bacillus cereus metode bam 2001_0003.pdf
TRANSCRIPT
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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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