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Title: Microbial Limit Test by Pour Plate Method SOP No: CPLR/SOP/MIC/005
Revision No.: 03
Supersedes Version No.: 02 Effective Date:
Review Date:
1.0 PURPOSE:
To lay down the procedure for microbial testing of Raw material and Finished Product, to determine the
microbial load and confirm the absence of pathogens.
2.0 SCOPE:
This Procedure is applicable for Microbial limit test in raw materials, in process and finished products in
microbiology section at Concept Pharmaceuticals Ltd., Roorkee.
3.0 RESPONSIBILITY:
Microbiologist / Officer / To follow laid down procedure.
Microbiology head All over responsibility
5.0 PROCEDURE:
5.1 Media:
Prepare the required media as per SOP No. CPLR/SOP/MIC/008.
Title: Microbial Limit Test by Pour Plate Method SOP No: CPLR/SOP/MIC/005
Revision No.: 03
Supersedes Version No.: 02 Effective Date:
Review Date:
Title: Microbial Limit Test by Pour Plate Method SOP No: CPLR/SOP/MIC/005
Revision No.: 03
Supersedes Version No.: 02 Effective Date:
Review Date:
5.5 Total aerobic microbial count (TAMC) and Total yeast and mould count (TYMC):
5.5.1 Preparation of the sample is as follows.
5.6 Water soluble products:
5.6.1 Dissolve or dilute 10g or 10ml of the product to be examined in buffered sodium
chloride-peptone solution pH 7.0 or in another suitable medium to make up volume to
100ml.
5.6.2 If the product is known to have antimicrobial activity, an inactivating agent shall be
added to the diluents, if necessary adjust the pH to about pH 7.0 and prepare further
serial tenfold dilution using the same diluents.
5.7 Non-fatty products insoluble in water:
5.7.1 Suspend 10g or 10ml of the product to be examined in buffered sodium chloride-
peptone solution pH 7.0 or in another suitable liquid and make up volume to 100ml.
5.7.2 A suitable surface-active agent such as 1 g / liter of polysorbate 80 may be added to
assist the suspension of poorly insoluble in water substance.
5.7.3 If the product is known to have antimicrobial activity, an inactivating agent shall be
added to the diluents, if necessary adjust the pH to about pH 7.0 and prepare further
serial tenfold dilution using the same diluents.
5.8 Fatty Products:
5.8.1 Homogenize 10g or 10 ml of the product in isopropyl myristate or specified, with 5 g of
sterile polysorbate 20 or polysorbate 80.
Title: Microbial Limit Test by Pour Plate Method SOP No: CPLR/SOP/MIC/005
Revision No.: 03
Supersedes Version No.: 02 Effective Date:
Review Date:
5.8.2 If necessary, heat to not more than 40 C. Mix carefully while maintaining the
temperature in waterbath.
5.8.3 Add 85ml of buffered sodium chloride peptone solution pH 7.0 or any other suitable
medium which does not have any antimicrobial agent heated to not more than 40C.
5.8.4 Maintain this temperature for the shortest time necessary for formation of emulsion and
in any case for not more than 30 min, if necessary adjust the pH to about pH 7.0 and
prepare further serial tenfold dilution using the same diluents.
5.9 If inhibitory substances are present in the sample used in activator as per Table-I given
below:
TABLE-I
Antimicrobial substances In activator Concentration
Phenolics,
Parahydroxybenzoates Polysorbate 80 30 g / liter
(parabens)
Iodine,Quaternary ammonium Lecithin 3 g / liter
compounds Sodium Lauryl sulphate 4 g / liter
Alcohol, Aldehydes, Sorbates Dilution -
Mercurial Halogens Sodium thiosulphate 5 g / liter
Title: Microbial Limit Test by Pour Plate Method SOP No: CPLR/SOP/MIC/005
Revision No.: 03
Supersedes Version No.: 02 Effective Date:
Review Date:
5.10.6 Incubate these SCA plates in inverted position at 20-25C for 5 days.
5.10.7 At the end of all sample analysis, perform the negative control test and positive control
test for the same media used in sample analysis.
5.10.8 For negative control, Aseptically and separately transfer 1.0 ml of sterilized diluents
solution in to sterile Petri plates and add 15-20 ml molten soybean casein digest agar
and Sabouraud chloramphenicol Agar Medium in separate Petri plates, maintained at
not more than 45C, swirl the plate to mix sample. Allow agar to solidify. Invert the
Petri dishes and incubate the Soyabean Casein Digest Agar plate at 30-35C for 3 to 5
days & Sabouraud Dextrose Agar plates at 20-25C for 5 to 7 days.
5.10.9 For positive control , aseptically add 1.0 ml of 10-100 cfu/ml of culture inoculums in
sterile petri plate and add add 15-20 ml molten soybean casein digest agar and
Sabouraud Dextrose chloramphenicol Medium in separate Petri plates, maintained at not
more than 45C, swirl the plate to mix sample. Allow agar to solidify. Invert the Petri
dishes and incubate the Soyabean Casein Digest Agar plate at 30-35C for 3 to 5 days &
Sabouraud Dextrose Agar plates at 20-25C for 5 to 7 days.
5.10.10 Total aerobic microbial count / g or ml = No. of colony obtained x dilution factor /
volume of sample taken
5.10.11 Total yeast and mould count / g or ml = No. of colony obtained x dilution factor /
volume of sample taken.
5.10.12 Interpretation of The Results:
5.10.12.1 The total aerobic microbial count (TAMC) is considered to be equal to the
number of cfu found using Soybean Casein Digest Agar; if colonies of fungi
are detected on this medium, they are counted as part of TAMC. The total
combined yeasts and molds count (TYMC) is considered to be equal to the
number of cfu found using Sabouraud Chloramphenicol Agar; if colonies of
bacteria are detected on this medium, they are counted as part of TYMC.
5.10.12.2 If the result of the enumeration is negative, it should be reported as "not
detected for a defined unit" or "less than the detection limit for a defined
unit". The result should not be
Title: Microbial Limit Test by Pour Plate Method SOP No: CPLR/SOP/MIC/005
Revision No.: 03
Supersedes Version No.: 02 Effective Date:
Review Date:
Title: Microbial Limit Test by Pour Plate Method SOP No: CPLR/SOP/MIC/005
Revision No.: 03
Supersedes Version No.: 02 Effective Date:
Review Date:
Title: Microbial Limit Test by Pour Plate Method SOP No: CPLR/SOP/MIC/005
Revision No.: 03
Supersedes Version No.: 02 Effective Date:
Review Date:
Title: Microbial Limit Test by Pour Plate Method SOP No: CPLR/SOP/MIC/005
Revision No.: 03
Supersedes Version No.: 02 Effective Date:
Review Date:
Title: Microbial Limit Test by Pour Plate Method SOP No: CPLR/SOP/MIC/005
Revision No.: 03
Supersedes Version No.: 02 Effective Date:
Review Date:
Title: Microbial Limit Test by Pour Plate Method SOP No: CPLR/SOP/MIC/005
Revision No.: 03
Supersedes Version No.: 02 Effective Date:
Review Date:
5.11.6. Clostridia:
5.11.6.1 Aseptically add 10g of substance to 100ml of buffered sodium chloride
peptone solution pH 7.0 (Solution A).
5.11.6.2 Take 2 equal portions corresponding to not less than 1g or 1ml of the
product to examine.
5.11.6.3 Heat 1 portion at 80C for 10 minutes and cool rapidly (I)
Title: Microbial Limit Test by Pour Plate Method SOP No: CPLR/SOP/MIC/005
Revision No.: 03
Supersedes Version No.: 02 Effective Date:
Review Date:
Title: Microbial Limit Test by Pour Plate Method SOP No: CPLR/SOP/MIC/005
Revision No.: 03
Supersedes Version No.: 02 Effective Date:
Review Date:
5.11.8 Shigella
5.11.8.1 Pipette out 10ml of the product from solution (A) to be examined and
inoculate 10ml or the quantity corresponding to 1 g to 90 ml of soyabean
casein digest broth (Solution B), homogenize and incubate at 30-35C for
18- 24 hrs.
5.11.8.2 Negative Control:
Shake the negative control container and inoculate in 100 ml GN broth
incubate at 30C to 35C for 18-24 hours.
5.11.8.3 After incubation shake the growth and transfer 1 ml to 100 ml of GN Broth
Medium 11) and incubate at 30 to 35 for 24 to 48 hours.
5.11.8.4 Subculture on a plate of Xylose lysine deoxycholate medium (Medium 12).
Incubate at 30 to 35 for 24 to 48 hours.
5.11.8.5 A red colored translucent colony without black centre indicates possibility of
presence of Shigella.
5.11.8.6 If colony found than perform indole test for confirmed:
5.11.8.7 If there is no growth of such colonies or if identification tests are negative,
it indicates absence of Shigella .
5.11.8.9 The test is not valid unless the positive control indicates the positive result
for respective organism.
Limits: As per specifications.
5.12 Precaution:
LAF bench should be validated.
All glassware should be dry and sterilized.
Autoclave should be validated.
Use positive culture carefully
Title: Microbial Limit Test by Pour Plate Method SOP No: CPLR/SOP/MIC/005
Revision No.: 03
Supersedes Version No.: 02 Effective Date:
Review Date:
6.0 ABBREVIATION (S ):
CPLR: Concept Pharmaceuticals Ltd Roorkee
SOP: Standard Operating Procedures
QA: Quality Assurance
QC: Quality Control
MIC: Microbiology
TAVC: Total Aerobic Viable Count
TAMC: Total Aerobic Microbial Count
TYMC: Total Yeast and Mould Count
Annexure-I
Title: Microbial Limit Test by Pour Plate Method SOP No: CPLR/SOP/MIC/005
Revision No.: 03
Supersedes Version No.: 02 Effective Date:
Review Date:
Title: Microbial Limit Test by Pour Plate Method SOP No: CPLR/SOP/MIC/005
Revision No.: 03
Supersedes Version No.: 02 Effective Date:
Review Date:
CPLR/MIC/005/F01-03
Title: Microbial Limit Test by Pour Plate Method SOP No: CPLR/SOP/MIC/005
Revision No.: 03
Supersedes Version No.: 02 Effective Date:
Review Date:
Title: Microbial Limit Test by Pour Plate Method SOP No: CPLR/SOP/MIC/005
Revision No.: 03
Supersedes Version No.: 02 Effective Date:
Review Date:
Title: Microbial Limit Test by Pour Plate Method SOP No: CPLR/SOP/MIC/005
Revision No.: 03
Supersedes Version No.: 02 Effective Date:
Review Date:
CPLR/MIC/005/F01-03
7) Test for Clostridium Species:-
Date: Date: Conclusion
Take two equal portions in tubes separately corresponding Take loop full from each tube &
to gm Or 1ml of the product to be examined. Heat one Sub-culture on Columbia agar & Clostridium
portion to 800C for 10 min & do not heat other portion. Lot No.ACM/________ Species
Transfer 10 ml of the each homogenized portions of Incubate under anaerobic
individual tube to two tubes containing 100 ml of conditions Present /Absent
Reinforced Medium individually. at 30C to 35C for 48 hrs. Checked By:
Incubate under anaerobic condition at 30C to 35C for 48 hours. Anaerobic growth observed/not Date and Sign
Negative control: observed
Incubator ID: If growth observed then
Done By: proceed Catalase test.
Date and Sign Done By:
8) Shigella
Date: Date: Date:
After incubation, add 0.1ml Take loopful from GN broth & Observation:-
from Inoculated SCD subculture on xylose, A red colored translucent Shigella
Medium to 100ml lysine,deoxycholate agar Lot colony without black centre
GN broth. Incubate at 30 to No.ACM/________ indicates possibility of Present / Absent.
presence of Shigella
35C for 18 to 24 hours. Incubate at 30 to 35C for Checked By:
Present / Absent.
Lot No.ACM/________ 18 to 48 hours. Date and Sign
(1) If growth of colonies
Negative control: Done By:
present then perform
Date and Sign
indole test
Incubator ID:
Done By:
Date and Sign
Done By:
Date and Sign
Positive control (SCDA) :-.. cfu/ml Negative control: Growth observed/ No growth
Title: Microbial Limit Test by Pour Plate Method SOP No: CPLR/SOP/MIC/005
Revision No.: 03
Supersedes Version No.: 02 Effective Date:
Review Date:
Remarks: - The Product / Material comply / not comply with the Microbial limit test as per
IH/IP/BP/USP/specification.
Done by Checked by
Sign & Date Sign & Date
CPLR/MIC/005/F01-03
Review Date:
Title: Microbial Limit Test by Pour Plate Method SOP No: CPLR/SOP/MIC/005
Revision No.: 03
Supersedes Version No.: 02 Effective Date:
Review Date:
Annexure III
Annexure-II
CONCEPT PHARMACEUTICALS LTD.
Khasra No. 104, Asaf Nagar, Roorkee
Title: Microbial Limit Test by Pour Plate Method SOP No: CPLR/SOP/MIC/005
Revision No.: 03
Supersedes Version No.: 02 Effective Date:
Review Date:
Annexure III
Title: Microbial Limit Test by Pour Plate Method SOP No: CPLR/SOP/MIC/005
Revision No.: 03
Supersedes Version No.: 02 Effective Date:
Review Date:
Annexure III
S. Date of Name of Batch GRN Mfg Exp. Date of Date of MLT No. Done Checked Remarks
No Sampling Material No. No. Date Date Testing Result by by
CPLR/MIC/005/F03-03
Review Date: