Microbial Limit Test by Pour Plate Method

CONCEPT PHARMACEUTICALS LTD.
Khasra No. 104, Asaf Nagar, Roorkee

STANDARD OPERATING PROCEDURE
Department: Quality Control ( Microbiology) Page : 1 of 24
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
4.0 DEFINITION (S) :

The estimation of the number of viable aerobic microorganisms present and for freedom from designated
microbial species in pharmaceuticals articles of all kinds from raw material to the finished forms. It may
be qualitative and quantitative or both.
5.0 PROCEDURE:
5.1 Media:
Prepare the required media as per SOP No. CPLR/SOP/MIC/008.
Prepared by Checked by Approved by

Name Nisha Bharti Manoj Grewal Shiv Singh
Designation Microbiologist Manager-QC Manager-QA
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Format No: CPLR/QA/001/F01-01
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5.2 Preliminary Testing:

Depending on the nature of the preparation being examined, dilute, dissolve, suspend or
emulsify it in a suitable liquid. Eliminate any antimicrobial properties of the preparation by
dilution, neutralization or filtration. Suitable neutralizers such as Soya Lecithin, Polysorbate,
Tween 80, Glycine, Thiosulfate are been added to neutralize certain interfering substances.
They may be added to the chosen diluents or medium preferably before sterilization. If
neutralizers / in activators are used for this purpose their efficacy and non toxicity versus
microorganisms must be demonstrated by carrying out a blank with neutralizer and without
product .
5.3 Sample Quantity used for the Test

5.3.1 Unless otherwise directed, use 10g or 10 ml of the product to be examined.
5.3.2 The amount to be tested may be reduced for active substances that will be formulated in
the following conditions: the amount per dosage unit (e.g., tablet, capsule) is less than
or equal to 1 mg, or the amount per g or ml (for preparations not presented in dose
units) is less than 1 mg. In these cases, the amount of sample to be tested is not less than
the amount present in 10 dosage units or 10g or 10 ml of the product.
5.3.3 For materials used as active substances where the sample quantity is limited or batch
size is extremely small (i.e., less than 1000 ml or 1000 g), the amount tested shall be 1
% of the batch unless a lesser amount is prescribed or justified and authorized.
5.3.4 For products where the total number of entities in a batch is less than 200 (e.g., samples
used in clinical trials), the sample size may be reduced to two units, or one unit if the
size is less than 100.
5.3.5 Select the sample(s) at random from the bulk material or from the available containers of
the preparation. To obtain the required quantity, mix the contents of a sufficient number
of containers to provide the sample.

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5.4 Sample Receiving and Preparation

5.4.1 After receiving the sample in microbiology section, record the sample details in the
Format No.: CPLR/MIC/005/F02-03 for finished products and Format
No.:CPLR/MIC/005/F03-03 for raw material. The method for sample preparation
depends on the physical characteristics of the product to be tested. If none of the
procedures described below can be demonstrated to be satisfactory, a suitable
alternative procedure must be developed.
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.

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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
5.10 Pour plate method:

5.10.1 Take 2 Pre-sterilized plates and pour out 1ml of sample (Solution A) in each plate.
5.10.2 Add about 15-20ml of sterilized, cooled (40-45C) Soybean casein digest agar medium.
5.10.3 Swirl the plates clockwise and anticlockwise to mix the sample with media.
5.10.4 Incubate the plate in inverted position at 30-35 C for 5 days.
5.10.5 Repeat this procedure with 2 plates of sterile Sabouraud Chloramphenicol agar (SCA).

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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

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5.10.12.3 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 given as "zero for a defined unit" unless it is a
regulatory requirement. Qualitative test results should be reported as
"detected/not detected/absent in, a defined quantity or volume". They may
also be expressed as "less than a specified number of organisms for a
defined unit" where the specified number of organisms exceeds the detection
limit of the method and this has been agreed with the client. In the raw data
the result should not be given as zero for a defined unit unless it is a
regulatory requirement. A reported value of "0" may be used for data entry
and calculations or trend analysis
5.10.12.4 Reporting of Total Aerobic Microbial Counts! Total Combined Yeasts and
Molds Count: After incubation count the number of colonies present in each
dilution. Take arithmetic average of the counts & Calculate number of
cfu/gm of sample tested.
5.11 Test for Specified Microorganism:
5.11.1 Escherichia coli :
5.11.1.1 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.
5.11.1.2 Negative Control:
Use 10 ml of same diluents used at the time of sample preparation to
inoculate a suitable amount of Soyabean casein digest Broth and mix and
incubate at 30-35C for 18-24 hrs.
5.11.1.3 Shake the container, transfer a quantity of the contents corresponding to 1g
of the product to be examined to 100ml of Mac-Conkey broth and incubate
at 42-44C for 24 hrs.

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Shake the negative control container, transfer 1ml of Soyabean Casein
Digest Broth to 100 ml of MacConkey Broth, and incubate at 42C to 44C
for 24 to 48 hrs. Subculture on a plate of MacConkey Agar at 30C to 35C
for 18 to 72 hours.
5.11.1.3 Subcultures on the plate of Mac-Conkey agar and incubate at 30-35C for
18-72 hrs.
5.11.1.4 Growth of red, non-mucoid colonies of gram-negative rods indicates the
possible presence of E. coli.
5.11.1.5 If no growth of microorganism is detected, the product passes the test.
5.11.1.6 Confirmative test: Indole Test:
Take red, non-mucoid colony of Gram ve rods and add to tube containing
5ml of 0.1% peptone water. Incubate at 42-44 C for 24 hours. Add 0.5 ml of
Kovacs reagent, shake well and allow standing for 1 minute. If a red colour
is produced in the reagent layer, indole is present.
For negative control use the sterile diluents instead of suspected colony and
then follow the section.
5.11.2 Salmonella :
18- 24 hrs.
5.11.2.2 After 18-24 hours, shake the tube and transfer 0.1ml of solution B to tubes
containing, 10ml of Rappaport vassiliadis salmonella enrichment broth
medium.
Negative Control:
Use 10 ml of same diluents used at the time of sample preparation to
inoculate a suitable amount of Soyabean casein digest Broth and mix.

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5.11.2.3 Mix and incubate at 30-35C for 18-24 hours.

5.11.2.4 Subculture on Xylose lysine deoxycholate agar.
5.11.2.5 Incubate at 30-35C for 18-48 hrs.
5.11.2.6 Growth of well-developed, red colonies, with or without black centre
indicates presence of Salmonella.
5.11.2.8 Confirmative test:
Transfer separately a few of the suspect colonies to triple sugar iron agar
slant tubes, using surface and deep inoculation and incubate at 30-35C for
24 hrs. The formation of red slant and yellow butt and usually formation of
gas with or without production of hydrogen sulphide in the butt indicates
presence of Salmonella.
5.11.3. Pseudomonas aeruginosa :
inoculate 10ml or the quantity corresponding to 1g to 90ml of soyabean
18- 24 hrs.
5.11.3.2 Subculture on a plate of Cetrimide agar and incubate at 30- 35C for 18-72
hrs.
Shake the negative control container and subculture on a plate of Cetrimide
Agar, and incubate at 30C to 35C for 18 to 72 hours.
5.11.3.4 Growth of generally greenish colonies, greenish under fluorescent UV light
and gram negative rods indicates presence of P. aeruginosa.

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5.11.3.6 Confirmative test:

Transfer colonies individually by means of an inoculating wire on to an
Oxidase test discs (N N-dimethyl-p-phenylene diamine oxalate). The test is
positive if purple colour is produced within 1 minute.
5.11.4 Staphylococcus aureus:
inoculate 10ml or the quantity corresponding to 1g to 90ml of soyabean
18-24 hrs.
5.11.4.2 Subculture on a plate of Mannitol salt agar and incubate at 30-35C for 18-
72 hrs.
Shake the negative control container and Subculture on a plate of Mannitol
Salt Agar, and incubate at 30C to 35C for 18 to 72 hours
5.11.4.4 Yellow/white colonies with yellow zones of gram-positive cocci; indicate
the presence of S. aureus.
5.11.4.6 Confirmative test: (Coagulase Test)
Transfer the suspected colony to tube containing 0.5ml of mammalian,
preferably rabbit or horse plasma. Incubate at 35C examining the tubes at 3
hours and subsequently at suitable interval up to 24 hours. If no coagulation
in any degree is observed the sample meets the requirements of the test for
absence of Staphylococcus aureus. The product passes the test if colonies of
the type described above do not appear on Mannitol salt Agar or if the
confirmatory biochemical tests are negative.
For negative control use sterile Buffered Sodium Chloride-Peptone Solution
instead of suspected colonies in 0.5 ml of Rabbit or Horse Plasma with or
without additives and follow section.

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5.11.5 Bile Tolerant Gram Negative Bacteria:

5.11.5.1 Aseptically add 10g of substance to 100ml of Buffered Sodium Chloride -
Peptone Solution pH 7.0 (Solution A).
5.11.5.2 Inoculate 10ml of the solution A in to 90ml of soyabean casein digest
medium (Solution B1).
5.11.5.3 Incubate the soyabean casein digest medium (Solution B1) at 20 to 25C for
a time sufficient to resuscitate the bacteria but not sufficient to encourage
multiplication of the organisms (2-5 hours).
5.11.5.4 Transfer 10ml of solution B1 to 90ml of soyabean casein digest medium
(Solution B2).
5.11.5.5 Shake the tube (Solution B2) transfer 10ml (0.1g or 0.1ml), 1ml (0.01g or
0.01ml) and 0.1ml (0.001g or 0.001ml) of the sample to 100ml of
Enterobacteria enrichment broth Mossel.
5.11.5.6 Incubate at 30-35C for 24-48 hrs. After incubation shake the container,
Subculture on plates of Violet Red Bile Glucose Agar.
5.11.5.7 Negative Control: Use 10 ml of same diluents used at the time of sample
preparation to inoculate a suitable amount of Enterobacteria Enrichment
Broth Mossel and incubate at 30C to 35C for 24 to 48 hours. After
incubation shake the container, Subculture on plates of Violet Red Bile
Glucose Agar. Incubate the plates at 30C to 35C for 18 to 24 hours.
5.11.5.8 The product complies with the test if there is no growth of colonies observed
on the plates.

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5.11.5.9 Quantitative Evaluation:

Inoculate suitable quantities of Enterobacteriaceae Enrichment Broth-
Mossel with quantities of solution, prepared in the section 5.7.1.1 and / or
dilutions of it, containing 0.1 g, 0.01 g and 0.001 g (or 0.1 ml, 0.01 ml and
0.001 ml) of the product to be examined. Incubate at 30C to 35C for 24 to
48 hours.
Subculture each of the cultures on a plate of Violet Red Bile Glucose Agar.
Incubate at 30C to 35C for 18 to 24 hours
5.11.5.10 Interpretation of Results:
Growth of well developed, generally red or reddish colonies of gram
negative bacteria constitutes a positive result. Note the smallest quantity of
the preparation that gives a positive result and the largest quantity that gives
a negative result. Determine from Table -I the probable number of bacteria.
After incubation subculture on plates of violet red bile glucose agar by
means of inoculating loop and incubate at 30-35 C for 18-24 hrs.
Results for each quantity of product
Probable number of bacteria
0.1 g or 0.1 0.01 g or 0.001g or per gram/ml of product
ml 0.01 ml 0.001 ml
+ + + More than 103
+ + - Less than 1032 and more than 102
+ - - Less than 10 and more than 10
- - - Less than 10
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)

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5.11.6.4 Do not heat the other portion.(II)

5.11.6.5 Transfer 10ml of (I) & (II) containers containing 100ml of reinforced
medium for clostridia.
5.11.6.6 Incubate under anaerobic condition at 30-35C for 48 hours.
Shake the negative control container and inoculate in 100 ml reinforced
medium and incubate at 30C to 35C for 48 hours anaerobically.
5.11.6.8 After incubation subculture from each tube to Columbia agar and incubate at
30 -35C for 48 hours.
5.11.6.9 Confirmative test: Catalase test:
Take suspected colony on the loop and transfer in a tube containing
hydrogen peroxide. The effervescent reaction in hydrogen peroxide indicates
positive. Upon examination the occurrence of anaerobic growth of gram
positive rods giving the negative catalase reaction indicate the presence of
clostridia. If no anaerobic growth of rods microorganism is detected on
Columbia agar or the catalase test is positive, the product complies the test.
5.11.7 Candida albicans:
5.11.7.1 Aseptically add 10g of substance to 100ml of buffered sodium chloride-
peptone Solution pH 7.0 containing 0.5% Polysorbate 80 (Solution A).
5.11.7.2 Inoculate 10.0ml of Solution A to 100ml of Sabouraud -dextrose broth
(Solution B3).
5.11.7.3 Incubate the Sabouraud-dextrose broth (SDB) at 30-35C for 72 hours.
Shake the negative control container and inoculate in 100 ml Sabouraud
-dextrose broth incubate at 30C to 35C for 72 hours.
5.11.7.5 After 3 days, shake the tube (Solution B3) and by means of inoculating loop
streak a portion of Sabouraud Dextrose agar medium.
5.11.7.6 Incubate Sabouraud Dextrose agar plate at 30-35C for 24-48 hours.

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5.11.7.7 Gram positive white colonies indicate the presence of C. albicans.
5.11.8 Shigella
18- 24 hrs.
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

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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
7.0 REFERENCE (S) : In-House, IP, BP, USP
8.0 ANNEXURE (S) :

8.1 Annexure I : Microbial Limit Test Report
8.2 Annexure II: Finished product log record
8.3 Annexure III : Raw material log record.
Annexure-I

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Khasra No. 104, Asaf Nagar,Roorkee

Title : Microbial Limit Test Report Page No 1 of
4
Name of Material/Product Batch No.
Mfg Date Exp. Date
Date of Sampling MLT No.
Date of Testing Date of Result
(A) TOTAL VIABLE AEROBIC COUNT: (Method used : Pour-plate)
Wt. taken & C.F.U CFU/gm/ ml Limit

Dilution TBC and TFC=
meanDF
1) TOTAL BACTERIAL COUNT Dissolve or dilute 10 1.
Medium: Soyabean casein Digest Agar, gm or ml sample
Lot No.. dilute in 100 ml 2.
Diluents : Buffered sodium chloride peptone sterilized buffered
water sodium chloride
Lot No.. peptone solution and
Incubation : 30-35C for 5 days pour one ml in two
sterilized plate.
2) TOTAL MOLD AND YEAST COUNT Dissolve or dilute 10 1.
Medium: Sabouraud Chlorophenicol Agar, gm or ml sample
Lot No.. dilute in 100 ml 2.
Diluent : Buffered sodium chloride peptone water sterilized buffered
Incubation : 20-25C for 5 to 7 days sodium chloride
peptone solution and
pour one ml in two
sterilized plate.
Dilution Factor :
C.F.U. = Colony Forming Unit

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(B) TEST FOR PATHOGENS.

Take 10ml Solution A in 90 ml sterile Soyabean Casein Digest Medium (SCDM), Lot no.ACM/____________ Incubate at 30 t-
35 C for 18 to 24 hours.
Positive Control:.. Negative control:..
CPLR/MIC/005/F01-03
1) Test for Staphylococcus aureus :

Date: Date: Conclusion
Observation:
After incubation, take loopful from SCD medium Yellow or white colonies surrounded by yellow zones. Staphylococcus
& subculture on Mannitol salt agar (MSA) Present / Absent aureus
Negative control:
If colonies of above characteristic are present perform Present / Absent
Incubate at 30 to 35C for 18 to 72 hrs. coagulase test as under Transfer a suspected colony
Incubator ID: using loop to tube containing 0.5ml mammalian Checked By:
Done By: plasma Incubate at 37C for 3 to 24 hours coagulation. Date and Sign:
Date and Sign: Incubator ID:
Done By:
Date and Sign
2) Test for Pseudomonas aeruginosa :

Date Date CONCLUSION

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After incubation, take loopful Observation :

from SCD medium & subculture Growth of colonies. Pseudomonas aeruginosa
on Cetrimide Agar, Present / Absent
Negative control: Present / Absent
Incubate at 30 to 35C for 18 to If present perform oxidase test as follows: with aid of
72 hours. inoculating loop transfer suspected colonies on disc or filter Checked By:
Incubator ID: paper impregnated with N, N-Dimethyl-p-phenylenediamine Date and Sign:
Done By: dihydrochloride. Purple color produced within 5 to 10 sec.
Date and Sign: Done By:
Date and Sign
3) Test for Escherichia coli.
Date Date Date Conclusion
After incubation, add 1ml from Take loopful from Observation:-
Inoculated SCDM to 100 ml MacConkeys Broth & Growth of colonies. Escherichia coli
Mac-Conkey Broth, Incubate at subculture on Mac-Conkey Present / Absent.
42 to 44C for 24 to 48 hrs. agar.Incubate at 30 to 35C (1) If growth of colonies Present / Absent.
Negative control: for 18 to 72 hours. present then perform indole Checked By:
Incubator ID: Incubator ID: test . Date and Sign:
Done By: Done By:
Date and Sign: Done By: Date and Sign
Date and Sign
4) Test for Salmonella species
Date Date: Date: Date CONCLUSION
After incubation, add 0.1ml Take loopful from Observation:-
from Inoculated SCD Rappaport Vassiliadis Well develop red colonies Observation:- Salmonella sps.
Medium to 10ml Salmonella enrichment with or without black TSI
Rappaport Vassiliadis broth & subculture on center. Slant Alkaline
Salmonella enrichment xylose, Present / Absent (Red) & butt
broth. lysine,deoxycholate agar If present streak & stab one acidic (Yellow) Present / Absent
Incubate at 30 to 35C for loofpul of suspected with or without
18 to 24 hours. Incubate at 30 to 35C colonies individually to blackening Checked By:
Negative control: for 18 to 48 hours . tube of triple sugar iron Date and Sign:
Incubator ID: agar slant (TSI).
Incubator ID: Incubate at 30 to 35C for
18 to 24 hours.

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Done By: Done By: Incubator ID:

Date and Sign: Date and Sign
Done By:
Date and Sign
5) Test for Candida albicans.
Date: Date: Date : CONCLUSION
Take 10 ml or the quantity Take loopful from of Sabouraud- Observation:
corresponding to not less than 1 g or dextrose Broth & subculture on White colonies. Candida
1ml of the product to inoculate 100 ml Sabouraud-dextrose agar. Present /Absent albicans
of Sabouraud-dextrose broth. Incubate at 30 to 35C for 24 If white colonies are present then
and incubate at 30-35C for 3-5 days. to 48 hours Prepare smear on slide & stain Present /Absent
Negative control: Incubator ID: with Lactophenol cotton blue. Checked By:
Incubator ID: Observe under microscope Date and Sign
Done By: Done By: Done By:
Date and Sign: Date and Sign Date and Sign
6) Test for Bile- tolerant gram-negative bacteria:-

Date: Date: Date: Date:
Take the sample Streak on the plate of Violet Inoculate suitable quantity of EE Observation
corresponding to 1 g or 1ml red bile glucose agar. Lot broth,Lot no.ACM/_______ from 0.1 gm 0.01 0.001
of the product from no.ACM/_____ SCD medium containing respectively or ml gm gm or
Incubate the plates at 30C 0.1 gm, 0.01 gm and 0.001 gm (or or ml
(Previously Inoculated & to 35C for 18 to 24 hours. 0.1ml, 0.01 ml, 0.00 1ml) of the product ml
incubated at 20-250C for 2- Observation:- to be examined. Incubate at 30C to
5 hrs) SCD Medium to 100 Growth of colonies. 35C for 24 to 48 hours. Incubator Id
ml EE broth Observed / not observed. No.
and incubate at 30-35C for 0.1 gm or ml : resent/Absent Result :
24 to 48 hours. Conclusion:- 0.01 gm or ml. Present/Absent Checked By:
Negative control: Present / Absent 0.1 gm or ml. Date and Sign
Present/Absent
Incubator ID: Done By: Refer SOP for table to determine the
Date and Sign probable number of bacteria
Done By:
Date and Sign

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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
Positive control (SCA): ..cfu/ml Negative control: Growth observed/ No growth

Sign. & Date
Revision No.: 03
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

Sign. & Date
Title: Microbial Limit Test by Pour Plate SOP No: CPLR/SOP/MIC/005

Method
Revision No.: 03
Review Date:

Revision No.: 03
Review Date:
Annexure III
Annexure-II
Title : Finished/Semi-Finished Product Log Record Page No. 1 of 1

S. No Date of Name of Batch Mfg Date Exp. Date of Date of MLT No. Done by Checked Remarks
Sampling Product No. Date Testing Result by
Sign. & Date

Revision No.: 03
Review Date:
Annexure III
Sign. & Date

Revision No.: 03
Review Date:
Annexure III

Title : Raw Material Log Record Page No. 1 of 1
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
Sign. & Date

Title: Microbial Limit Test by Pour Plate SOP No: CPLR/SOP/MIC/005

Method
Revision No.: 03
Review Date:
9.0 REVISION HISTORY:
Serial No. Revision No. Date of preparation Reason for revision
01 NIL 20/04/2013 Original Issue

Periodic review/new pathogen
02 01 21/04/2015
added
Add microbial preservatives
and its ancativator, Sampling
03 02 17/05/2016 quantity used for the test and
change in format of Microbial
Limit test
SOP Changed according to
04 03 26/10/2016 QA SOP ON SOP
CPLR/SOP/QA/001
10.0 DISTRIBUTION RECORDS:
10.1 Master Copy: Quality Assurance
10.2 Controlled copy (S): Microbiology (Quality Control).

Sign. & Date

Microbial Limit Test by Pour Plate Method

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CONCEPT PHARMACEUTICALS LTD.

Khasra No. 104, Asaf Nagar, Roorkee

Department: Quality Control ( Microbiology) Page : 1 of 24

4.0 DEFINITION (S) :

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5.2 Preliminary Testing:

5.3 Sample Quantity used for the Test

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5.4 Sample Receiving and Preparation

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5.10 Pour plate method:

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5.10.12.3 If the result of the enumeration is negative, it should be reported as "not

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5.11.1.4 Negative Control:

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5.11.2.3 Mix and incubate at 30-35C for 18-24 hours.

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5.11.3.6 Confirmative test:

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5.11.5 Bile Tolerant Gram Negative Bacteria:

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5.11.5.9 Quantitative Evaluation:

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5.11.6.4 Do not heat the other portion.(II)

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5.11.7.7 Gram positive white colonies indicate the presence of C. albicans.

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7.0 REFERENCE (S) : In-House, IP, BP, USP

8.0 ANNEXURE (S) :

CONCEPT PHARMACEUTICALS LTD.

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Khasra No. 104, Asaf Nagar,Roorkee

(A) TOTAL VIABLE AEROBIC COUNT: (Method used : Pour-plate)

Wt. taken & C.F.U CFU/gm/ ml Limit

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(B) TEST FOR PATHOGENS.

1) Test for Staphylococcus aureus :

2) Test for Pseudomonas aeruginosa :

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After incubation, take loopful Observation :

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Done By: Done By: Incubator ID:

6) Test for Bile- tolerant gram-negative bacteria:-

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