5/19/2018 Bacillus cereus metode BAM 2001_0003.pdf

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1U

Bacillus

cereus

Page

3 of

8

C.

Sample

preparation

Using

aseptic

technique,

weigh

50

g

of

sample

into

sterile

blender

jar.

Add

450

ml

Butterfield s

phosphate-buffered

dilution

water

(1:10

dilution)

and

blend

for

2

min at

high

speed

(18,000-21,000

rpm).

Using

the

1:10 dilution,

make

serial

dilutions of

sample

for

enumeration

of

B.

cereus

as

described

in D or

E, below.

D.

Plate

count

of

B. cereus

Prepare

serial

ditutions

from

10-2

to

t0-6

by

transferring

10

ml

homogenized

sample

(1:10

dilution)

to

90

ml dilution

blank,

mixing

well

with

vigorous

shaking,

and

continuing

until

t0-6

dilution

is reached.

Inoculate

duplicate

MYP

agar

plates

with

each

dilution

of

sample

(including

1:10)

by

spreading

0.1

ml

evenly

onto

surface

of

each

plate

with

sterile

glass

spreading

rod. Incubate

plates 24 h

at

30oC and

observe

for

colonies

surrounded

by

precipitate zone,

which

indicates

that

lecithinase

is

produced . B. cereus

colonies

are

usually

a

pink

color

which

becomes

more

intense

after

additional

incubation.

If reactions

are

not

clear,

incubate

plates

for additional

24

h before

counting

colonies.

Select

plates

that

contain

an

estimated

15-150

eosin

pink,

lecithinase-producing

colonies.

Mark bottom

of

plates

into

zones

with

black

felt

pen

to

facilitate

counting

and

count

colonies

that are

typical

of

B.

ce:reus.

This

is the

presumptive

plate

count

of

B.

cereus.

Pick

5

or

more presumptive positive

colonies

from

the

MYP

agar plates

and

transfer

to

nutrient

agar

slants

for

confirmation

as

B. cereus.

Confirm

isolates

as

B.

cereus

as

described

in

F and

G,

below.

Calculate

number

of

B. cereus

cells/g

of

sample,

based

on

percentage

of

colonies

tested

that

are

confirmed

as

B. cereus.

For

example,

if

average

count

obtained

with

10-4

dilution

of

sample

was

65

and

4 of 5

colonies

tested

were

confirmed

as

B.

cereLtst

the

number

of

B.

cereus

cells/g

of

food

is 65

x4/5

x 10,000

x

10

=

5,200,000.

(NOTE: Dilution

factor

is

tenfold

higherthan

sample

dilution

because

only

0.1

ml was

tested).

E.

Most

probable number

(MPN)

of B.

cereus

The

MpN

technique

is

recommended

for

enumerating

B. cereus,in

foods

that

are

expected

to

contain

fewer

than

10

B.

cereus

organismslg.

It

may

also

be

preferred

for examining

certain

dehydrated

starchy

foods

for

which

the

plate

count

technique

is

inappropriate.

Inoculate

3-tube

MPN

series

in

trypticase

soy-polymyxin

broth,

using

1

ml

inoculum

of

10-1,

LO-Z,

and

10-3

dilutions

of

sample

with 3

tubes

at

each

dilution.

(Additional dilutions

should

also

be

tested

if

B.

cereus

population

is expected

to

exceed

LO3/g.)

Incubate tubes

48

+

2 h

at

30o

C

and

observe

for

dense

growth, which

is typical

of

B. cereus.

Streak

cultures

from

positive

tubes

onto

separate

MYP

agar

plates

and

incubate

6l7l12009

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