SOP for Microbial analysis of Raw Material Finished Products

1.0 OBJECTIVE
1.1 To lay down the procedure for Microbial analysis of Raw Material, Finished Products.
2.0 SCOPE
2.1 This SOP is applicable for Microbial analysis of Raw Material, Finished Products
3.0 RESPONSIBILITY
3.1 Microbiologist QC
4.0 ACCOUNTABILITY
4.1 Q.C. Manager

5.0 PROCEDURE
5.1 Collect the Raw Material, Finished Products for microbiological analysis and record the sample details in Raw material

and Finished product sampling record.
5.2 Prepare required quantity of Soyabean casein Digest agar and Broth medium, Sabouraud Dextrose Agar,

Buffer sodium chloride peptone solution pH 7.0 ± 0.5 as per media.
5.3 Sample Preparation:
5.3.1 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.
5.3.2 Water-Soluble Products: Dissolve or dilute (usually 1 in 10 dilution is prepared) the product to be examined in Buffered

Sodium Chloride Peptone Solution pH 7.0, Phosphate Buffer Solution pH 7.2, or Soybean–Casein Digest Broth. If necessary,

adjust to a pH of 6 to 8. Further dilutions, where necessary, are prepared with the same diluents.
5.3.3 Non fatty Products Insoluble in Water: Suspend the product to be examined (usually a 1 in 10 dilution ) in Buffered

Sodium Chloride Peptone Solution pH 7.0, Phosphate Buffer Solution pH 7.2, or Soyabean Casein Digest Broth.

A surface-active agent such as 1 g per L of Polysorbate 80 may be added to assist the suspension of poorly

wet table substances. If necessary, adjust to a pH of 6 to 8. Further dilutions, where necessary, are prepared with the same diluent.
5.3.4 Fatty Products: Dissolve in isopropyl myristate sterilized by filtration, or mix the product to be examined with the minimum

necessary quantity of sterile Polysorbate 80 or another non-inhibitory sterile surface-active reagent. Heated if necessary,

to not more than 40oC or, in exceptional cases, to not more than 45oC. Mix carefully and if necessary maintain

the temperature in a water bath. Add a sufficient quantity of the pre-warmed chosen diluent to make a 1 in 10 dilution

of the original product. Mix carefully, while maintaining the temperature for the shortest time necessary for the formation

of an emulsion. Further serial 10-fold dilutions may be prepared using the chosen diluent containing a suitable

concentration of sterile Polysorbate 80 or another non-inhibitory sterile surface-active reagent.
5.4 Sample Amount Used for the Test:
5.4.1 Unless otherwise directed, use 10 g or 10 ml of the product to be examined.
5.4.2 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 10 g or 10 ml of the product.
5.4.3 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.
5.4.4 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.

5.4.5 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.
5.4.6 Microbial Enumeration Test (Tests for Total Aerobic Microbial Count / Total Combined Yeast and Molds Count).

5.5 Membrane filtration method
5.5.1 Collect & start the process of filtration.
5.5.2 Use sterile membrane filter of 47 mm diameter & having pore size of 0.45.
5.5.3 Sterilize the membrane filtration assembly & assemble the filtration assembly under Laminar Air Flow.
5.5.4 Dilute the pretreated preparation so that counts of 100 colony-forming units (cfu) may be expected.
5.5.5 Pipette out 10 ml prepared sample and transfer it into the filtration assembly by using micropipettes or graduated pipettes.
5.5.6 Wash the membrane, with 100 ml of suitable diluents like buffered sodium chloride peptone solution to

remove out the traces of residue of the sample to be tested.
5.5.7 For determination of total aerobic microbial count, transfer the membrane filter on the surface of Soyabean Casein Digest Agar plate.
5.5.8 For determination of total combined yeast and molds count, transfer the membrane filter on surface of Sabouraud Dextrose Agar plates.
5.5.9 At the end of all sample analysis, perform the negative control test for the same media used in sample analysis.

For negative control filter the 100 ml of sterilized Buffered sodium chloride peptone saline solution through the

sterile membrane filtration units containing sterile membrane filter paper of 0.45 pore size.
5.5.10 Carefully remove the funnel from the base of the holder. Transfer the filter membrane by using a sterile

forceps to the surface of the used Culture media in membrane filtration method.
5.5.11 Invert the Petri dishes and Incubates the Soyabean Casein Digest Agar Plate at 30-350C for 24-72 hours

and Sabouraud Dextrose agar Plates at 20 –250C for 5 days.
5.6 Pour plate method
5.6.1 Plate count shall be carried out by using 9-10 cm diameter petri dish.
5.6.2 Prepare the sample as per section No. 5.3.1.
5.6.3 Pipette out 1 ml prepared sample into four Petri dishes by using micropipettes or graduated pipettes.
5.6.4 Pour 15-20 ml of previously sterilized and maintained at not more than 45ºC Soyabean casein Digest agar

in two Petri plates for bacteria & Sabouraud Dextrose agar for fungal growth in another two petri plates and gently

rotate the plate for uniform distribution of the sample.
5.6.5 At the end of all sample analysis, perform the negative control test for the same media

used in sample analysis. Aseptically and separately transfer 1.0 ml of sterilized Buffered Sodium Chloride Peptone solution

in to sterile petri dish, Add 15- 20 ml molten soybean casein digest agar and Sabouraud Dextrose Agar

Medium in separate Petri plates, maintained at not more than 45ºC, swirl the plate to mix sample. Allow agar to solidify.
5.6.6 Invert the Petri dishes and Incubate the Soyabean Casein Digest Agar plate at 30-350C for 24-72 hours

and Sabouraud Dextrose Agar plates at 20-250C for 5 days.

5.6.7 Interpretation of the Results: 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 Dextrose Agar; if colonies of bacteria are detected

on this medium, they are counted as part of TYMC. When the TYMC is expected to exceed the acceptance

criterion due to the bacterial growth, Sabouraud Dextrose Agar containing antibiotics may be used.
5.6.8 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.
5.6.9 Calculations : Total colonies forming units/gm of sample = Total no. of observed colonies X dilution factor

If no microbial colonies are recovered from the dishes representing the 1:10 dilution the count is given as

“less than 10 microorganisms per gram of sample”. When the number of cfu per plate exceeds

250 for TAMC and 50 for TYMC, for all dilutions, record the counts as too numerous to count (TNTC).
5.7 Tests for Specified Microorganisms
5.7.1 All the raw material, semi finished/in process samples, finished product and stability samples which

fall under the “Tests for Specified Microorganisms” shall be analyzed for absence of Escherichia coli or as per their respective specification.
5.8 Test for absence of Escherichia coli
5.8.1 Sample preparation and pre-incubation: Prepare the sample as per section No. 5.3.1.
Use 10 ml or the quantity corresponding to 1 g or 1ml, to inoculate a suitable amount of

Soyabean casein digest Broth, mix, and incubate at 30°C to 35°C for 18 to 24 hours.
5.8.2 Selection and subculture: Shake the container, transfer 1ml of Soyabean Casein Digest Broth to 100 ml of MacConkey Broth,

and incubate at 42°C to 44°C for 24 to 48 hrs. Subculture on a plate of MacConkey Agar at 30°C to 35°C for 18 to 72 hours.
5.8.3 Interpretation: Upon examination, growth of colonies as non mucoid, pink in colour with or without surrounding zone

of precipitated bile, indicate the possible presence of Escherichia coli in the sample.

5.9 Test for absence of Salmonella species
5.9.1 Sample preparation and pre-incubation: Prepare the sample as per section No. 5.3.1 ,
Use 10 ml or the quantity corresponding to 1 g or 1ml, to inoculate a suitable amount of

Soyabean casein digest Broth, mix, and incubate at 30°C to 35°C for 18 to 24 hours.
5.9.2 Selection and subculture: Shake the container, transfer 0.1ml of Soyabean Casein Digest Broth to 10 ml of

Rappaport Vassiliadis Salmonella Enrichment Broth, and incubate at 30°C to 35°C for 18 to 24 hrs. Subculture on

a plate of Xylose Lysine Deoxycholate Agar and incubate at 30°C to 35°C for 18 to 48 hours.
5.9.3 Interpretation: The possible presence of Salmonella species is indicated by the growth of well-developed, red colonies, with or without black centers.
5.10 Test for absence of Pseudomonas aeruginosa.
5.10.1 Sample preparation and pre-incubation: Prepare the sample as per section No. 5.3.1.
Use 10 ml or the quantity corresponding to 1 g or 1ml, to inoculate a suitable amount of

Soyabean Casein Digest Broth, mix. When testing transdermal patches, filter the volume of sample

corresponding to one patch of the preparation through a sterile filter membrane, and place in 100 ml of

Soybean–Casein Digest Broth. Incubate at 300C to 350C for 18 to 24 hours.
5.10.2 Selection and subculture: Subculture on a plate of Cetrimide Agar, and incubate at 300C to 350C for 18 to 72 hours.
5.10.3 Interpretation: Growth of greenish colonies indicates the possible presence of Pseudomonas aeruginosa.
5.11 Test for absence of Staphylococcus aureus
5.11.1 Sample Preparation and Pre-incubation: Prepare the sample as per section No. 5.3.1.
Use 10 ml or the quantity corresponding to 1 g or 1ml, to inoculate a suitable amount of

Soyabean casein digest Broth, mix. When testing transdermal patches, filter the volume of sample

corresponding to one patch of the preparation through a sterile filter membrane,

and place in 100 ml of Soybean Casein Digest Broth. Incubate at 30ºC to 35ºC for 18 to 24 hours.
5.11.2 Selection and Subculture: Subculture on a plate of Mannitol Salt Agar, and incubate at 30ºC to 35ºC for 18 to 72 hours.
5.11.3 Interpretation: The possible presence of Staphylococcus aureus is indicated by

the growth of yellow or white colonies surrounded by a yellow zone.

6.0 ABBREVIATIONS

Abbreviation used	Full form of abbreviation used
QA	Quality Assurance
QC	Quality Control
MLT	Microbial Limit Test
°C	Degree Centigrade
IP	Indian Pharmacopeia
USP	United State Pharmacopeia