Bacillus cereus

detection
According to ISO Method & PerkaBPOM
2019 Standard

For PT. Ariake Europe Indonesia

2022

Gintang Prayogi
Sales Manager of Microbiology for
F&B Business

The life science business of Merck operates

as MilliporeSigma in the U.S. and Canada.
Microbial Burden Limit on Finished Good
According to PerkaBPOM No.13 th 2019

Peraturan badan pengawas obat dan makanan

tentang batas maksimal cemaran mikroba
dalam pangan olahan No. 13 tahun 2019.

Ruang lingkup
Setiap Orang yang memproduksi, memasukkan,
dan/atau mengedarkan Pangan Olahan ke dalam
wilayah Negara Republik Indonesia wajib memenuhi
persyaratan keamanan, mutu, dan gizi Pangan.

Persyaratan keamanan, mutu, dan gizi Pangan yang

diatur dalam Peraturan Badan ini berupa batas
maksimal Cemaran Mikroba dalam Pangan Olahan
Microbial Burden Limit on Finished Good
According to PerkaBPOM No.13 th 2019

Dari 5 sampel yang diambil dan diuji, hanya 2 sampel yang boleh mengandung jumlah Bacillus cereus antara 10⁴ sampai
10⁵ koloni per gram, sedangkan 3 sampel yang lainnya jumlah Bacillus cereus harus kurang dari 10⁴ koloni per gram.
Sample Preparation
According to ISO 6887:2017
Dehydrated and other low-moisture products

Mix powdered products thoroughly in their container and then weigh out
using aseptic techniques. Other products may require breaking or cutting up
into small pieces with sterile tools before sampling.

For dehydrated and other low-moisture products, it is important to weigh

the diluent and then add the test portion to reduce osmotic shock on
any microorganisms present.

Low-moisture products may require a period (up to 1 h) soaking in the

diluent used for the initial suspension to soften them before homogenization
and subsequent manipulations. See ISO 6887-4 for detailed requirements for
the different types of low moisture products.

Automatic dilutor Food Homogenizer

Bacillus cereus detection
Importance on Food Production

Nature of bacteria

• Bacillus cereus is an environmentally widespread Gram-positive, spore-

forming, motile rod, which can cause gastrointestinal (e.g. diarrhea) as
well as non-gastrointestinal diseases (e.g. septicemia, endocarditis,
infections of the central nervous system) in humans.

• The illness is normally self-limiting and of short duration, although a few

fatal cases have occurred.

• Bacillus cereus is isolated from foods relatively frequently, making it an

important environmental indicator organism for the food industry. Foods
bearing contamination risk include meat and milk products, vegetables,
Colony morphology
soups, spices and especially baby food. of Bacillus species grown on
modified mannitol egg yolk
• Almost all Bacillus cereus strains possess the ability to produce one or more polymyxin (mMYP) agar plate
toxins. Cytotoxic enterotoxins are produced by almost 95% of isolates. without the egg yolk. Colony
diameter is about 1 mm
• In addition to this, some Bacillus cereus strains produce a different, heat
stable toxin known as Bacillus cereus emetic toxin. This toxin is assumed to (Images, courtesy of Atul
be pre-formed in the food and is often associated with starchy products such Singh and Arun Bhunia)
as rice and pasta.
Bacillus cereus detection
According to ISO 7932:2004

Principle.

• Specified quantity of test sample is surface plated on a solid selective culture

medium in petri dishes

• The plates are incubated under aerobic conditions at 30°C for 18h to 48h

• The Number of B. Cereus per gram or per mililitre of sample is calculated from the
number of confirmed colonies obtained on plates at dilution levels chosen
Bacillus cereus detection
According to ISO 7932:2004

Medium for Analysis

Dilution Media
• See ISO 6887-1
Base Media
• Using MYP Agar Base

• Supplement Polymixin B Solution (Antibiotic) & Egg Yolk Sterile

Confirmation Media

• Using Blood Agar Base + Defibrinated Sheep Blood

Bacillus cereus detection
According to ISO 7932:2004
Method for Analysis
Take two sterile Petri dishes. Transfer to each dish, by means of a sterile
pipette, 0.1 ml of the test sample if liquid, or 0.1 ml of the initial
suspension. Repeat the procedure using further dilution if necessary

Carefully spread the innoculum as quickly as possible, leave theplate with

the lids on for 15min at ambient termperature for the inoculum to be absorbed

Invert the plate and incubate for 18h to 24h at 30°C, if the colonies are
not clearly visible, incubate the plate for an additional 24h. after incubation
count the presumptive colonies

Select at least five presumtive colonies from each plate for

confirmation, if there are less than five colonies, take all presumptive
colonies present. Perform haemolysis test on sheep blood agar
Bacillus cereus detection
According to ISO 7932: 2004
Interpretation – Presumptive B. cereus
• The presumptive colonies are large, pink (negative mannitol
fermentation) and generally surrounded by zone of precipitation
(positive lecithinase activity)

Bacillus cereus Bacillus cereus

ATCC® 11778 on MYP Agar Haemolytic activity on Blood Agar

Interpretation – Confirmation test

• Streak, Stabs or spot the selected colonies onto the
surface of sheep blood agar in a manner which allows
good interpretation of the haemolysis reaction
Result Expression and Calculation
According to ISO 7218: 2007

Method of calculation: after identification

When the method used requires identification, a given number A (generally 5) of presumptive colonies is
identified from each of the dishes retained for the colony counting. After identification, calculate, for
each of the dishes, the number a of colonies complying with identification criteria, using Equation:

Where:
A is the number of identified colonies for confirmation test (generally 5 colony at least)
b is the number of colonies complying with identification criteria among the identified colonies A;
C is the total number of presumptive colonies counted on the dish.

Round off the calculated result to the nearest whole number. When doing this, if the first figure after the
decimal point is less than 5, do not modify the preceding figure; if the first figure after the decimal point
is greater than or equal to 5, increase the preceding figure by one unit.
Result Expression and Calculation
According to ISO 7218: 2007
𝑏
a = xC
𝐴
6
a = x 66 = 50
8
Ordering Information

Catalogue No Description Packing Size

1.12535.0500 GranuCult™ Maximum Recovery Diluent (Peptone Salt Solution) 500 g

1.07228.0500 GranuCult™ Buffered Peptone Water 500 g

1.05267.0500 GranuCult™ MYP (Mannitol Egg Yolk polymyxin) Agar (Base) acc. ISO 7932, ISO 21871 500 g

1.03784.0001 Egg-Yolk Emulsion sterile, 50%, for microbiology 10 x 100 ml

1.09875.0010 Bacillus Cereus Selective Supplement 10 x 1 vial

1.03879.0500 GranuCult® plus Blood agar (BA) (base) 500 g

1.04146.0001 Duopath® Cereus Enterotoxins 25 Test

Duopath® Cereus Enterotoxins

Thank You For Your Attention

If you need further assistance, kindly contact us at

Sales Manager for Biomonitoring:
gintang.prayogi@merckgroup.com/ 08118392902
Customer Service:
CS.Merck.MCLS@merckgroup.com
Visit our website:
Merckmillipore.com/Sigmaaldrich.com