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

• 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:

• 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:

• 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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