SOP on Procedure for microbiological monitoring of Purified water in pharmaceutical company

by
  • OBJECTIVE  
  • To lay down a procedure for microbiological monitoring of raw water and purified water
  • SCOPE:
  • This SOP shall be applicable for sampling and microbial analysis of raw and purified water from all user points.
  • RESPONSIBILITY
  •  Quality Control Executive / Officer
  • ACCOUNTABILITY
  • Quality Assurance Head.
  • PROCEDURE:
  • Sampling of water
  • Sampling of water from various points shall be done as per the sampling plan.
  • For sampling of raw water, sanitize the point to be sampled externally with filtered 70% IPA solution.
  • Open the valve and drain water from the point for about 30 seconds for purified water and 1 minute for raw water.
  • In sterile containers/sterile sampling bags collect about 250 ml of water and label it appropriately with the type of water, sampling point number and date of sampling.
  • Bring the samples to the microbiological lab for testing.
  • Methods of testing:
  • Prepare the media required for testing and sterilize it in steam sterilizer
  • If the sampled water cannot be tested within two hours after sampling, refrigerate the water at 2- 8 degree Centigrate and test within 24 hrs.   DO NOT FREEZE THE WATER SAMPLE
  • For analysis purpose, mark all the petri plates and test tubes with sampling point code and date of analysis.
  • Test the water sample for the following parameters:
  • Total viable aerobic microbial count (TVAMC)
  • coli
  • Salmonella
  • aeruginosa
  • aureus
  • Test for TVAMC
  • Aseptically pipette out 1 ml of sample into a sterile petri plate and add 20-22 ml of sterilize and cooled soybean casein digest agar (SCDA) to the plates.
  • Swirl the plates gently to mix the media and sample, allow the media to solidify.
  • For testing of pathogens place a sterile 0.45µ membrane filter on the perforated base of a sterile filtration assembly, transfer 100 ml of water to it and filter it with the aid of vacuum.
  • Upon completion of filtration, transfer the membrane filter to a tube containing 100ml of sterile Soybean Casein Digest Medium (SCDM) and incubate the tubes at 35-37°C for 18 to 48 hrs.
  • Test for E. coli
  • If any turbidity observed then transfer 1 ml of 48 hrs. SCDM to 100 ml of Mac Conkey broth and incubate at 43 to 45°C for 18 to 24 hrs
  • Streak a loop full of enrichment media on the plates of MacConkey’s agar(MCA) and Eosin Ethylene Blue (EMB) agar plates. Preserve the rest of enrichment media.
  • Incubate the plates at 35-37 for 18-48 hrs.
  • Upon observation growth of red,non mucoid colonies on Mac Conkey’s agar plates or translucent colonies surrounded with metallic sheen on Eosin Ethylene Blue agar plates indicating possible presence of E.coli
  • In presence of characteristic colonies, perform confirmatory test .
  • Confirmatory test:  Add 0.1ml of the Mac Conkey Broth for the previous step, to 5ml of sterile tryptone water and incubate the tubes at 42-44°C for 24hrs.
  • After completion of incubation, add 0.5ml of Kovac’s reagent to the tubes containing tryptone water and allow it to stand for 1 minute. Appearance of red coloration in the reagent layer indicate confirmation of E.coli.
  • Test for Salmonella:
  • Transfer 1.0ml of 48 hrs culture from SCDM  to 10ml of Tetra Thionate Bile Brilliant Green (TTBG) broth and incubate at 41-43°Cfor 18-24 hrs.
  • After incubation streak a loop full from the TTBG onto Brilliant Green Agar(BGA) and Xylose Lysine Deoxycholate Agar (XLDA).
  • Incubate the plates at 35-37°C for 18-72 hrs.
  • The probable presence of salmonella is indicated by the growth of colonies with the following characters:
MEDIUM Colony Characteristics
BGA Small, transparent, colorless or pink or opaque white colonies often surrounded by a pink or red zone.
XLDA Red colonies with or without black centers
  • In case of presence of characteristic colonies, perform confirmatory test.
  • Confirmatory Test: – Transfer the suspected colony onto Triple Sugar Iron(TSI) slants at 36-38°C for 18-24 hrs. The formation of acid or gas in the stab culture(with or Without concomitant blackening) and the absence of acidity from the surface growth in the TSI, indicates the presence f salmonella in the specimen.
  • Test for Pseudomonas aeruginosa:
  • From the 48 hrs old SCDM tube streak a loopfull of the medium on the surface of sterile Cetrimide Agar. Incubate the plates at 35-37°C for 18-72 hrs.
  • The growth of greenish colonies on the surface of Cetrimide agar indicate the presence of Pseudomonas in sample tested.
  • In case of presence of characteristic colonies, perform the confirmatory test.
  • Confirmatory Test: – Perform the oxidase test by smearing the suspected colony onto a filter paper wetted with saturated solution of N,N,N,N-tetra methyl-diphenyl dihydrochloride or oxidase disc. If there is no development of pink color changing to purple on the paper or disc, the sample passes the test for absence of Pseudomonas.
  • Test for S. aureus:
  • From the 48hrs old SCDM tube ,streak a loopful of  medium on the surface of sterile Mannitol Salt Agar(MSA) and    incubate the plates at 35-37°C for 18-72 hrs.
  • The occurrence of yellow colonies with yellow zones on the agar surface indicate the presence of S aureus.
  • If the characteristic colonies are observed, confirm the presence of S.aureus by performing the Coagulase Test (confirmatory test)
  • Confirmatory Test:- Transfer a small portion of the suspected colony within the aid of nichrome wire loop onto a slide/test tube containing 0.5ml of mammalian plasma. Incubate the test tube or slide at 37°C for 24 hrs and observe the presence of coagulation. If no coagulation is observed, the test passes for the absence of aureus.
  • Record the results of analysis in annexure-I. (Note: The annexure shall be finalized after the completion of one year of validation study)
  • Limits:
PURIFIED WATER RAW WATER
Alert Limit 30CFU/ml 100CFU/ml
Action Limit 80CFU/ml 200CFU/ml
  • If any of the result exceeds the alert limit then inform Head QC/QA, followed by Head Production & Engg.
  • If any of the result exceed the alert limit on two consecutive days then inform Head QC/QA, followed by Head Production & Engg., investigate the probable cause and sanitize the system
  • If any of the result exceed the action limit on two consecutive days showing an upward trend, then inform Head QC/QA, followed by Head Production & Engg,, stop the supply of water for production activities and sanitize the system.
  • Forms and Records (Annexures)
  • Not Applicable
  • Distribution
  • Master copy –  Quality Assurance
  • Controlled copies- Quality Assurance, Production, Quality Control.engineering
  • History
    Date Revision Number Reason for Revision
    – 00 New SOP

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