OBJECTIVE:To lay down a procedure for microbiological monitoring of raw water and purified water.

SCOPE:This SOP shall provide the procedure for sampling and testing of raw water and purified water from all the user points and all the points across the critical functions in the water purification process.

RESPONSIBILITY :Microbiologist



Sampling of water:

Sampling of water from the various points shall be done as per the sampling plan.

Sanitize the point to be sampled externally with filtered 70%v/v IPA solution.

Open the valve and drain water from the point for about 3 minutes.

In sterile containers/ sterile sampling bags collect about 250 ml of water and and label it appropriately with the type of water, sampling point number, date of sampling.

Bring the sample to the microbiology lab for testing.


Method of testing:

Prepare the media required for testing as per SOP.

Operate the autoclave as per SOP.

If the sampled water cannot be tested within 2 hours after sampling, refrigerate the water and test within 24 hrs. DO NOT FREEZE THE WATER SAMPLE.

Test the water samples for the following parameters;

  • Total viable aerobic microbial count (TVAMC)
  • E coli
  • Salmonella
  • aeruginosa
  • S aureus

Test for TVAMC

Place a sterile 0.45 µ membrane filter on the perforated base of the  sterile filtration assembly. Prepare two such assemblies.

  • Transfer 100 ml of water sample to each of the two assemblies and filter the water with the aid of vacuum.
  • Upon completion of filtration, transfer one of the membrane filter intended for the enumeration of TVAMC onto the surface of sterile Soyabean Casein Digest Agar (SCDA) in such a manner that the bottom surface of the membrane is in contact with the agar.
  • Incubate the plates at 30 – 35 oC for 5 days.
  • Transfer the other membrane filter to 100 ml of sterile Soyabean Casein Digest Medium (SCDM) and incubate the tube at 35 – 37 oC for 18 to 48 hrs.

Test for E.coli

Transfer 1 ml of the 48 hr SCDM to 100 ml of Mac Conkey’s broth and  incubate at 43 to 45 oC for 18 to 24 hrs.

Streak a loopfull of the enrichment media on the plates of Mac Conkey’s agar (MCA)and Eosin Methylene Blue(EMB)  agar plates. Preserve the  rest of the enrichment media.

Incubate the plates at 35 – 37 oC for 18 – 24 hrs.

Upon observation, growth of red, non mucoid colonies on MacConkey’s agar plates or translucent colonies surrounded with metallic sheen on Eosin Methylene Blue agar plates indicates possible presence of E.coli.

In case of presence of characteristic colonies, perform confirmatory test.

Confirmatory Test: Add 0.1 ml of the MacConkey’s broth from the previous step, to 5ml of sterile Tryptone water and incubate the tubes at 42 – 44o C for 24hrs.

After completion of incubation, add 0.5 ml of Kovac’s reagent to the tubes containing Tryptone water and allow it to stand for 1 minute. Appearance of a red coloured  in the reagent layer indicates confirmation of  E coli.

Test for Salmonella:

Transfer 1ml of 48 hrs SCDM  to 10ml of TetraThionate Bile Brilliant Green(TTBG) Broth and incubate at 41- 43oC for  18 – 24 hrs.

After incubation, streak a loopfull from the TTBG onto the surface of Brilliant Green Agar (BGA) and Xylose Lysine

Deoxycholate Agar(XLDA).

Incubate the plates at 35 – 37OC for 18 – 72 hrs.

The probable presence of Salmonella is indicated by the growth of colonies with the following characteristics.

Medium Colony Characteristics
BGA small, transparent, colourless 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 and incubate slants at 36-38oC for  18 – 24 hrs.The formation of acid and 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 of Salmonella in the specimen.

Test for Ps. aeruginosa

From the 48hr old SCDM tube, streak a loopful of the medium on the surface of sterile Cetrimide agar.

Incubate the plates at  35 – 37oC for 18 – 72 hrs.

The growth of greenish colonies on the surface of cetrimide agar indicates the presence of Pseudomonas in the sample tested.

In case of presence of characteristic colonies, perform 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-diethylene dihydro bromide 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 48hr old SCDM tube, streak a loopful of the medium on the surface of sterile Mannitol Salt Agar and incubate the plates at 35 – 37oC for 18 – 72 hrs.

The occurance of yellow colonies with yellow zones on the agar surface indicates the presence of S. aureus.

If 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 with the aid of Ni wire loop onto a slide/ test tube                      containing 0.5ml of mammalian plasma.

Incubate the slide or test tube at 37oC for 24 hrs and observe the plasma intermittently for the presence of coagulation.

If no coagulation is observed, the test passes for the absence of aureus.

Record the results of analysis .


Purified Water Raw Water
Alert Limit 30 CFU/m 100 CFU/ml
Action Limit 80 CFU/ml 200 CFU/ml

If any of the results exceed the alert limit on two consecutive days then inform Manager QC/QA ,followed by Manager production & Engg to take necessary corrective action.

If any of the results exceed the action limit then inform Manager QC/QA ,followed by Manager production & Engg, investigate the probable cause and sanitize the system.



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About Pharmaceutical Guidanace

Ms. Abha Maurya is the Author and founder of pharmaceutical guidance, he is a pharmaceutical Professional from India having more than 18 years of rich experience in pharmaceutical field. During his career, he work in quality assurance department with multinational company’s i.e Zydus Cadila Ltd, Unichem Laboratories Ltd, Indoco remedies Ltd, Panacea Biotec Ltd, Nectar life Science Ltd. During his experience, he face may regulatory Audit i.e. USFDA, MHRA, ANVISA, MCC, TGA, EU –GMP, WHO –Geneva, ISO 9001-2008 and many ROW Regularities Audit i.e.Uganda,Kenya, Tanzania, Zimbabwe. He is currently leading a regulatory pharmaceutical company as a head Quality. You can join him by Email, Facebook, Google+, Twitter and YouTube

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