International Journal

of Food Microbiology
International Journal of
Food Microbiology 25 (1995) 131-139

Prevalence of Bacillus cereus in selected foods

and detection of enterotoxin using TECRA-VIA
and BCET-RPLA
Gulam Rusul * , Nur Hayati Yaacob
Departmtnt of Food Science, Faculty of Food Science and Biotechnology, University Pertanian Malaysia,
43400 Serdang, Selangor D.E., Malaysia

Received 9 February 1994; revision received 3 June 1994; accepted 16 June 1994

Abstract

Enterotoxigenic Bacillus cereus was detected in cooked foods (17), rice noodles (31, wet
wheat noodles (2), dry wheat noodles (lo), spices (81, grains (41, legumes (11) and legume
products (3). One hundred ninety-four (42.3%), 70 (15.3%) and 23 (5.2%) of the 459
presumptive B. cerm.s colonies isolated from PEMBA agar were identified as B. cereus,
Bacillus hringiensis and B. mycoides, respectively. B. cereus isolates were examined for
growth temperature, pH profile and enterotoxin production using both TECRA-VIA and
BCET-RPLA kits. One hundred seventy-eight (91.8%) and 164 (84.%) of the strains were
enterotoxigenic as determined using TECRA-VIA and BCET-RPLA, respectively. Eighty-
two (50%) of the enterotoxigenic strains were capable of growing at 5”C, and 142 (86.6%)
grew at :W within 7 days of incubation. The enterotoxigenic strains did not grow at pH 4.0
but 69 (42.0%) of the strains were able to grow at pH 4.5 within 7 days at 37°C. The isolates
were resllstant to ampicillin (98.8%), cloxallin (100%) and tetracycline (61.0%), and suscepti-
ble to chloroamphenicol (87%), erythromycin (77.4%), gentamycin (100%) and streptomycin
(98.7%).

Keyw0rd.r: Bacillus cereus; Enterotoxin; Food; VIA

1. Introduction

Bacillus cereus is a gram-positive facultative anaerobic spore-forming rod-shaped

bacterium. It is known to produce many types of toxins, two (diarrhoegenic and

* Corresponding author. Tel. 603-9486101 ext. 3421. Fax 603-9485970.

016%1605/95/$09.50 0 1995 Elsevier Science B.V. All rights resewed

SSDI 0168-1605(94)00086-7
132 G. Rusul, N.H. Yaacob /ht. .I. Food Microbiology 25 (1995) 131-139

emetic) of which are known to cause foodborne illness (Johnson, 1984; Kramer and
Gilbert 1989). Besides causing foodborne illness, B. cereus is also responsible for
the spoilage of a variety of food products, particularly pasteurized milk and cream.
Bacillus species are ubiquitous in nature, being present in high numbers, often as
vegetative cells and endospores, in soil and in a variety of dried foods such as
grains, legumes, starches and spices. Presence of certain species of Bacillus in high
numbers in these foods may pose a threat to human health, especially when added
as ingredients to foods that are subsequently subjected to minimal heat treatment.
Endospores of certain strains of B. cereus often survive pasteurization tempera-
tures and are able to grow in milk and foods stored at 6-8°C (Coghill and Juffs,
1979; Christiansson et al., 1989; van Netten et al., 1990).
In Malaysia, no information is available on the incidence of enterotoxigenic
strains of B. cereus in foods. Dried foods such as rice, noodles, soybeans, lentils
and spices are used extensively. Among the foods sold by street vendors are fried
wheat or rice noodles and rice cooked in coconut milk (nasi lemak), which is
packed in plastic wrapped with newspapers or polystyrene containers or sold in
open trays. These foods are sold for breakfast or morning tea in school canteens,
hawker stalls and restaurants. Meat dishes (chicken, beef and fish) containing
various spices are also extensively sold in these facilities. These foods are often
prepared and stored for several hours at ambient temperature (28-30°C) before
eating.
The objectives of this study were to determine the prevalence of enterotoxigenic
strains of B. cerew in selected foods, to determine the efficacy of PEMBA agar
(Oxoid, UK) as a selective medium for B. cereus and to compare the performance
of TECRA VIA (Bioenterprises Pty. Ltd., Australia) and BCET-RPLA kits (De-
nka Seiken, Japan) for detecting enterotoxin.. In addition, the influence of pH and
temperature on growth, and antibiotic resistance of B. cereus was determined.

2. Materials and methods

2.1. Samples

Sixty-nine foods were analysed for the presence of enterotoxigenic B cereus.

Test foods consisted of dried rice noodles (31, wet wheat noodles (2), dried wheat
noodles (lo), spices (81, grains (41, legumes (ll), legume products (3) and a variety
of cooked foods (28). Dried foods and wet wheat noodles were purchased from
retail stores and wet-markets in Serdang, respectively. The cooked foods were
purchased from canteens on the University Pertanian Malaysia campus. All foods
were analysed within 2 h of purchase.

2.2. Isolation and enumeration of B. cereus

B. cereus was isolated according to the method of Holbrook and Anderson

(1980). Dried foods were rehydrated by soaking 20 g of sample in 90 ml of tryptone
 G. Rmul, N.H. Yaacob /ht. J. Food Microbiology25 (1995) 131-139 133

water (Toxoid, CM87) for 50 min at room temperature. An additional 90 ml of

sterile 0.1% peptone water was added before homogenizing the mixture for 30 s in
a Stomacher 400 (Seward, UK). Cooked foods (10 g) or wet wheat noodles (10 g>
were homogenized in 90 ml of 0.1% peptone water for 30 s in a Stomacher 400; 0.1
ml of diluted sample was surface-plated in duplicate on Bacillus cereus Selective
Agar (Olxoid, CM 617) which is the polymyxin pyruvate egg yolk manitol bromothy-
mol blue agar (PEMBA) as described by Holbrook and Anderson (1980). Plates
were incubated at 37°C for 24 h plus an additional 24 h at room temperature to
facilitate the development of turquiose to peacock blue colonies typical of B.
cereus.

2.3. Identification of B. cereus

All colonies that were rough in texture, turquiose to peacock blue in color,
surrounded by greyish zones of egg yolk precipitate and mannitol negative were
picked from plates with the highest dilutions for identification and confirmation.
These isolates belonging to the ‘Bacillus cereus group’ were identified using
staining procedures described by Holbrook and Anderson (1980) for detecting lipid
globules and spore location. Differential biochemical tests were carried out as
described by Harmon (1982). Motility, hemolytic activity on trypticase soy sheep
blood agar, rhizoid growth on nutrient agar and the presence of toxin crystals were
determined.

2.4. Determination of growth temperature and pH profile

The growth temperature profile of isolates was determined by streaking

overnight cultures grown on Tryptose soy agar (TSA) (Oxoid) slants onto TSA
plates and incubating at 5, 7, 45 and 55°C for 7 days. Plates were examined daily
for visible colonies. pH profiles were determined by streaking on TSA plates at pH
3.5, 4.0, 4.5, 5.0, 6.0, 8.0, 8.5, 9.0 and 9.5. The pH of the TSA plates was adjusted
aseptically with 5N NaOH or HCL after autoclaving the medium and before
pouring the plates. Streaked plates were incubated at 37°C and examined daily for
growth for 7 days.

2.5. Determination of antibiotic suscepitibility

Antibiotic susceptibility was determined by the disk agar diffusion method in

accordance with the instructions of the antibiotic disk supplier (BBL, Becton
Dickinson).

2.6. Determination of enterotoxin production

Isolates that were confirmed as B. cereus were tested for enterotoxin produc-
tion. Enterotoxin production was determined using TECRA VIA (Bioenterprises
Pty, Australia) and BCET-RPLA (Denka Seiken, Tokyo) kits. The TECRA kit is a
134 G. Rusul, N.H. Yaacob /ht. J. Food Microbiology 25 (1995) 131-139

visual immunoassay, whereas the BCET-RPLA is a reverse passive latex agglutina-

tion (RPLA) test.
Overnight cultures of B. cereus strains grown on TSA slants were transferred to
Brain Heart Infusion broth (BHI) (Oxoid) and incubated with agitation (110 rpm)
for 12-14 h at 32°C. Cultures were centrifuged (3000 X g for 10 min at room
temperature) and the supernatant fluids were tested for enterotoxin. Enterotoxin
determination was performed according to the directions supplied by the manufac-
turers.

3. Results

B. cereus was present in 17 of the cooked foods and in all dried foods tested
(Table 1). Of the 459 presumptive B. cereus isolates examined, 194 (42.31, 70
(15.3%), and 23 (5.2%) of the isolates were confirmed as strains of B. cereus, B.
thuringiensis and B. mycoides, respectively. One hundred fifty-two (33.1%) of the
isolates were not identified, as the biochemical tests proposed by Harmon (1982)
were inadequate. These results indicate that PEMBA agar is not exceptionally
selective for the isolation and enumeration of B cereus as only 42.3% of the
isolates were confirmed to be B. cereus. Other Bacillus spp. were also able to grow.
Cooked foods, legumes, spices and rice noodles yielded the highest numbers of B.
cereus .
Results of enterotoxin analysis using both visual immunoassay (TECRA) and
reversed-passive latex agglutination (RPLA) kits are presented in Table 1. One
hundred seventy-eight (91.8%) and 164 (84.5%) of the isolates tested were positive
for enterotoxin production using TECRA and RPLA kits, respectively; 155 of the
isolates were positive for enterotoxin production both using TECRA and RPLA

Table 1
Prevalence of enterotoxigenic B. cereus in different types of foods obtained from retail stores near
Serdang, Malaysia
Types of foods No. of Total counts on B. cereus No. (%I enterotoxigenic.
samples PEMBA (g-r) a
No. pre- No. (%) TECRA RPLA
sumptive confirmed
Rice noodles 3 2x10’-3~10~ 39 16 (41) 13 (18.2) 16 (100)
Wheat noodles (wet) 2 2x103-3x lo4 17 3 (17.6) 2 (66.7) 3 (100)
Wheat noodles (dried) 10 2x 102-5.2 x103 97 33 (34) 29 (87.9) 25 (75.8)
Spices 8 7x102-9x103 98 45 (45.9) 45 (100) 43 (95.6)
Grains 4 3.2~ lo’-4x lo3 37 11 (29.7) ll(100) 6 (54.5)
Legumes 11 2x 102-1.2 x106 102 49 (46.7) 47 (95.9) 47 (95.9)
Legume products 3 8x103-1~10~ 27 9 (33.3) 8 (88.9) 8 (88.9)
Cooked foods b 17 1 x 102-5x 10’ 42 28 (70.3) 23 (82.1) 16 (57.1)
Total 58 459 194 (42.3) 178 (91.8) 164 (84.5)
a Range of CFU g-’ present in all the samples of foods examined in each category.
b Eleven of the cooked food samples did not contain B. cereus.
 G. Rusul, N.H. Yaacob /ht. J. Food Microbiology 25 (1995) 131-139 135

Table 2
Growth temperature profile of enterotoxigenic strains of B. cerew isolated from different foods
incubated at 5, 7, 45 and 55°C for 7 days
Temp. Incubation time (days) No. (%) of isolates
“C i 2 3 4 5 6 7 that did not grow

5 NG a NG NG NG NG 20 (12.2) b 62 (37.8) 82 (50.0)

7 NG NG NG 29 (17.7) 10 (6.1) 72 (43.9) 31 (18.9) 22 (13.4)
45 160 (97.6) 3 (1.8) l(0.6) - - - - -
55 NG 7 (4.3) 13 (7.9) 9 (5.5) 94 (57.3) 5 (3.0) 18 (11.0) 18 (11.0)
a No growth.
b No. (%) of isolates that grew.

kits. Nine of the isolates that were positive for enterotoxin using the RPLA kit
were negative when tested with the TECRA kit; 23 isolates positive for enterotoxin
production using the TECRA were negative when tested with RPLA. Seven of the
cultures were negative for enterotoxin when tested with TECRA and RPLA kits.
Cultures that were positive for enterotoxin production using the RPLA kit were
also examined for their gbility to grow at 5, 7, 45 and 55°C (Table 2) and at pH 3.5,
4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 8.0, 9.0 and 9.5. Twenty (12.2%) and 62 (37.8%) of the
isolates were able to grow after 6 and 7 days of incubation at 5”C, respectively. At
7”C, 29 1:17.7%), 10 (6.1%), 72 (43.9%) and 31 (18.9%) of the isolates grew at 4, 5, 6
and 7 days of incubation, respectively; 82 (50.0%) and 22 (13.4%) of the isolates
did not grow at 5 and 7”C, respectively. All isolates were able to grow within 3 d at
45°C. Growth at 55°C was staggered, with 94 (57.3%) isolates growing at day 5 and
18 (11.0%) of the isolates not growing after 7 days of incubation. No isolates grew
at pH 4.0 or below (data not shown); however, 69 (42.0%) enterotoxigenic strains
were able to grow at pH 4.5 within 7 d of incubation. At pH 5.0 and above, all the
strains tested were able to grow within 3 days of incubation.
Isolates were resistant to ampicillin (98.8%), cloxallin (100%) and tetracycline
(61.0%) (Table 3) and were susceptible to chloramphenicol (87%), erythromycin
(77.4%), gentamycin (100%) and streptomycin (98.7%).

Table 3
Antibiotic profile of enterotoxigenic B. cerem strains isolated from different foods obtained from retail
stores near Serdang, Malaysia
Type of antibiotic Cont. Resistant Intermediate Moderately Susceptible
(mcg) susceptible
Ampicillin 10 162 (98.7) a - 1 (0.60) 1 (0.6)
Chloramphenicol 10 4 (2.4) 73 (44.5) - 87 (53.0)
Cloxallin 1 164(100) - _
Erythromycin 15 2 (1.2) 35 (21.3) _ 127 (77.4)
Gentamycin 10 _ _ 164 (100)
Streptomycin 10 1 (0.6) 1 (0.6) _ 162 (98.7)
Tetracycline 30 100 (61.0) 58 (35.4) - 6 (3.6)
a (%I.
136 G. Rusul, N.H. Yaacob / ht. J. Food Microbiology 25 (1995) 131-139

4. Discussion

B. cereus is becoming an important food poisoning organism partly because of

its wide distribution in nature. Its presence in food products can be traced to the
environment. B. cereus can be transmitted to food products when contaminated
ingredients are used to formulate these foods. The presence of B. cereus in nearly
all foods tested in this study is of great concern. Wheat and rice noodles are widely
consumed in Malaysia. Dried rice and wheat noodles are soaked for few hours
before being stir-fried for few minutes. The noodles are served either hot or cold.
Fried noodles are retailed at school canteens, hawkers stalls and restaurants in
trays or packed in small packets in plastic sheets surrounded with newspaper. The
noodles are eaten during breakfast or morning tea. Nasi lemak (rice cooked in
coconut milk), served with cucumber slices, anchovies cooked hot chili paste, is
also retailed in a similar fashion.
All of these foods that are served for breakfast and morning tea but are
normally cooked at dawn. Cooked foods are retailed at ambient temperatures and
remain exposed for several hours. Some restaurants will reheat leftovers from
lunch and serve for dinner. The method of food preparation, cooking and retailing
does not assure inactivation of B. cereus endospores and subsequent growth during
retailing at ambient temperature. Heat-resistant endospores of B. cereus are
known to survive cooking or frying processes, multiply rapidly and produce
sufficient amounts of toxin in cooked rice stored at room temperature to cause
food poisoning (Gilbert et al., 1974; Byran et al., 1981).
Mean generation times for four strains of B. cereuS in cooked chicken breast
and leg meat have been reported to vary from 0.4 to 0.5 h at 37”C, 1.1 to 1.5 h at
22°C and from 2.6 to 4.1 h at 15°C. (Sooltan et al., 1987). Blakey and Priest (1980)
reported that B. cereus present in red lentils and kidney beans was able to survive
boiling for 30 and 35 min, respectively. Populations increased to 10*/g after 48 h
when stored at 22 and 37°C. The DloO and D,,., values for six strains of B. cereus
isolated from rice in rice broth were 4.2-6.5 min and 16-36 min, respectively
(Chung and Sun, 1986), whereas strains isolated from dairy products had D,oooc
values ranging from 2.0 to 5.4 min (Chung and Sun, 1988).
Spices are used extensively in Malaysian cuisine. The most common vehicles of
B. cereus include cooked meat and meat products to which contaminated spices
have been added (Gilbert, 1979; Konuma et al., 1988). Konuma et al. (1988)
reported that 119 (39.7%), 29 (37.7%) and 21 (56.8%) samples of spices, seasoning
and starch, respectively, used in the preparation of meat products were contami-
nated with B. cereus. Spices used in a large variety of food products revealed a
high prevalence of B. cereus (53%), (Powers et al., 1976) which confirms the role
played by these additives as a common source of contamination in many countries.
Eighty-two (50%) and 142 (86.6%) of the enterotoxigenic strains were able to
grow at 5 and 7”C, respectively. The presence of a high proportion psychrotrophic
strains of B. cereus poses a serious threat to the safety of foods stored at
refrigeration temperatures, especially the newer types of cooked-chilled meals
promoted as refrigerated pasteurized foods of extended durability (REPFEDS).
 G. Rusul, NH. Yaacob/Int. J. Food Microbiology 25 (1995) 131-139 137

van Netten et al. (1990) reported that psychrotrophic strains of B. cereus produced
enterotoxin in BHI after 24 days, 12 days and 48 h when incubated at 4, 7 and
17°C re.spectively. Coghill and Juffs (1979) observed that the sporeforming and
psychrotrophic characterictics of B. cereus enables it to survive pasteurization and
grow in milk at refrigerated storage temperature. Christiansson et al. (1988)
reported that 94 of 136 strains of B. cereus isolated from milk and cream exhibited
human embryonic lung (HEL) cell cytotoxicity when cultured under aeration
(agitatbn, 200 rpm) at 30, 15 and 8°C in BHIG and milk. These strains did not
produce cytotoxins when cultured in unaerated milk at 8°C. In Malaysia, the use of
cold chain for the storage and sale of many types of processed and cooked foods is
rapidly expanding. The high isolation rate of psychrotrophic strains of B. cereus
suggest that ready to eat cooked foods should be stored below 4°C to avoid
germination, outgrowth of spores and enterotoxin production.
Results of our study demonstrate that PEMBA is not a particularly suitable
medium for selectively isolating and enumerating B cereus, as only 194 (42.3%) of
459 were confirmed as B. cereus. Similar observations have being made by other
investigators. Harmon et al. (1984) reported that there was no difference in the
number of B. cereus recovered from bean sprouts and mung beans when plated on
mannitol egg-yolk polymyxin (MYP), PEMBA and trypticase-soy-polymyxin blood
agar, except that B. cereus could be differentiated more easily from other microor-
ganisms on MYP agar and required fewer confirmatory tests. Sooltan et al. (1987)
examined 102 samples of raw and cooked poultry and observed that only 6.9% of
the samples contained detectable populations of B. cereus, with counts ranging
from log,, 1.4 to 3.5 g- ‘. They also observed that large numbers of other
microorganisms, up to log,, 7.7 were occasionally present in samples. Out of 20
presumptive colonies tested, only seven were confirmed as B. cereus. They ob-
served that the presence of large numbers of contaminating microorganisms
adversely affected the recovery of B. cereus on PEMBA, MYP and Donovan’s
medium.
Presently, there are only two commercial kits available for the rapid detection of
B. cerem enterotoxin. The RPLA kit was evaluated by Granum et al. (1993) by
comparing it with the Western immunoblot and vascular permeability reaction
(VPR). They reported that 104 (94.5%) of 110 enterotoxigenic isolates tested for
enterotoxin production were positive both by RPLA and Western immunoblot.
Three of the isolates that tested negative with RPLA were weakly positive by
Western immunoblot and this was attributed to the fact that the culture super-
natant fluids used for Western blot was 20 times more concentrated than the
culture supernatant fluids used for RPLA. The RPLA and Western immunoblot
also compared favourably with VPR, as 23 of 25 isolates tested were positive for
enterotoxin by all the three methods. These investigators concluded that the
BCET-RPLA kit provides a simple and reliable method for detecting B. cereus
enterotoxin culture filtrates and food extracts. Schultz and Buchanan (1993)
concluded that, based on biological activity, the TECRA kit possesses superior
characteristics for the detection of B. cerem diarrhea1 toxin. In their study, 9 of 12
(75%) iisolates were positive using the BCET-RPLA kit and two of the isolates that
138 G. Rusul, N.H. Yaacob /ht. J. Food Microbiology 25 (1995) 131-139

were negative for RPLA were positive with TECRA and cytotonicity. These
investigators compared the two kits with biological activity against CHO cells. In
our study, 91.8% of the isolates tested positive for enterotoxin with TECRA
compared to 84.5% by RPLA. Our results tend to concur with the findings of
Granum et al. (1993) because a large number of isolates were tested and,
furthermore, Western immunblot and VPR techniques are sensitive for the detec-
tion of enterotoxin.
More than 90% of the isolates were susceptible to chloroamphenicol, ery-
thromycin and gentamycin which are used therapeutically in humans. A large
number of the isolates were resistant to ampicillin, cloxallin and tetracycline.
Similar results were also observed by Chung and Sun (1986, 1988). The resistance
to tetracycline is associated with a plasmid (Bernhard et al., 1978).
The results of this study indicate that B. cereus could be a significant etiological
agent of food poisoning in Malaysia, especially among school children. There have
been numerous outbreaks of food poisoning among school children reported in
local newspapers. Often the causative agent is not identified. This can be at-
tributed to the fact that by the time symptoms develop, no food is available for
analysis. Also, the ecology of foodborne pathogens in Malaysian foods is not
well-understood due to a lack of surveillance and epidemiological studies. Results
also suggest that serious attention should be devoted to the sanitary and tempera-
ture conditions under which foods are prepared, cooked and marketed. As large
numbers of enterotoxigenic B. cereus are required to cause food poisoning (> lo5
cfu/g), therefore it is essential that control measure should be directed at
preventing the germination and outgrowth of spores.

Acknowledgements

This project was funded by the Malaysian Government through the IRPA
mechanism. The authors wish to express their sincere gratitude and appreciation
to Prof. Larry Beuchat, Department of Food Science and Technology, Center for
Food Safety and Quality Enhancement, University of Georgia, USA, for his
invaluable comments and assistance in the preparation of this manuscript.

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