- To lay down a procedure for microbiological monitoring of raw water and purified water
- This SOP shall be applicable for sampling and microbial analysis of raw and purified water from all user points.
- Quality Control Executive / Officer
- Quality Assurance Head.
- 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)
- 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:
||Small, transparent, colorless or pink or opaque white colonies often surrounded by a pink or red zone.
||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)
- 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
- Master copy – Quality Assurance
- Controlled copies- Quality Assurance, Production, Quality Control.engineering
||Reason for Revision
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