An improved selective and diagnostic medium for the isolation and enumeration of Bacillus

cereus in foods
R. HOLBROOK
A N D JUDITH
M. ANDERSON
Utlileuo Reserrrch Lnhorcrrory, Colnjorrh Hortse. Shnrtlbrook, Berlfordshire, M K 4 4 ILQ, E t ~ g l n t ~ d
Accepted March 25. 1980

HOLBROOK. R., and J . M. ANDERSON. 1980. An improved selective and diagnostic medium for the
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by YORK UNIV on 08/11/14

isolation and enumeration of Bncillrrs certrrs in foods. Can. J. Microbiol. 26: 753-759.
The use and performance of an improved diagnostic and selective medium, PEMBA (polymy-
xin pyruvate egg yolk mannitol bromothymol blue agar), for the detection of B(rci1lus cereus in
foods is described. The distinct colonial appearance of B. cererrs on PEMBA permitted the
recognition of both strains: those that d o precipitate egg yolk and those that d o not react with egg
yolk. A staining procedure, used to demonstrate microscopically both the presence of lipid
globules in vegetative cells and spore morphology of isolates. proved a rapid and reliable
confirmatory test whichgave completeagreement with a battery ofbiochemical testsusedfor this
purpose. The quantitative recovery of B . cereus on PEMBA from 143 food samples was not
significantly different from counts on KG (Kim and Goepfert), MYP (mannitol egg yolk phenol
red), and McClung's media, and the selectivity of PEMBA was generally superior.

HOLBROOK, R., et J . M. ANDERSON, 1980. An improved selective and diagnostic medium for the
isolation and enumeration of Bncillrrs crrrrrs in foods. Can. J. Microbiol. 26: 753-759.
Nous decrivons I'utilisation et la performance d'un milieu diagnostique et selectif ameliore. le
PEMBA (polymyxin pyruvate egg yolk mannitol bromothymol blue agar). pour la detection de
For personal use only.

Bncillrrs cerc,rrs dans les aliments. L'apparence distincte des colonies de B. cereus sur PEMBA
permet de reconnaitre a lafois les souches qui precipitent le jaune d'ceuf et celles qui ne reagissent
pas avec ce dernier. Une technique de coloration qui est utilisee en microscopie pour reveler la
presence de globules lipidiques dans les cellules vegetatives et la morphologie des spores des
isolats, s'est averee un test rapide et fiable de confirmation en complet accord avec une ensemble
de tests biochimiques utilises a cette fin. La recouvrement quantitatifde B . crrcrrs sur PEMBA ne
differait pas sensiblement, pour 143 echantillons d'aliments, des dknombrements sur milieux KG
(Kim et Goepfert), MYP (mannitol egg yolk phenol red) et McClung; la selectivite du PEMBA
leur etait generalement superieure.
[Traduit par le journal]
Introduction principal diagnostic characteristic used in all three
Bacillus cereus is widely distributed in nature media is the egg yolk reaction, precipitation of hy-
and is commonly found in cereals, milk, and dried drolysed lecithin. However, all three media have
food ingredients such as spices and herbs (Mossel some drawbacks. Differentiation of mannitol fer-
et al. 1967; Kim and Goepfert 1971~;Davies and menting colonies on MYP agar is poor after 40 h of
Wilkinson 1973). This organism can cause food- incubation at 32°C (Kim and Goepfert 1971b). The
borne illness through the ingestion of enterotoxin egg yolk reaction of KG agar is often weak and
produced during its growth to high numbers frequently there is poor differentiation ofB. cereus
(> 106/g)in certain foods (Goepfert et al. 1972). The colonies from those of other organisms on KG agar
source of contamination is from spores, naturally and McClung's medium. These media give only
present in a food, that are able to survive normal presumptive counts of B. cereus and suspect col-
cooking procedures. Blood agar is suitable for the onies require further biochemical tests for
detection of large numbers of B. cereus in foods confirmation.
implicated in food poisoning incidents (Hauge This paper describes the formulation and per-
1955; Gilbert and Taylor 1976) but a selective formance of a new selective and diagnostic
medium is necessary for the detection or enumera- medium, PEMBA (polymyxin pyruvate egg yolk
tion of low numbers of B. cereus in the presence of mannitol bromothymol blue agar) for the iso-
other organisms. Two such media have been de- lation of B. cereus from foods. This medium was
scribed: MYP (mannitol egg yolk phenol red) developed to meet the requirements for a medium
medium of Mossel et al. (1967) and KG medium of that is sufficiently selective to be able to detect
Kim and Goepfert (1971b). Also the nonselective small numbers of B. cereus cells and spores in the
but diagnostic McClung medium (McClung et al. presence of large numbers of other organisms, and
1946) is used for detecting B. cereus in food. The sufficiently diagnostic that colonies of B. cereus
0008-4 166/80/070753-07$01.OO/O
GI980 National Research Council of Canada/Conseil national de recherches du Canada
 754 C A N . J. MICROBIOL. VOL. 26. 1980
were readily identified and this identity confirmed
by a rapid test.
Materials and methods
B(rc~torir11 strains
Thirty-one strains of B . c,ererts were used in this study. These
included 13 enterotoxigenic strains of B . cereus isolated from
food poisoning incidents, kindly provided by Dr. R. J . Gilbert,
Central Public Health Laboratory, Colindale, London.
Twenty-two other Bacilllts species (60 strains including desig-
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nated strains of all species) were also examined. These are listed
below with the number of strains in parentheses: B . nlycoidts
(I), B . thurirlgiensis (3). B. trlegnterirrnl (9), B . lichenfirmis (6),
B . srrbtilis (l5), B.prrrnilus (5). B . j r n l u s (I), B . coagulrrns(l), B .
polyn7y.ra (3), B . mrrcerar~s( I ) , B . rrlvei (2). B . lnterosporrrs ( I ) ,
B . breuis (2), B . circrrlnns (I), B . sphaericrrs (2). B . badirrs (I), B .
globisporrrs (I), B . lentus (I), B . insolitlrs ( I ) , B . psychrophilus
(I), B . psychrosncchnrolyticus ( I ) , and B. pantotherzticus (I).
Ten other bacterial species capable of hydrolysing egg yolk were
also examined. These were Achrot?lobrrctrr brrtyri, Achrotno-
bnctrr lipolytic~rrn,Aerotnotras sp., CIostridirrt7r pc~rfringc~ns,
Protrus urrlgaris, Psc~udornonas aerugirlosrr, Psercdotrlurlas
Juorescens. Stnphylococcus arrrerrs, Strrphyloccrts rpidermidis,
and Serrrrtia mnrcescens.
Cor?~positiorla r ~ d p r e p n m t i o rof
~ PEMBA
The basal medium contained (grams per litre) peptone
For personal use only.
(Evans Medical Ltd., Speke. Liverpool. England), 1.0; D-
mannitol. 10; M g S 0 , . 7 H 2 0 , 0.1; NaCI, 2.0; Na,HPO,, 2.5;
KH,PO,. 0.25; bromothymol blue (water soluble) (BDH Chemi-
cals. Poole, England), 0.12; and agar (Difco). 18. The medium
was steamed to dissolve the ingredients and adjusted to pH 7.4.
The medium was dispensed in 90-mL amounts into screw-
capped bottles and autoclaved at 12I0Cfor 15 min (final p H 7.2).
For use, the following filter sterilized solutions were added to
the molten cooled (48-50°C) base: 20% wlv sodium pyruvate
(Koch Light Laboratories Ltd., Colnbrook, Buckinghamshire,
England). 5 mL; polymyxin (Aerosporin) (Wellcome La-
boratories, Beckenham, Kent, England) to a final concentra-
tion of 100 units/mL (= 10 pg/mL); and egg yolk emulsion (see
below), 5 mL. A filter sterilized aqueous solution of 0.4% w/v
actidione (I mL) was added to the molten cooled medium when
foods suspected of containing large numbers of moulds were
examined (Sinell and Baumgart 1967). Eight plates (9.0-cm
diameter) were poured from each bottle and the plates driedfor a
minimal time before inoculation. Surface inoculated plates were
incubated at 3 P C and examined after 24 h of incubation, and
then left at room temperature for a further day and re-examined.
Suspect colonies were marked on the plates each day.
McClung's medium
The basal medium was prepared as described by McClung et
al. (1946) except that the agar concentration was reduced to
1.5%. Egg yolk emulsion (10%) was added to the molten cooled
agar immediately before use; the medium was used a s pour
plates. Plates were incubated at 30°C for 3 days.
Kim and Goepfert's K G medium
The medium was prepared and used a s described by Kim and
Goepfert (197 lb). Surface inoculated plates were incubated at
3 P C for 24 h.
Mossel's M Y P Medium
The formula described by Mossel et al. (1967) was followed.
Dried plates were surface inoculated and incubated at 30°C (not
32°C a s recommended) and examined after 1 and 2 days' incuba-
tion.
Owens' E Y ( e g g yolk) broth rrrlrl EY agar
These were prepared and inoculated a s described by Owens
(1974). The hydrolysis of the egg yolk w a s observed during 5
days' incubation at 3 P C .
Egg yolk c,mulsion
Egg yolk emulsion was prepared in sterile distilled water
following the method of Billing and Luckhurst (1957). Each
preparation was checked for sterility and used within 3 days o r
sterilized by filtration through a Carlson Ford HPIEKS Sterimat
(Carlson Ford Sales Ltd., Ashton-under-Lyne. Lancashire, En-
gland) and used within 4 weeks.
Staining procedrrre
The objective was t o determine vegetative cell, sporangium,
and spore morphology, and demonstrate the presence of lipid
globules within the vegetative cell (Gordon 1973). The method
was developed by combining the spore stain of Ashby (1938)
with the intracellular lipid stain of Burdon (1946). Films were
made from the centre of I-day-old colonies or from the edge of
2-day-old colonies. Films were air dried and fixed with minimal
flaming. The slide w a s placed over boiling water and flooded
with 5% wlv malachite green. After 2 min the slide was washed,
blotted dry, and stained with 0.3% w/v Sudan black B in 70%
ethyl alcohol for 15 min. The slide w a s washed in xylol f o r 5 s
and blotted dry before counterstaining with 0.5% w/v safranin
for 20 s.
Enrrrneratiorl of B. cereus irl food srrn~ples
Samples ( l o g ) of various meat products were homogenized
for 30s in 90-mL amounts of 0.1% peptone water using a
Stomacher 400 (A. J. Seward, Bury St. Edmunds, Suffolk, En-
gland). Dried foods of commercial origin, including flour, rice,
herbs and spices, dehydrated vegetables, and dried milk and egg
powder, were rehydrated by soaking 20 g of sample in 90 rnL of
tryptone salt solution for 50 min at room temperature. A further
90 mL of 0.1% peptone water was added to give a final dilution of
1/10 before homogenizing for 30 s in a Stomacher 400. Locally
purchased bottled pasteurized milk w a s examined periodically
over a 4-month period. The cream from the top of each bottle
w a s sampled without mixing the contents.
Decimal dilutions of the milk and further decimal dilutions of
the homogenates were made in 0.1% peptone water. One-
tenth-millilitre amounts of the 1/10 and higher dilutions were
inoculated onto the surface of KG, MYP, and PEMBA; 1.0 -mL
amounts were used to prepare pour plates of McClung's
medium.
Biochemical tests
The following tests (Gordon 1973) were performed t o confirm
the identity of B. cereus isolates. Aerobic and anaerobic dis-
similation of glucose with acid production was determined using
Hugh and Leifson's method a s described by Baird-Parker
(1963); inoculated tubes were incubated at 3 P C for 14 days.
Tests to demonstrate acid production from mannitol, xylose,
and arabinose, hydrolysis of soluble starch, and citrate utiliza-
tion (Christensen's medium) were performed a s described by
Cowan and Steel (1965). Tests for t h e hydrolysis of gelatin,
hydrolysis of casein, and the production of acetylmethylcar-
binol were performed a s described by Smith et a l . (1952).
Nitrite production was tested after growth in nitrate broth
(Cowan and Steel 1%5) by adding I m L of broth to 1 mL of 2%
wlv sulphanilamide in 1 N HCI and 0.2 m L of naphthyl reagent
(0.1% wlv N-(2-naphthy1)ethylenediamine dihydrochloride in
distilled water) (Hanson 1973). The presence of nitrite was
shown by the development of a pale red-brown colour. Nega-
tive results were confirmed by reducing nitrate to nitrite using
spongy cadmium. Growth on chloral hydrate agar was deter-
 HOLBROOK A N D ANDERSON 755

1. Egg yolk reaction on PEMBA and microscopical appearance of 3 1 strains of B. cerer~s

TABLE

Microscopy of stained
films from PEMBA at 48 h
Egg yolk reaction
Comments on PEMBA at 48 hb Sporesc Fat globulesd

F H 3 100,48 10,3605
4429,4430,4433 Implicated in food poisoning episodes
F H 2426,3463,
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4431,4432 Implicated in food poisoning episodes

F H 1443,3552 Implicated in food poisoning episodes
F H 1444 Implicated in food poisoning episodes
F H 3502 Implicated in food poisoning episodes f Scanty
F H 4096 Implicated in food poisoning episodes Negative Nil
2 Heat resistant
CBCC 318, 325,605,
622,629, 1580,1940,
1942, 1977, 1978,
1979,1980,2245 Nil ++ ++
CBCC 33 1 Asporogenous mutant ++ Nil
CBCC 2280 Nil Negative Scanty
oFH, enterotoxigenic strains, received from R . J. Gilbert; strain 7, received from Bradshaw cr a/. (1975); CBCC, Colworth Bacterial Culture
Collection.
bEgg yolk reactions: -;-- i - , wide ~recipitate;-i-, narrow dense precipitate; 2 ,weak narrow precipitate; negative, no precipitate aroundcolony.
'Spores: 4-i-,numerous spores and sporangia; -i-, small numbers of spores and sporangia; scanly, several fields scanned to find any spores or
For personal use only.

sporangia.
d - t , fat globules present in vegetative cells.

m~neduslng Kn~sely'smethod (Knlsely 1965). Plates were In-

both mannitol utilizing and nonmannitol utilizing
oculated w ~ t ha s~nglestreak of culture and Incubated at 30°C
organisms. Finally polymyxin, at 100 unitslml,
overn~ght
was added to improve the selectivity of the medium
Results but actidione (40 pglml) was also found to be neces-
Deueloptnetzt cind performance of PEMBA sary to supress the growth of moulds when large
Examination of the colonial appearance of numbers of these were present in an inoculum.
strains of B. cereus on MYP, KG, and McClung's Thirty-one strains of B. cereus (Table 1 ) after
media showed obvious differences. On KG medium 18-24 h of growth on PEMBA ranged from crenate
B. cereus formed markedly rhizoid colonies, > 1 cm or fimbriate colonies to slightly rhizoid forms (2- to
in diameter, and the egg yolk precipitate was much 5-mm diameter) turquoise to peacock blue incolour
weaker than on the other media; however, sporula- with flat ground-glass surfaces. After a further 24 h
tion on KG medium occurred during 24 h of incu- of incubation at room temperature all colonies were
bation. On MYP and McClung's medium the non- peacock blue with some strains developing a
rhizoid colonies of B. cereus gave good zones of egg greyish raised centre. Egg yolk precipitin reac-
'
yolk precipitation but sporulation was absent. tions, forming zones up to 5 mm wide around each
The lack of sporulation on McClung's medium colony, were evident from the majority of strains
was related to the high (4%) peptone content and after 24 h of incubation; these were grey to tur-
when this was reduced to 0.1%, sporulation of B. quoise blue and turned to a peacock blue colour
cereus readily occurred, but surface colonies were after 48 h. Six B. cereus strains giving weak or
now rhizoid and surrounded by a weak egg yolk negative egg yolk reactions on PEMBA (Table 1)
precipitate. Addition of sodium pyruvate to the developed typical coloured colonies and were
medium containing 0.1% peptone considerably re- treated as suspect colonies for further examination.
duced the rhizoid colonial form and markedly im- Other Bacillus species inoculated onto PEMBA
proved the egg yolk reaction, but still permitted showed that three strains of B. thuritzgietzsis
sporulation to occur after 24 h of incubation at formed colonies that were indistinguishable from
37°C. The replacement of glucose with mannitol, those of B. cereus. Bacillus mycoides grew over the
addition of bromothymol blue, and adjustment of whole agar surface in 24 h as a rhizoid turquoise
the concentration of phosphates in the medium blue colony. Other Bacillus species able to grow on
permitted good differentiation of B. cereus from PEMBA, B. aluei, B . subtilis, B. lichenifortnis, B .
 CAN. J. MICROBIOL. VOL. 26, 1980

TABLE2. Colonial characters of Bacilliis spp. on PEMBA after 48 h incubation

Colonial appearance

No. of strains Diameter, Egg yolk

Species examined mm Form Colour precipitatea

B. cererrs Crenate to slightly rhizoid Peacock blue

B. r/~irringiensis Crenate to slightly rhizoid Peacock blue
B. rl~ycoides Markedly rhizoid Turquoise blue
Flat, crenated edge Green
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B. /icIzenijbr~~ris
B. srrbrilis Flat, entire edge Grey-green
B. polynryxn Flat, entire edge Pale green
B. alvei Convex, entire edge Dark green
B. breuis Convex. entire edge Dark green
'+, positive; -, n o precipitate formed; k ,weak narrow precipitate.
*Egg yolk negative strains also described, see Table 3 and text.

TABLE3. Type of egg yolk reaction given by 20 weak or negative egg yolk reacting
strains of B. cererrs on four selective media

Numbers of strains giving reaction o n :

Type of egg yolk reaction McClung's MYP KG PEMBA

Definite narrow precipitate around colony 10 10 4 10

For personal use only.

Weak narrow precipitate around colony 5 7 2 8

Weak clearing only 1 1 9 0
N o observable reaction 4 2 5 2

polymyxa, and B. breuis, were distinguished from
B. cereus by colony form, colour, and lack of an egg
yolk precipitin reaction. (Table 2). The remaining
Bacillus species listed above failed to grow. Other
egg yolk reacting organisms able to grow on
PEMBA, including Straphylococcus aureus, Ser-
ratia marcescens and Proteus vulgaris, were dis-
tinguished from B. cereus by colony form and col-
our, together with the production of an egg yolk
clearing reaction in contrast to the egg yolk pre-
cipitate produced by B. cereus.
Dectection of weak or negative egg yolk reacting
strains of B. cereus
Twenty strains of B. cereus with weak or nega-
tive egg yolk reactions, including strains F H 3502,
F H 4096, F H 3552, F H 1443, F H 1444, and CBCC
2280 (Table l), and 14 strains isolated from foods
during this study were inoculated into Owens' EY
broth and onto the four selective media and Owens'
EY agar to give isolated colonies after incubation.
The types of egg yolk reaction observed and the
number of strains producing each type of reaction
on the selective media are given in Table 3. KG
medium performed least satisfactorily, five strains
failed to give any observable egg yolk reaction,
whilst nine strains showed some degree of clearing
of the egg yolk. McClung's medium failed to show
an egg yolk reaction with four strains of B. cereus.
MYP medium and PEMBA performed equally,
each medium failing to detect an egg yolk precipi-
tate from the egg yolk negative strains FH 4096 and
CBCC 2280. These strains also failed to give the
typical reactions of egg yolk hydrolysis described
by Owens (1974); strains FH 4096 and CBCC 2280
failed to show any degree of egg yolk hydrolysis
after 5 days' incubation at 37°C whilst the weak egg
yolk reacting strains produced very weak precipi-
tates on EY agar, and growth in EY broth resulted
in some opalescence but no fatty curd, the product
of egg yolk hydrolysis shown by typical strains of
B. cereus.
Quantitative recovery of B. cereus
Duplicate viable counts (lop6 dilution) of 13
strains of B. cereus from overnight culture in nutri-
ent broth inoculated onto nutrient agar, McClung's
medium, MYP, KG, and PEMBA gave log mean
counts of 1.86, 1.66, 13 1 , 1.78, and 1.79, respec-
tively. Analysis of these results showed that there
was a significant difference (P = 0.01) between
McClung's medium and nutrient agar but no
significant difference between counts on the other
media.
Two hundred and thirty-four food samples were
inoculated onto the four isolation media. Bacillus
cereus was not detected in 153 samples (detection
limit 100/g) and no samples contained > 104/g.
 HOLBROOK A N D ANDERSON 757

TABLE
4. Recovery of B.cereus from 39 food materials on four media

B. cererrs count x 103/ga

No. of
Food material samples McClung's MYP KG PEMBA
Flour 20 I .I-2.7b 1 .O-2.0 1.2-1.9 1.0-2.5
Milk (pasteurized) 6 1.0-5.7 1.9-6.8 1.9-5.8 1.9-7.0
Herbs 5 1.6-3.9 0.8-3.8 I .l-3.2 0.9-2.5
Spices 3 1.3-2.9 0.62.5 0.6-2.6 0.6-2.4
Asparagus (dehydrated) 3 0.6-1.3 1.2-1.9 0.6-1.9 0.7-1.6
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Mushrooms (dehydrated) 1 1.3 1.6 1.8 1.5

OMean of duplicate counts on each sample.
bRange of the counts obtained from all the samples comprising the food material indicated.

Thirty-nine samples contained between 10' and
lo4 B. cereuslg (Table 4), and analysis of variance
of the B. cereus counts on PEMBA and B. cereus
counts on MYP, KG, or McClung's medium
showed no significant difference ( P = 0.01) be-
tween the counts on each medium.
Cornpnrative selectivify of PEMBA
The examination of raw food materials with high
total counts (> IOS/g)showed that all the selective
For personal use only.
PEMBA and counts obtained on plates poured and
stored for 4 days at 4°C before use. The quality of
both the egg yolk reaction and the intensity of the
peacock blue colour of colonies grown on plates
stored for longer than 4 days at 4°C was inferior to
their appearance on freshly prepared plates.
Rapid confirmatory tesffor B. cereus colonies from
PEMBA
Well-isolated colonies of strains of B. cereus

media permitted the growth of organisms other

than B. cereus. Pour plates of McClung's medium (Table I) grown on PEMBA were examined after 1
and 2 days' growth using the staining method de-
were generally the least selective and gave rise to
the largest number of suspect colonies requiring scribed above. The spores stain pale green to mid-
confirmation as B. cereus by further tests. MYP green, depending upon their stage of development,
lipid globules are black, and the vegetative cyto-
medium was more selective but when moderate
plasm red. Vegetative cells of B. cereus generally
numbers of mannitol utilizing organisms were pres-
ent the identification of B. cereus became difficult had a characteristic appearance being 4-5 pm long
and 1.O- 1.5 pm wide with square ends and rounded
due to the poor buffering capacity of the medium.
corners; lipid globules were present in vegetative
KG medium showed slight improvement in selec-
cells of all the B. cereus strains examined (Table I).
tivity over MYP, but the weak egg yolk reaction
given by colonies of B. cereus was frequently Spores present in sporangia were central or para-
masked by the overgrowth of other organisms due central in position and did not obviously swell the
to their large colony size. PEMBA was generally sporangium (Smith et al. 1952). Two strains of B.
more selective than either MYP or KG medium. cereus, FH 4096, an egg yolk negative en-
terotoxigenic strain, and CBCC 33 1, an asporoge-
Flora other than B. cereus on PEMBA showed
nous mutant, failed to produce spores after 48 h of
about a 10-fold decrease in numbers compared with
MYP, and their smaller colony size compared with growth on PEMBA. The microscopical appearance
of B. mycoides and B. fhuringierzsis was indistin-
colonies on KG resulted in the easier recognition of
B. cereus on PEMBA. However, the distinctive guishable from B. cereus. Crystalline inclusion
bodies were not found in films of the two strains of
peacock blue colour of colonies of B. cereus on
PEMBA produced by the majority of strains after B. thuringiensis studied after growth on PEMBA
24 h of incubation was occasionally retarded by the for 48 h. None of the other Bacillus species (listed
presence of other organisms. In these cir- in Table 2) able to grow on PEMBA contained lipid
cumstances further incubation for a further 20-24 h granules in their vegetative cells.
at room temperature enhanced the colour de- The staining procedure was extensively
evaluated during the examination of food samples
velopment which, together with the use of the rapid
inoculated onto PEMBA. Colonies resembling B.
confirmatory staining precedure, confirmed their cereus after 24 and 48 h of incubation, suspect egg
identification as B. cereus. yolk negative colonies otherwise resembling B.
Effect of sforageon performance of PEMBA cereus, and a wide range of other nonmannitol fer-
There was no significant difference between the menting colonies were examined. All turquoise or
B. cereus counts obtained from 12 pure cultures and peacock blue coloured colonies, including egg yolk
10 flour samples on freshly prepared plates of negative variants, had the vegetative cell and spore
 758 C A N . J. MICROBIIOL. VOL. 26, 1980

TABLE5. Biochemical characteristics of B. cereris isolates tests, such as inability to ferment mannitol, spore
formation, and the presence of lipid globules in
Strains examined vegetative cells. It must also have good selectivity.
We have attempted to incorporate these features
Designated Food
Tests strains isolates
into a new selective medium for the isolation of B.
cereus from foods. Typical colonies of B. cereus
Anaerobic fermentation of glucose growing on PEMBA have a distinctive turquoise to
Acid production peacock blue colour surrounded by agood egg yolk
Glucose precipitate of similar colour, features that distin-
Mannitol
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Xylose
guish B. cereus from other Bacilllls species except
Arabinose B. thuritzgiensis. Bacillus thuringiensis cannot be
Hydrolysis differentiated from B. cereus on any of the other
Gelatin media examined. Other egg yolk reacting or-
Casein ganisms growing on PEMBA have colonial appear-
Starch ances that distinguish them from B , cereus. Four-
Nitrate reduced to nitrite teen weak or negative egg yolk reacting strains of
~cetylmethylcarbinolproduced B. cereus were isolated on PEMBA from 243 food
Citrate utilised samples. These organisms were recognized by
Growth on 0.25%chloral hydrate agar their characteristic peacock blue colonies.
-

.Percent o f strains tested giving positive results. The value of microscopical examination a s a n aid
to the identification of B. cereus growing on
morphology described above, and all contained PEMBA was evaluated using the staining method
lipid granules in their vegetative cells. Other non- described. Lipid globules in vegetative cells oc-
For personal use only.

mannitol utilizing Bacillus species able to grow on curred in all food isolates and type strains of B.
PEMBA differed from B. cereus in the absence of cereus, including an enterotoxigenic stain that was
lipid granules in the vegetative cells; these isolates not egg yolk reacting and was weakly sporulating.
were also shown by biochemical tests not to be B. No lipid globules were found in other Bacillus
cereus. species growing on PEMBA.
Sporangia formation and free spores mor-
Biochemical characteristics c$B. cereus lsolates phologically typical of B. cereus occurred in all
Comparisons (Table 5) among the biochem'ical food isolates and in all but two laboratory strains of
reactions of 31 type strains and 107 strains of B. B. cereus. The identification of B . cereus food iso-
cereus isolated from foods on PEMBA showed lates showing these characteristics were confirmed
over 99% correlation for glucose, mannitol, xylose, by further biochemical tests. Microscopical exami-
and arabinose utilization, gelatin hydrolysis, casein nation is therefore a rapid and simple confirmatory
hydrolysis, nitrate reduction, and growth on test. The quantitative recovery of B. cereus on
chloral hydrate agar. Citrate utilization and PEMBA did not differ significantly from the B.
acetymethylcarbinol production were more vari- cereus counts obtained on the three other media
able features but both characters were present in used. For the food samples examined the selectiv-
over 90% of isolates; starch hydrolysis was the ity of PEMBA is better than the selectivity of the
most variable, 45% of type strains and 87% of food other three media studied.
isolates giving positive results.

Discussion ASHBY,G. K. 1938. Simplified Schaeffer spore stain. Science,

The primary diagnostic character currently used
in selective media for the isolation and enumeration
of B. cereus is the precipitation of hydrolysed
lecithin, the so-called egg yolk reaction (Kushner
1957). However, some strains ofB. cereus may give
only weak or negative egg yolk reactions, and if
these strains are not to be overlooked, the isolation
medium must allow other reliable features, such as
a distinctive colony appearance, to occur. Ideally
the medium should also promote other characteris-
tics which can be used as the basis for confirmatory
87: 433-435.
BAIRD-PARKER, A. C . 1963. A classification of Micrococci and
Staphylococci based on physiological and biochemical tests.
J. Gen. Microbiol. 30: 409-427.
BILLING.E . , and E. R. LUCKHURST. 1957. Asimplified method
for the preparation of egg yolk media. J . Appl. Bacteriol. 20:
90.
BRADSHAW, J . G., J. T. PEELER,and R. M. TWEDT.1975. Heat
resistance of ideal loop reactive Bacilllis cereus strains iso-
lated from commercially canned food. Appl. Microbiol. 30:
943-945.
BURDON, K . L. 1946. Fatty material in bacteria and fungi re-
vealed by staining fixed dried slide preparations. J. Bacteriol.
52: 665-678.
 HOLBROOK AND ANDERSON
COWAN, S. T., and K. J . STEEL.1965. Manual for identification
of medical bacteria. Cambridge University Press, London.
DAVIES, F. L., and G. WILKINSON. 1973. Bacill~rscereus in milk
and dairy products. 111 The microbiological safety of food.
Edited by B. C. Hobbs and J . H. B. Christian. Academic
Press, New York and London.
GILBERT, R. J., and A. TAYLOR.1976. Bncill~rscereus food
poisoning. 111 Society of applied bacteriology symposium
series no. 4. Editedby F. A. Skinner and J. C. Carr. Academic
Press, London.
GOEPFERT, J. M.. W. M. SPIRA,and H. U. KIM. 1972. Bacill~rs
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by YORK UNIV on 08/11/14
cereus: food poisoning organism. A review. J . Milk Food
Technol. 35: 2 13-227.
GORDON,R. 1973. The genus Bacill~rs.III CRC Handbook of
microbiology. Vol. 1. Edired by A. I. Laskin and H . A.
Lechevalier. CRC Press, Cleveland, OH, U.S.A.
HANSON,P. W., (Editor.) 1973. Determination of nitrite and
nitrate in meat and meat products. 111Official standardised and
recommended methods of analysis. 2nd ed. Society for
Analytical Chemists. London.
HAUGE,S. 1955. Food poisoning caused by aerobic spore-
forming bacilli. J. Appl. Bacteriol. 18: 591-595.
K I M ,H. U., and J. M. GOEPFERT.197la. Occurrence of Bncil-
For personal use only.
759
/us cereus in selected dry food products. J . Milk Food
Technol. 34: 12-15.
1971b. Enumeration and identification of Bacill~rscerelrs
in foods. Appl. Microbiol. 22: 581-587.
KNISELY. R. F. 1965. Differential media for the identification of
Bncill~rsntlthrncis. J. Bacteriol. 90: 1778- 1783.
KUSHNER, D. J. 1957. An evaluation of the egg yolk reaction as a
test for lecithinase activity. J. Bacteriol. 43: 297-302.
MCCLUNG,L . S.. P. H E I D E N R E I Cand H , R. TOABE.1946. A
medium for the Nagler plate reaction for the identification of
certain Clostiridirr. J. Bacteriol. 51: 751-752.
MOSSEL,D. A. A,, M. J . KOOPMAN, and E. JONGERIUS. 1967.
Enumeration of Bncillrrs cereus in foods. Appl. Microbiol. 15:
650-653.
OWENS,J . J. 1974. The egg yolk reaction produced by several
species of bacteria. J. Appl. Bacteriol. 37: 137-148.
S I N E L LH.
. J.. and J. BAUMGART. 1967. Selektivnahrboden mit
Eigelb zur lsolierung von pathogenen Staphylokokken aus
Lebensmittein. Zentralbl. Bakteriol. Parasitenk. Infek-
tionskr. Hyg. Abt. 1 Orig. 203: 248-264.
SMITH,N. R., R. E. GORDON, and F. E. CLARK.1952. Aerobic
spore-forming bacteria. U.S. Dep. Agric., Agricultural
Monograph No. 16, Washington, DC.