1. Objective
To lay down a procedure for microbiological analysis of water.
2. Scope
This procedure is applicable for analysis of potable, purified and distilled water.
3. Responsibility
Microbiology department
4. Procedure
4.1 Limits: Total viable count:

Pathogens: Escherichia coli, Salmonella species, Pseudomonas aeruginosa and Staphylococcus aureus must be absent
4.2 Before start of the activity, engineering department shall confirm the readiness of the water system for sampling.
4.3 Sampling shall be performed as per sampling plan.
4.4 Sampling location and frequency of sampling should be available with microbiologist at the time of sampling.
4.5 Samples shall be collected in clean pre-sterilised neutral glass stoppered bottle from each sampling point.
4.6 Carry the sampling bottles to the sampling points in a tray.
4.7 Use water proof marking pen to write the identity of the point of use/sampling point and sampling date on the sampling bottle.
4.8 Sampling bottle shall not be opened except at the time of sampling.
4.9 Remove the cover of sampling point.
4.10 Spray the sampling point using 70% IPA solution and wait for 5 minutes.
4.11 Open the valve of sampling point slowly to full and drain water for approx. 1 minute.
4.12 Control the valve of sampling point to avoid splashing.
4.13 Carry out sampling step 4.14 and 4.19 as rapidly as possible to avoid exposure of the sample to the external environment. Microbiologist shall wear sterilised gloves during the sampling activity.
4.14 Take sampling bottle near the sampling point.
4.15 Microbiologist carrying the sampling activity must hold the bottle at the base with one hand such that it faces slightly away from him/her.
4.16 Remove the aluminium foil and stopper, hold them in hand.
4.17 Avoid touching the bottom of the stopper and passing hands over the open neck of the bottle during sampling.
4.18 Collect 300 ml sample in bottle.
4.19 Stopper the sampling bottle immediately and cover with the aluminium foil.
4.20 Return the sample bottle to the tray and proceed to the next sampling point.
4.21 Repeat the procedure till the designated points have ben sampled.
4.22 If a hose pipe is attached to any point of use, the end of hose pipe shall be considered as sampling point.
4.23 If a hose pipe is not attached to a sampling point but is routinely used at that sampling point, it must be attached to the point of use prior to sampling.
4.24 In both the above cases, step Nos. 4.22 and 4.23, drain the water from the hose pipe for approx. 1 minute before drawing the sample.
4.25 For potable water and at all stages of purification prior to the SMBS dosing stage aseptically add 2 ml of 0.5% filter sterilised Sodium thiosulphate to the sample bottle prior to sampling bottle.
4.26 Solution of Sodium thiosulphate should be prepared and consumed as required.
4.27 Record the preparation of Sodium thiosulphate.
4.28 Analysis of samples should be preferably commenced within two hours of sampling.
4.29 If, for any reason, it is not possible to commence analysis within this period, store the sample at 2°C – 8°C and ensure that analysis is carried out within 24 hours of sampling.
4.30 Discard the sample if not tested within above specified time interval.
4.31 Resample and retest the water from the concerned sampling point/s immediately.
4.32 Method of testing for Total Viable Count:

4.32.1 Pour plate method for Total Viable Count (TVC) – Potable water:
4.32.1.1 TVC shall be performed in duplicate, for each sample using R2A agar as the medium.
4.32.1.2 Write the name of media, sampling point under test and date on bottom of each petri dish.
4.32.1.3 Aseptically transfer 1 ml of sample on each petri dish.
4.32.1.4 Pour the sterile melted and cooled media into petri dishes and mix well, avoiding formation of bubbles.
4.32.1.5 Allow the agar media to set. Transfer the petri dishes to incubator and incubate at 30C – 35C for 5 days.
4.32.1.7 After incubation, count the number of colonies recovered.
4.32.2 Membrane Filtration Method for Total Viable Count (TVC) for Purified/Distilled water IP/EP:
4.32.2.1 Write the name of the medium, sampling point under test and date on the bottom of petri dish containing 15-20 ml solidified sterile R2A agar.
4.32.2.2 Filter 10 ml sample from each sampling point of the ambient loop of distilled water/purified water through a sterile 0.45-micron pore size cellulose acetate membrane filter.
4.32.2.3 Filter 100 ml sample of distilled water/purified water from the hot loop through a sterile 0.45-micron pore size cellulose acetate membrane filter.
4.32.2.4 Aseptically transfer the filter to the surface of medium in the petri dish.
4.32.2.5 Transfer petri dish to incubator and incubate at 30C – 35C for 5 days.
4.32.2.6 After incubation, count the number of colonies recovered.
4.33 Detection of pathogens:
4.33.1 Aseptically transfer 100 ml of the water sample to a conical flask containing sterile 100 ml double strength casein soyabean digest broth.
4.33.2 Incubate the above flask at 30C – 35C for 24 hours.
4.33.3 If growth (turbidity) is observed in the flask, proceed for further testing to confirm the presence or absence Escherichia coli, Salmonella species, Pseudomonas aeruginosa and Staphylococcus aureus
4.33.4 If no growth is observed in the flask of casein soyabean digest medium, Escherichia coli, Salmonella species, Pseudomonas aeruginosa and Staphylococcus aureus are absent from the sample under test.
4.33.5 Record the results of testing for absence of the specified pathogens.
4.34 Interpretation of results:
4.34.1 The sample under test comply with microbiological standards if the results comply with the alert limits for TVC and pathogen absent.
4.34.2 If the result of the TVC exceeds the alert/action limit, inform the concerned department heads for carrying out appropriate corrective action.
4.34.3 If any of the specified pathogens are detected or TVC found above target limit from any sampling point, inform the QC manager and concern department heads. Operation of the purified water generation and distribution system should be suspended and detailed investigation shall be performed. OOS shall be logged wherever applicable.
4.35 Identification of isolates:
4.35.1 Any organism detected whilst testing for the total viable count for purified water/atypical colonies detected on any specific medium must be checked for gram nature and identified to minimum genus level.
5. Abbreviations:
SOP: Standard operating procedure
OOS: Out of specification
TVC: Total viable count
QC: Quality control
IP: Indian Pharmacopoeia
EP: European Pharmacopoeia
USP: United States Pharmacopoeia
CFU: Colony forming unit
SMBS: Sodium metabisulphite
R2A agar: Reasoner’s 2A agar
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