SOP On Procedure For Microbiological Monitoring of Purified Water in Pharmaceutical Company

SOP on Procedure for microbiological monitoring of Purified water in
pharmaceutical company
Posted By: Pharma Editor on: December 12, 2016 In: QA & QC, Quality Control, SOP No Comments
OBJECTIVE
To lay down a procedure for microbiological monitoring of raw water and puri ed water
SCOPE:
This SOP shall be applicable for sampling and microbial analysis of raw and puri ed water from all user points.
RESPONSIBILITY
Quality Control Executive / O cer
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 ltered 70% IPA solution.
Open the valve and drain water from the point for about 30 seconds for puri ed 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 lter on the perforated base of a sterile ltration assembly, transfer 100 ml of 
water to it and lter it with the aid of vacuum.
Upon completion of ltration, transfer the membrane lter 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 con rmatory test .
Con rmatory 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 con rmation 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
Small, transparent, colorless or pink or opaque white colonies often surrounded by a

BGA
pink or red zone.
XLDA Red colonies with or without black centers
In case of presence of characteristic colonies, perform con rmatory test.
Con rmatory 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 con rmatory test.
Con rmatory Test: – Perform the oxidase test by smearing the suspected colony onto a lter 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, con rm the presence of S.aureus by performing the Coagulase Test (con rmatory test)
Con rmatory 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 nalized 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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SOP on Procedure for microbiological monitoring of Purified water in

Quality Control Executive / O cer

Quality Assurance Head.

number and date of sampling.

Bring the samples to the microbiological lab for testing.

hrs. DO NOT FREEZE THE WATER SAMPLE

Test the water sample for the following parameters:

Total viable aerobic microbial count (TVAMC)

Test for TVAMC

(SCDA) to the plates.

and incubate the tubes at 35-37°C for 18 to 48 hrs.

Test for E. coli

rest of enrichment media.

Incubate the plates at 35-37 for 18-48 hrs.

In presence of characteristic colonies, perform con rmatory test .

42-44°C for 24hrs.

Test for Salmonella:

Incubate the plates at 35-37°C for 18-72 hrs.

MEDIUM Colony Characteristics

Small, transparent, colorless or pink or opaque white colonies often surrounded by a

XLDA Red colonies with or without black centers

In case of presence of characteristic colonies, perform con rmatory test.

the presence f salmonella in the specimen.

Test for Pseudomonas aeruginosa:

for 18-72 hrs.

In case of presence of characteristic colonies, perform the con rmatory test.

or disc, the sample passes the test for absence of Pseudomonas.

Test for S. aureus:

at 35-37°C for 18-72 hrs.

PURIFIED WATER RAW WATER

Alert Limit 30CFU/ml 100CFU/ml

Action Limit 80CFU/ml 200CFU/ml

investigate the probable cause and sanitize the system

Forms and Records (Annexures)

Master copy – Quality Assurance

Controlled copies- Quality Assurance, Production, Quality Control.engineering

Date Revision Number Reason for Revision

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