.
According to Euzéby (1998) B. anthracis is a feared
bacterium due to its high potential for use as a
biological weapon. It causes a disease that is known as
anthrax, which affects herbivorous animals (particularly
ruminants) and which may be transmitted to man,
mainly through contact with infected animals. Transmis-
sion occurs through exposure to respiratory, cutaneous
or gastrointestinal secretions, and poses an impor-
tant risk to animal health professionals, breeders and
shearers. Differentiation from B. cereus (Tallent et al.,
7007TS-DASILVA-Book.indb 119
lytic after 24 h of incubation. A few B. cereus strains
are also nonmotile but the cultures usually are strongly
hemolytic and produce a 2–4 mm zone of complete (β)
hemolysis surrounding growth after 24 h.
B. thuringiensis is pathogenic to insects, and
as such has been used as a biological control agent
(bio-insecticide) in agriculture for more than 30 years
(Valadares-Inglis et al., 1998). During the sporeforming
process, the cells produce crystalline inclusions (par-
asporal crystals), composed of one or more polypeptides
(delta-endotoxins), which are encoded by Cry genes. The
toxins are active against the larvae of the insects of the
Lepidoptera, Coleoptera and Diptera orders, as well as
against nematodes, mites and ticks (Dean, 1984). They
do not affect man, animals or plants (Souza et al., 1999).
B. cereus does not produce protein toxin crystals.
B. mycoides exhibits a typical colony morphology
on solid culture media (rhizoid colonies), reminding
fungal growth. From the point of inoculation onwards,
multiplication occurs in chains of cells linked end to
end, forming long radial filaments bending to the right
or to the left (Di Franco et al., 2002). The colonies have
the appearance of roots, from which derives the term
rhizoid.
B. pseudomycoides is a new species, proposed by
Nakamura (1998), formed by a group of strains of B.
mycoides with a different fatty acids composition. The
morphological, physiological and growth characteristics
are indistinguishable from those of B. mycoides, includ-
ing the rhizoid growth on solid culture media.
B. weihenstephanensis is a new species, proposed by
Lechner et al. (1998). It is formed by psychrotrophic
strains of B. cereus, separated from the mesophilic
strain, which continue belonging to the cereus species.
It exhibits all typical characteristics of B. cereus, from
11/26/2012 11:15:27 AM
 120 Microbiological examination methods of food and water

Table 11.1 Differential characteristics of the species of Bacillus cereus group (Tallent et al., 2012)a.

Characteristic B. cereus B. thuringiensis B. mycoides B. weihenstephanensis B. anthracis

Gram reaction +(b) + + + +

Catalase + + + + +
Motility +/−(c) +/− −(d ) + −
Reduction of nitrate + + + + +
Tyrosine decomposed + + +/− + −(e)
Lysozyme-resistant + + + + +
Egg yolk reaction + + + + +
Anaerobic utilization of glucose + + + + +
VP reaction + + + + +
Acid produced from mannitol − − − − −
Hemolysis (Sheep RBC) + + + ND −(e)
Observation Produces Produces endotoxin Rhizoidal Growth at 6°C; Pathogenic to
enterotoxins crystals, pathogenic growth no growth at 43°C animals and
to insects humans
a
The data were taken from Chapter 14 of BAM Online (Tallent et al., 2012) which does not deal with B. mycoides and B. pseudomycoides
separately.
b
+, 90–100% of strains are positive.
c
+/−, 50–50% of strains are positive.
d
−, 90–100% of strains are negative.
e
−, Most strains are negative. ND, not determined.

which it differs only by its capacity to grow between 4
and 7ºC and its inability to grow at 43ºC. According
to Euzéby (2003) no intoxications have been formally
attributed to B. weihenstephanensis, but it is likely that
the pathogenicity of this species is comparable to that
of B. cereus.
11.1.2 Main characteristics
of B. cereus
The strains of B. cereus are Gram positive, usually motile
rods, occurring singly, in pairs and long chains. They
form ellipsoidal, sometimes cylindrical, subterminal,
sometimes paracentral, spores which do not swell the
sporangia. Catalase-positive, oxidase-negative, nitrate
is reduced by most strains. Egg yolk reaction is posi-
tive and tyrosine is decomposed. Resistant to 0.001%
lysozyme. Facultative anaerobic, acid without gas is
produced from glucose and a limited range of other
carbohydrates. B. weihenstephanensis is phenotypically
similar (Logan and De Vos (2009).
According to ICMSF (1996) which does not sepa-
rate B. weihenstephanensis from B. cereus, the optimal
growth temperature is between 30 and 40ºC, with
a minimum of 4ºC and a maximum of 55ºC. The
optimal pH value lies between 6.0 and 7.0, with a
minimum of 5.0 and a maximum of 8.8. The mini-
mum water activity is 0.93. According to Logan and De
Vos (2009), who separate B. weihenstephanensis from
B. cereus, the minimum temperature for B.cereus growth
is usually 10–20°C, but psychrotolerant strains grow-
ing at 6°C have been isolated. The maximum growth
temperature is 40–45°C, with the optimum at 37°C.
B. weihenstephanensis characteristically grows at 7°C
and does not grow at 43°C.
The spores of B. cereus have a level of heat resistance
comparable to that of other spores of mesophilic bacte-
ria, with a D121ºC value between 0.03 and 2.35 min and
a z value between 7.9 and 9.9ºC (in 0.067 M phosphate
buffer). In rice broth at 100ºC, they resist for 4.2 to
6.3 min (ICMSF, 1996).
The diseases caused by B. cereus are intoxications,
which result from the ingestion of toxins formed in the
food as a result of the multiplication of cells. Two types
of diseases are known:
One is the diarrheic syndrome, characterized by
abdominal pain and diarrhea, with an incubation period
from eight to 16 hours and onset of symptoms 12 to
24 hours after exposure. This disease is caused by the

7007TS-DASILVA-Book.indb 120 11/26/2012 11:15:27 AM


diarrheic toxin, a heat-sensitive protein, inactivated by
heating at 56°C/5 min (Bennett and Belay, 2001).
The other disease known to be caused by B. cereus
is the emetic syndrome, characterized by nausea and
vomiting, beginning between one and five hours after
the contaminated food is consumed. Diarrhea is not
the predominant symptom in this case, although it may
occur. It is caused by the emetic toxin, a heat-resistant
peptide which resists cooking and, also, much more
severe heat treatments, such as 120°C for more than
one hour (Bennett and Belay, 2001). The optimal tem-
perature for the production of the emetic toxin in rice is
25–30ºC (ICMSF, 1996).
According to Bennett and Belay (2001) the presence
of B. cereus in foods does not represent a health hazard,
unless it is allowed to multiply and reach populations
greater than 105 viable cells per gram. The foods most
frequently implicated in outbreaks are either cooked
products or products containing cooked ingredients,
particularly those rich in starch or proteins, such as
cooked rice, cooked pasta, cooked vegetables, soups,
vegetable salads, seed sprouts, puddings and cooked
meats. Cooking activates the spores and, if refrigeration
is not appropriate, these spores may germinate and pro-
duce toxins.
11.1.3 Methods of analysis
B. cereus counts in foods can be done by the direct plate
count method, which is the most commonly used, or
by the most probable number method, the latter being
recommended for cases in which counts lower than
103 CFU/g are expected.
In direct plating, the most frequently used medium
is Mannitol Egg Yolk Polymyxin Agar (MYP), which
combines polymyxin as selective agent and egg yolk
and mannitol as differential agents. The production of
colonies with a strong reaction of egg yolk (lecithinase
activity), characterized by a large precipitation halo, is
typical for bacilli of the B. cereus Group. The non-fer-
mentation of mannitol gives the halo surrounding the
colony a milky pink color.
Another recommended medium is Kim-Goepfert
(KG) Agar, which has the same level of sensitivity and
selectivity as MYP, but is much less used. The colonies
that grow on the KG medium are identical to those
growing on MYP, but do not show the typical color,
since the medium does not contain mannitol. Formu-
lated to stimulate the formation of free spores after
7007TS-DASILVA-Book.indb 121
Bacillus cereus 121
20–24 h incubation, it allows for immediate confir-
mation of the identity of the colonies (directly from
the incubated plates), by staining both the spores and
intracellular lipid globules (rapid confirmatory test
developed by Holbrook & Anderson). To apply this
test to the colonies obtained on MYP, it is necessary to
subculture the culture on Nutrient Agar. This way, KG
is a faster alternative for the enumeration of B. cereus.
Another advantage of KG is that other Bacillus species
that produce lecithinase, such as B. polymyxa, are unable
to form lecithinase on this nutritionally poor medium.
Confirmation of typical colonies includes two groups
of testes, the first to verify whether the isolated culture
belongs to the B. cereus Group and the second to differ-
entiate B. cereus from the other bacilli of the Group.
To confirm the culture as pertaining to the B. cereus
Group, the 4th Edition of the Compendium of Methods
for the Microbiological Examination of Foods (Bennett &
Belay, 2001) recommends the rapid confirmatory test of
Holbrook & Anderson (1980), a technique that com-
bines the Ashby’s spore stain and the Burdon’s intracel-
lular fat stain. In the Holbrook & Anderson method,
isolation of B. cereus is achieved in PEMBA (Polymyxin
Pyruvate Egg-Yolk Bromothymol Blue Agar). According
to the authors, only B. cereus, among the Bacillus spe-
cies capable of growth on PEMBA, present intracellular
lipid globules. Due to the similarity in composition and
differential characteristics, PEMBA may be substituted
by KG, in the method described by the Compendium.
Colonies presenting typical characteristics on MYP
or KG can also be confirmed as pertaining to the
B. cereus Group by biochemical assays. The most typical
characteristics of the group are determined, including
the test of anaerobic utilization of glucose, the tyrosin
decomposition test, the VP test, the nitrate test and the
lysozyme resistance test.
To differentiate B. cereus from the other bacilli of the
Group, the tests to be used are those that verify respec-
tively the following conditions: the production of intra-
cellular toxin crystals, rhizoid growth, and hemolytic
activity.
11.2 Plate count method APHA 2001
for Bacillus cereus in foods
Method of the American Public Health Association
(APHA), as described in Chapter 32 of the 4th Edition
of the Compendium of Methods for the Microbiological
Examination of Foods (Bennett & Belay, 2001).
11/26/2012 11:15:27 AM
 122 Microbiological examination methods of food and water
Before starting activities, carefully read the guide-
lines in Chapter 3, which deals with all details and care
required for performing plate counts of microorgan-
isms, from dilution selection to calculating the results.
The procedure described below does not present these
details, as they are supposed to be known to the analyst.
11.2.1 Material required for analysis
Preparation of the sample and
serial dilutions
• Diluent: 0.1% Peptone Water (PW) or Butterfield’s
Phosphate Buffer
• Dilution tubes containing 9 ml 0.1% Peptone Water
(PW) or Butterfield’s Phosphate Buffer
• Observation: consult Annex 2.2 of Chapter 2 to
check on special cases in which either the type or
volume of diluent vary as a function of the sample
to be examined.
Direct plate count method
• Mannitol Egg Yolk Polymyxin (MYP) Agar plates or
Kim-Goepfert (KG) Agar plates
• Laboratory incubator set to 30–32°C
B. cereus confirmation by Holbrook &
Anderson test
• Spore Stain Reagents (Malachite Green Dye and
Safranin Dye)
• Sudan Black B Solution (0.3% w/v in 70%
ethanol)
• Xylene (reagent grade)
B. cereus confirmation by biochemical tests
• Mannitol Egg Yolk Polymyxin (MYP) Agar (plates)
(if KG were used for the direct plating)
• Nutrient Agar (NA) (slants and plates)
• Phenol Red Carbohydrate Broth with 1% Glucose
(tubes)
• Tyrosine Agar (tubes)
• Voges Proskauer (VP) Broth Modified for Bacillus
(tubes)
• Nitrate Broth (tubes)
• Nutrient Broth with 0.001% lysozyme (tubes)
• Motility Medium for B. cereus
• Trypticase Soy Agar (TSA) with 5% Sheep Blood
• Anaerobic atmosphere generation system (Anaero-
gen from Oxoid, Anaerocult A from Merck, GasPak®
from BD Biosciences, or equivalent)
7007TS-DASILVA-Book.indb 122
• Voges-Proskauer (VP) Test Reagents (5% α-naphthol
alcoholic solution, 40% potassium hydroxide aque-
ous solution, creatine phosphate crystals)
• Nitrate Test Reagents (sulfanilic acid solution,
α-naphthol solution)
• Coomassie Brilliant Blue Solution
• Laboratory incubator set to 30°C
• Laboratory incubator set to 35°C
11.2.2 Procedure
A general flowchart for the enumeration of Bacillus
cereus in foods using the plate count method APHA
2001 is shown in Figure 11.1.
a) Preparation of the samples, inoculation, and
incubation: For the sample preparation and
serial dilutions, follow the procedures described
in Chapter 2. Select three appropriate dilu-
tions of the sample and inoculate 0.1 ml each
on Mannitol Egg Yolk-Polymyxin (MYP) Agar
or Kim-Goepfert (KG) Agar (using the spread
plate technique). Incubate the plates (inverted) at
30–32°C/20–24 h.
Note a.1) For solid samples with low count, inoculate 1 ml of
the first dilution and 0.1 ml of the two subsequent
dilutions (distribute 1 ml of first dilution to four
plates: 0.3 ml, 0.3 ml, 0.3 ml, and 0.1 ml). For liq-
uid samples with low count, start with 1 ml of the
sample without dilution.
Note a.2) If is necessary to count exclusively spores of
B. cereus, apply a heat shock (70°C/15 min) to the
sample.
b) Colony counting: Examine the plates for typical
B. cereus colonies. The most commonly colonies
seen on KG Agar are round, flat, dry, translucent or
creamy white, surrounded by a wide precipitate zone
of lecithinase activity. Less commonly the colonies
may have irregular edges. The colonies on MYP are
similar except that colonies and surrounding medium
are pink (mannitol not fermented). Select for count-
ing the plates containing 10 to 100 colonies.
Caution: MYP and KG allow the growth of B.
anthracis and its colonies can not be distinguished
from B. cereus colonies. The plates and all the cul-
tures isolated during confirmation should be han-
dled with care.
c) B. cereus group confirmation: Select five or more
presumptive positive colonies from the KG or MYP
agar plates for confirmation. If there are fewer than
11/26/2012 11:15:28 AM
 -1 -2 -3
10 10 10
Homogenization 1ml 1ml

9ml 9ml
PW PW

25g of sample +
225ml of Peptone Water (PW) 0.3ml 0.3ml 0.3ml 0.1ml 0.1ml 0.1ml

Manitol Egg Yolk Polymyxin (MYP) Agar or Kim-Goepfert (KG) Agar

o
30-32 C/20-24h

KG typical colonies
Typical colonies (5)

MYP typical colonies

Nutriente Agar (NA)

o
30 C/24h

SPORE AND LIPID

GLOBULES STAIN
(optional)

BIOCHEMICAL SERIE (1) BIOCHEMICAL SERIE (2)

Nutrient Agar Trypticase Soy Agar

with Sheep Blood
Nutrient Broth Phenol Red Nitrate Broth VP Broth Tyrosine Agar Nutrient Agar Motility Medium
with Lysozyme Carbohydrate Broth Modified for Bacillus for Bacillus cereus
1% Glucose o
30 C/24h+
o o o
o
35 C/48h o
o
35 C/24h 35 C/48h o
35 C/48-72h 3 days at room 30oC/18-24h 30 C/48-72h 30-32 C/24h
35 C/24h
Anaerobic temperature

LYSOZYME GLUCOSE USED NITRATE VP TEST TYROSINE CRYSTAL PROTEIN MOTILITY RHIZOID HEMOLYTIC
RESISTANCE IN ANAEROBIC TEST (+) DECOMPOSITION STAINING TEST GROWTH ACTIVITY
TEST CONDITION (usually +) (+) Bacillus cereus Bacillus cereus Bacillus cereus Bacillus cereus
(+) (+) (-) (usually +) (-) (+)

B. cereus group confirmation Differentiation of species of B. cereus group

Confirmed cultures Confirmed cultures

Bacillus cereus
CFU/g

Figure 11.1 Scheme of analysis for the enumeration of Bacillus cereus in foods using the plate count method APHA 2001 (Bennett & Belay,
2001).

7007TS-DASILVA-Book.indb 123 11/26/2012 11:15:28 AM

 124 Microbiological examination methods of food and water
five colonies, select all. Transfer each colony to
nutrient agar slants and incubate at 30°C/24 h to
inoculate the test media.
The Compendium of Methods for the Microbiologi-
cal Examination of Foods (Bennett & Belay, 2001)
suggests two ways to confirm the cultures as mem-
bers of the B. cereus group, the biochemical tests of
anaerobic glucose fermentation, tyrosine decompo-
sition, Voges-Proskauer, nitrate reduction, and lys-
ozyme resistance or the Holbrook & Anderson rapid
confirmatory test (spore and lipid globules stain).
c.1) Anaerobic glucose fermentation: From
each culture, inoculate a tube of Phenol
Red Carbohydrate Broth with 1% glucose
(exhaust oxygen from the tubes before inocu-
lation). Incubate the tubes at 35°C/24 h in
an anaerobic jar, using an anaerobic atmos-
phere generation systems (Anaerogen from
Oxoid, Anaerocult A from Merck, GasPak®
from BD Biosciences, or equivalent). A color
change from red to yellow indicates a posi-
tive reaction (acid produced anaerobically).
The lack of the color change indicates a nega-
tive reaction. B. cereus and the other mem-
bers of the B. cereus group ferment glucose
anaerobically.
Note c.1) Use positive and negative control tubes
because a partial color change from red
to orange/yellow may occur even in non-
inoculated tubes (pH reduction upon
exposure of media to CO2 formed in the
anaerobic jars).
c.2) Tyrosine decomposition: From each culture
inoculate the surface of a Tyrosine Agar slant
and incubate the tubes at 35°C/48–72 h. A pos-
itive test is indicated by a clearing zone immedi-
ately under the growth (tyrosine decomposed).
B. cereus and the other members of the B. cereus
group (except B. anthracis) decompose tyrosine.
c.3) Voges-Proskauer (VP) test: From each cul-
ture, inoculate a tube of Voges Proskauer
(VP) Broth Modified for Bacillus and incu-
bate the tubes at 35°C/48 h.To test for acetyl-
methylcarbinol, transfer 1 ml of culture to a
test tube and add 0.2 ml of 40% KOH solu-
tion and 0.6 ml of 5% α-naphthol alcoholic
solution. Shake, and add a few crystals of cre-
atine. Observe results after 15 min at room
temperature. Development of a pink or violet
color indicates positive result. Lack of pink
color indicates negative result. B. cereus and
7007TS-DASILVA-Book.indb 124
the other members of the B. cereus group are
VP positive.
c.4) Nitrate reduction test: From each cul-
ture, inoculate a tube of Nitrate Broth and
incubate the tubes at 35°C/24 h. To test for
nitrate reduction to nitrite, add 0.25 ml each
of nitrate test reagents (sulfanilic acid solu-
tion and α-naphthol solution) to each cul-
ture. The development of an orange color
within 10 min, indicates a positive reaction
(nitrate reduced to nitrite). If no color devel-
ops (nitrite absent), test for residual nitrate
by adding a small amount of zinc dust. An
orange color indicates a negative reaction
(nitrate is present, has not been reduced) and
the absence of color indicates a positive reac-
tion (nor nitrate or nitrite present, nitrate has
been completely reduced to N2). B. cereus and
the other members of the B. cereus group usu-
ally reduce nitrate to nitrite.
c.5) Lysozyme resistance: From each culture,
inoculate a tube of Nutrient Broth (NB)
with 0.001% lysozyme and a tube of NB
without lysozyme (control). Incubate the
tubes at 35°C/48 h and observe for growth
in the presence of lysozyme (resistant strain)
or only in NB without lysozyme (sensitive
strain). B. cereus and the other members of
the B. cereus group are resistant to lysozyme.
c.6) Holbrook & Anderson rapid confirmatory
test (spore and lipid globules stain): Colo-
nies from KG may be tested directly from the
KG plates. Colonies from MYP should be
subcultured on NA slants (30°C/24 h) before
testing. Stain procedure:
• Prepare a smear from each colony.
• Place the slide over a boiling water and
flood with the Spore Stain Reagent Mal-
achite Green Dye for two minutes. An
acceptable alternative is heating the slide at
least twice at 1 min interval with a Bunsen
burner until steam is seen.
• After 2 min, wash the slide, blot dry, and
stain for 20 min with a Sudan Black B
Solution (0.3% w/v in 70% ethanol).
• Pour the stain off, blot dry the slide and
wash with reagent grade xylene for 5–10s.
• Blot dry the slide immediately and coun-
terstain for 20s with the Spore Stain Rea-
gent Safranin Dye.
11/26/2012 11:15:28 AM

• Wash the slide, blot dry and examine
microscopically under oil immersion. The
members of the B. cereus group will show:
a) Lipid globules within the cytoplasm,
stained dark blue. b) Central-to-subtermi-
nal spores that do not obviously swell the
sporangium, stained pale to mid-green. c)
Vegetative cells stained red.
d) Differentiating members of the B. cereus group:
Presumptively identify as B. cereus those isolates
which 1) produce large Gram-positive rods with
spores that do not swell the sporangium; 2) pro-
duce lecithinase and do not ferment mannitol on
MYP agar; 3) grow and produce acid from glucose
anaerobically; 4) reduce nitrate to nitrite (a few
strains may be negative); 5) produce acetylmethyl-
carbinol (VP-positive); 6) decompose L-tyrosine;
and 7) grow in the presence of 0.001% lysozyme.
Use the tests described below to differentiate spe-
cies within the B. cereus group.
d.1) Motility Test: From each culture, inoculate a
tube of Motility Medium for B. cereus by stab-
bing. Incubate the tubes at 30°C/18–24 h.
Motile strains grow away from the stab and
non-motile strains growth only in and along
the stab. Alternatively, the motility can be
observed by microscope. Add 0.2 ml sterile
distilled water to the surface of a Nutrient
Agar (NA) slant and inoculate the slant with a
loopful of the culture. Incubate the NA slants
at 30°C/6–8 h and suspend a loopful of the liq-
uid culture from the base of the slant in a drop
of sterile water on a microscope slide. Apply a
cover glass and examine microscopically under
oil immersion. Most strains of B. cereus and B.
thuringiensis are actively motile. B. anthracis
and B. mycoides are nonmotile. A few B. cereus
strains are also non-motile.
d.2) Rhizoid growth: From each culture, inocu-
late a pre-dried plate of Nutrient Agar (NA)
by gently touching surface of medium near
the center of each plate. Incubate the plates
at 30°C/48–72 h and examine for devel-
opment of rhizoid growth, which is char-
acterized by production of colonies with
root-like structures extending from the point
of inoculation. This property is characteristic
only of B. mycoides.
d.3) Hemolytic activity: Use one plate of Tryp-
ticase Soy Agar (TSA) with 5% Sheep Blood
7007TS-DASILVA-Book.indb 125
Bacillus cereus 125
to inoculate eight cultures. Mark the bottom
of the plate into eight sections and inoculate
each section by gently touching the medium
surface with a loopful of the culture. Incu-
bate the plates at 30–32°C/24 h and examine
for hemolytic activity, indicated by a clear
zone of complete hemolysis surrounding
the growth. B. cereus is hemolytic, B. thur-
ingiensis and B. mycoides are often weakly
hemolytic (produce hemolysis zone smaller
than B. cereus or restricted to the region
under the growth) and B. anthracis is usually
nonhemolytic.
d.4) Crystal protein staining (method Sharif &
Alaeddinoglu, 1988): Inoculate the culture
onto a slant of Nutrient Agar (NA), incubate
at 30°C/24 h and then held at room tempera-
ture for two or three days. Prepare a smear
from the culture and heat fix with minimal
flaming. Dip the slide into a small container
containing a Coomassie Brilliant Blue Solu-
tion (coomassie brilliant blue 0.25 g + abso-
lute ethanol 50 ml + glacial acetic acid 7 ml
+ water 43 ml) for 3 min. Wash the slide
with tap water, dry and examine microscopi-
cally under oil immersion. B. thuringiensis
will show free spores and toxin crystals. The
released crystals can be distinguished from
the spores since they stain purple and display
a unique diamond (tetragonal) shape, while
spores remain white and elliptical in appear-
ance. Vegetative cells appear as purple rods.
Crystals and spores appear as white bodies
within purple stained cells. B. cereus does not
produce crystals.
Note d.4.1) The procedure described in the Compen-
dium is different, more laborious, and uti-
lizes methanol – a toxic material that must
be handled with care. Prepare a smear from
the culture, fix with minimal flaming and
further fix by flooding the slide with meth-
anol. After 30s, pour off the methanol and
dry the slide by passing it through a flame.
Flood the slide with 0.5% aqueous Basic
Fuchsin Solution or TB Carbol Fuchsin
ZN (Mycobacterium tuberculosis carbol
fuchsin Ziehl-Neelsen stain) and gently heat
the slide until steam is seen. After 1–2 min
heat the slide again until steam is seen, held
for 30s, and pour off the stain. Rinse the
slide in water, dry and examine under oil
immersion. The strains of B. thuringiensis
11/26/2012 11:15:28 AM
 126 Microbiological examination methods of food and water
will present free spores and a large quantity
of red-stained, tetragonal-shaped crystals.
e) Calculation of results: Calculate number of
B. cereus cells/g of sample, based on percentage of
colonies tested that are confirmed as B. cereus.
Example: The presumptive count obtained with
10−4 dilution of sample was 65. Four of five col-
onies tested (80%) were confirmed as B. cereus.
The number of B. cereus cells/g of food is
(65 × 0.8 × 104 × 10) = 5.2 × 106 CFU/g (dilu-
tion factor is tenfold higher than sample dilution
because only 0.1 ml was tested).
11.3 Most probable number (MPN)
method APHA 2001 for
Bacillus cereus in foods
Method of the American Public Health Association
(APHA), as described in Chapter 32 of the 4th Edition
of the Compendium of Methods for the Microbiological
Examination of Foods (Bennett & Belay, 2001).
The MPN method is a suitable alternative to the
direct plate count for examining foods that are expected
to contain fewer than 1000 B. cereus per gram.
Before starting activities, carefully read the guidelines
in Chapter 4, which deals with all the details and care
required for MPN counts of microorganisms, from
dilution selection to calculating the results. The proce-
dure described below does not present these details, as
they are supposed to be known to the analyst.
11.3.1 Material required for analysis
Preparation of the sample and
serial dilutions
• Diluent: 0.1% Peptone Water (PW) or Butterfield’s
Phosphate Buffer
• Dilution tubes containing 9 ml 0.1% Peptone Water
(PW) or Butterfield’s Phosphate Buffer
• Observation: consult Annex 2.2 of Chapter 2 to
check on special cases in which either the type or
volume of diluent vary as a function of the sample
to be examined.
Presumptive counting
• Trypticase Soy Broth (TSB) with Polymyxin (tubes)
• Laboratory incubator set to 30°C
7007TS-DASILVA-Book.indb 126
Confirmation
• The same items required as for the plate count
method APHA 2001 (11.2.1)
11.3.2 Procedure
A general flowchart for the enumeration of Bacillus
cereus in foods using the Most Probable Number (MPN)
method APHA 2001 is shown in Figure 11.2.
a) Preparation of the samples and inoculation. For
the samples preparation and serial dilutions (10−1,
10−2 and 10−3) follow the procedures described in
Chapter 2. Inoculate three 1 ml portions of each
dilution into three tubes containing 10 ml of Tryp-
ticase Soy Broth (TSB) with Polymyxin. Incubate
tubes at 30°C/48 h and examine for dense growth
typical of B. cereus.
b) Confirmation. Streak the cultures from pre-
sumptive positive tubes on Mannitol Egg Yolk
Polymyxin (MYP) Agar and incubate plates at
30–32°C/20–24 h. Examine plates for characteris-
tic colonies (described in the plate count method
APHA 2001 (11.2.2.b) and select one or more typ-
ical colonies of each plate for confirmation. Con-
tinue procedure for confirmation, as described in
the plate count method APHA 2001 (11.2.2.c-d).
In the absence of suspected colonies, consider the
tube negative for B. cereus.
c) Calculation of results. Record the number of con-
firmed tubes and determine the MPN/g or MPN/
ml as detailed in Chapter 4, using one of the MPN
tables.
11.4 References
Bennett, R.W. & Belay, N. (2001) Bacillus cereus. In: Downes, F.P.
& Ito, K. (eds). Compendium of Methods for the Microbiological
Examination of Foods. 4th edition. Washington, American Public
Health Association. Chapter 32, pp. 311–316.
Dean, D.H. (1984) Biochemical genetics of the bacterial insect-
control agent Bacillus thuringiensis: basic principles and prospect
for genetic engineering. Biotechnology and Genetic Engineering
Reviews, 2, 341–363.
Di Franco, C., Beccari, E., Santini, T. Pisaneschi, G. & Tecce, G.
(2002) Colony shape as a genetic trait in the pattern-forming
Bacillus mycoides. BMC Microbiology, 2 (1), 33–48.
Euzéby, J.P. (1998). Bacillus anthracis. In: Euzéby, J.P. Dictionnaire
de Bactériologie Vétérinaire. [Online] France. Available from:
11/26/2012 11:15:28 AM
 -1 -2 -3
10 10 10
Homogenization 1ml 1ml

9ml 9ml
PW PW
25g of sample +
225ml of Peptone Water (PW)

1ml 1ml 1ml 1ml 1ml 1ml 1ml 1ml 1ml

Trypticase Soy
Broth (TSB)
with Polymyxin

30oC/48h

Tube with growth

Streak a loopful

KG typical colonies Mannitol Egg Yolk Polymyxin (MYP) Agar or

Kim-Goepfert (KG) Agar
30-35oC/24h

MYP typical colonies

Nutriente Agar (NA)

30oC/24h

SPORE AND LIPID

GLOBULES STAIN
(optional)

BIOCHEMICAL SERIE (1) BIOCHEMICAL SERIE (2)

Nutrient Agar Trypticase Soy Agar

with Sheep Blood
Nutrient Broth Phenol Red Nitrate Broth VP Broth Tyrosine Agar Nutrient Agar Motility Medium
with Lysozyme Carbohydrate Broth Modified forBacillus for Bacillus cereus
1% Glucose 30oC/24h+
35oC/48h 35oC/24h 35oC/48h 35oC/48-72h 3 days at room 30oC/18-24h 30oC/48-72h 30-32oC/24h
35oC/24h
Anaerobic temperature

LYSOZYME GLUCOSE USED NITRATE VP TEST TYROSINE CRYSTAL PROTEIN MOTILITY RHIZOID HEMOLYTIC
RESISTANCE IN ANAEROBIC TEST (+) DECOMPOSITION STAINING TEST GROWTH ACTIVITY
TEST CONDITION (usually +) (+) Bacillus cereus Bacillus cereus Bacillus cereus Bacillus cereus
(+) (+) (-) (usually +) (-) (+)

B. cereus group confirmation Differentiation of species of B. cereus group

Confirmed cultures Confirmed cultures

Bacillus cereus
MPN/g

Figure 11.2 Scheme of analysis for the enumeration of Bacillus cereus in foods using the Most Probable Number (MPN) method APHA
2001 (Bennett & Belay, 2001).

7007TS-DASILVA-Book.indb 127 11/26/2012 11:15:28 AM

 128 Microbiological examination methods of food and water
http://www.bacterio.cict.fr/bacdico/bb/anthracis.html [Accessed
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Euzéby, J.P. (2003). Systématique des espèces placées dans le “groupe
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naire. [Online] France. Available from http://www.bacterio.cict.
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Holbrook, R. & Anderson, J.M. (1980) An improved selective
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ICMSF (International Commission on Microbiological Specifica-
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Schleifer, K. & Whitman, W.B. (eds). Bergey’s Manual of Sys-
tematic Bacteriology. 2nd edition, Volume 3. New York, Springer,
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Nakamura, L.K. (1998) Bacillus pseudomycoides sp. nov. Interna-
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Tallent, S.M., Rhodehamel, E.J., Harmon, S.M. & Bennett, R.W.
(2012) Bacillus cereus. In: FDA (ed.) Bacteriological Analytical
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enceResearch/LaboratoryMethods/BacteriologicalAnalytical-
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