GENERAL TESTING PROCEDURE
TITLE: TEST FOR TOTAL AEROBIC MICROBIAL COUNT
GTP No.
GTP013 -00
Effective Date
Reference
USP/EP/BP/JPc
Page No.
1.
1 of 5
INTRODUCTION:
Total aerobic microbial count is one type of microbiological purity testing, which is used to count
the number of colony forming unit present in an article complying with monograph standards.
2.
PREPARATION OF CULTURE MEDIA:
Sodium hydroxide 1N: Dissolve 4.0 g of sodium hydroxide in water to make 100 mL.
Hydrochloric Acid 1N: Dissolve 8.5 ml of Hydrochloric Acid in water to make 100 mL.
Soyabean-Casein Digest Broth Medium (SCDM)/ Soyabean-Casein Digest Agar(SCDA)/
Plate Count Agar(PCA): Reconstitute dehydrated media as directed by the manufacturer and
sterilize in an autoclave at 121°C for 20 minutes.
3.
PROCEDURE:
Sample preparation:
Water soluble products: Aseptically add 10g of specimen if it’s a solid or 10ml accurately
measured, if the specimen is a liquid to make 100ml of sterile Soybean Casein Digest broth
Medium unless otherwise specified in the individual standard testing procedure, shake and mix
the sample preparation to achieve a homogeneous solution/suspension or dilute the specimen in
a suitable quantity of diluter unless otherwise specified in the individual standard testing
procedure.
Water insoluble products: Aseptically add 10g of specimen in 100ml of sterile Soybean Casein
Digest broth Medium with 0.1ml of Polysorbate 80, unless otherwise specified in the individual
standard testing procedure. Shake and mix the sample preparation to achieve a homogeneous
solution/suspension or dilute the specimen in a suitable quantity of diluter unless otherwise
specified in the individual standard testing procedure.
Capsules: Aseptically add 10g of capsules in 100ml of sterile Soybean Casein Digest broth
Medium unless otherwise specified in the individual standard testing procedure, subject the
GENERAL TESTING PROCEDURE
TITLE: TEST FOR TOTAL AEROBIC MICROBIAL COUNT
GTP No.
GTP013 -00
Effective Date
Reference
USP/EP/BP/JPc
Page No.
2 of 5
dilution to a water bath at temperature NMT 45 ºC for 15 min. shake every 5 min to achieve a
homogeneous solution/suspension or dilute the specimen in a suitable quantity of diluter unless
otherwise specified in the individual standard testing procedure.
POUR PLATE METHOD:
Note: For specimen that is sufficiently soluble or translucent perform Pour Plate Method,
otherwise use the Multiple-tube Method or Membrane filtration method.
Test preparation:
Pipette 1 mL of the final diluted sample into each of two sterile petri dishes.
Transfer to each dish 15-20mL of Soyabean -Casein Digest Agar Medium that previously has
been melted and cooled to approximately 45°C.
For water samples add 1ml of sample in to each of two Petri dishes and pour 15-20 mL of Plate
Count Agar Medium that previously has been melted and cooled to approximately 45°C.
Cover the petridishes, mix the agar by tilting or rotating the dishes and allow the contents to
solidify at room temperature.
Negative Control:
Transfer 15-20mL of Soyabean -Casein Digest Agar Medium or Plate Count Agar Medium (for
water samples) without any sample that previously has been melted and cooled to approximately
45°C.
Cover the petridishes, mix the agar by tilting or rotating the dishes, and allow the contents to
solidify at room temperature.
Positive Control:
Pipette 1mL of <100 cfu/ ml of Escherichia coli or Staphylococcus aureus culture suspension into
a sterile Petri dish.
Transfer 20mL of Soyabean -Casein Digest Agar Medium or Plate Count Agar Medium (for water
samples) without any sample that previously has been melted and cooled to approximately 45°C.
GENERAL TESTING PROCEDURE
TITLE: TEST FOR TOTAL AEROBIC MICROBIAL COUNT
GTP No.
GTP013 -00
Effective Date
Reference
USP/EP/BP/JPc
Page No.
3 of 5
Cover the petridishes, mix the suspension with agar by tilting or rotating the dishes and allow the
contents to solidify at room temperature.
Incase of water samples incubate all the petridishes at 30-35°C for 48-72 hours at inverted
position for products incubate at 30-35°C for 3-5 days at inverted position.
After incubation, examine the plates for growth.
MOST PROBABLE NUMBER METHOD (MPN):
Prepare three consecutive 1:10 dilutions (1:10, 1:100 & 1:1000) of the sample (as taken from the
sample preparation) to 9 ml Soyabean-Casein Digest broth medium, each dilution should have
triplicate as mentioned in below.
Transfer each 1 ml of sample preparation in to three 9 ml of Soyabean-Casein Digest broth
medium (1:10 dilution).
Add each 1 ml of 1:10 dilution in to three 9 ml of Soyabean-Casein Digest medium broth (1:100
dilution).
Add each 1 ml of 1:100 dilution in to three 9 ml of Soyabean-Casein Digest medium broth (1:1000
dilution).
Incubate all 9 tubes and one negative control and one positive control tube for 3-5 days at 3035ºC.
Record for each level of dilution the number of tubes showing microbial growth.
If the results is difficult to read, subculture the tube into fresh sterile Soyabean-Casein Digest
broth medium or Soyabean-Casein Digest Agar and incubate for 24 to 48 hrs at 30-35ºC.
Determine the most probable number of micro organisms per gm or per ml from table 1
Table-1
3 tubes at each level of dilution
Number of positive tubes
MPN
per
95 %
gm
Confidence
0.1 g
0.01 g
0.001g
/per
Limits
ml
0
0
0
<3
0-9.4
0
0
1
3
0.1-9.5
0
1
0
3
0.1-10
0
1
1
6.1
1.2-17
0
2
0
6.2
1.2-17
0
3
0
9.4
3.5-35
GENERAL TESTING PROCEDURE
TITLE: TEST FOR TOTAL AEROBIC MICROBIAL COUNT
GTP No.
GTP013 -00
Effective Date
Reference
USP/EP/BP/JPc
Page No.
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
0
0
0
1
1
2
2
3
0
0
0
1
1
1
2
2
2
3
3
0
0
0
1
1
1
1
2
2
2
2
3
3
3
3
0
1
2
0
1
0
1
0
0
1
2
0
1
2
0
1
2
0
1
0
1
2
0
1
2
3
0
1
2
3
0
1
2
3
3.6
7.2
11
7.4
11
11
15
16
9.2
14
20
15
20
27
21
28
35
29
36
23
38
64
43
75
120
160
93
150
210
290
240
460
1100
>1100
4 of 5
0.2-17
1.2-17
4-35
1.3-20
4-35
4-35
5-38
5-38
1.5-35
4-35
5-38
4-38
5-38
9-94
5-40
9-94
9-94
9-94
9-94
5-94
9-104
16-181
9-181
17-199
30-360
30-380
18-360
30-380
30-400
90-990
40-990
90-1980
200-4000
-
MEMBRANE FILTRATION METHODS:
Membrane filtration method only applicable to the sample where more than 1 ml of sample has
to be used for total aerobic microbial count.
Arrange sterile filtration apparatus and place membrane with 0.45µm nominal pore size in to the
filtration cup. Filter the sample in to membrane filter and rinse the membrane filter with
appropriate volume diluent (100 to 300ml). Transfer the membrane filter to the surface of the
soybean casein digest agar or Plate count agar for total aerobic microbial count (use one
GENERAL TESTING PROCEDURE
TITLE: TEST FOR TOTAL AEROBIC MICROBIAL COUNT
GTP No.
GTP013 -00
Effective Date
Reference
USP/EP/BP/JPc
Page No.
5 of 5
membrane filter).Incubate the plate at 30°- 35°C for 3 to 5 days. After examine the plates for
growth and record the results.
4.
INTERPRETATION:
Growth should not be observed in negative control.
Growth should be observed in positive control.
Select the plate corresponding to a given dilution and showing highest number of colonies less
than 250 cfu/ plate, Count the number of colonies and express the average for the two plates in
terms of Colony forming unit per gram or per mL of specimen.
If the count is carried out by the most probable number method report the result as MPN per gm
or per mL.
If the count is carried out by the membrane filtration method report the results cfu/ gm or per mL
If the colonies of fungal count are detected on the same medium, they are counted as part of total
aerobic microbial count.
If no microbial colonies are recovered from the dishes representing the initial 1:10 dilution of the
specimen, express the results as "less than10 micro-organisms per g or per mL of specimen" or
express the result as per the dilution specified in the individual standard testing procedure.