MPK | SOP FOR MICROBIOLOGICAL EXAMINATION OF WATER SAMPLES | MPK

SOP FOR MICROBIOLOGICAL EXAMINATION OF WATER SAMPLES

Examination of water

1.0.      Purpose

To carry out the Microbiological examination of water samples.

 2.0.      Scope:

This procedure is applicable for the Microbiological examination of different water samples i.e. Potable water, De-ionized (D.I) water, Reverse Osmosis(R.O)water, Purified Water and Water For Injection(W.F.I) in Micro lab.

 3.0.      Responsibilities

Action	Responsibility
Training over this procedure	Manager
Implementation of this procedure	Manager
Ensure effective implementation of this procedure	Head of Department

 4.0.      General Requirements:

4.1.   Glassware

4.2  Sterile Petri plates

4.3  Sterile Pipettes

4.4  Sterile caped sample bottles

4.2. Media and Reagents

4.5  R2A agar (Low nutrient media for slow-growing oligotrophic bacteria, stressed and chlorine damaged bacteria)

4.6  Standard Plate Count Agar SPC (High nutrient media for heterotrophic bacteria)

4.7  Tryptic Soy Broth

4.8  MacConkey’s agar

4.9  Eosin Methylene Blue (EMB) Agar

4.0.1  Pseudomonas Cetrimide agar

4.0.2  Sterilized Sodium Thiosulfate  Solution (100gm/L)

4.0.3  Sterile water blanks

4.3.   Sampling Criteria

Drain out water at full flow for about two minutes before collecting water samples from any sampling points. Then carefully collect sample in a sterilized capped bottle.

4.4.   Sampling Points

         4.4.1. Potable Water

·               Underground tank

·               Main supply line

·               Basic dept.

·               Liquid manufacturing area

·               Purified water plant

·               Canteen

·               D.I water plant from 

        4.4.2. D.I Water

·              bed of D.I water plant

·               D.I Storage Tank of D.I  plant

·               Water town water plant system

·               Basic department

·               Ointment Manufacturing

·               Tablet Section

·               A mixed bed of purified water plant

         4.4.3. D.I Water

·               Mixed bed

·               Oral Antibiotic Area.

         4.4.4. D.I water from Purified Water Plant

·               Mixed bed

         4.4.5. Process Water

·               Process water

·               Process water from the storage tank

         4.4.6. Purified water

·               After Ozonation

·               After U.V1

·               Purified Water Storage Tank

·               After loop & UV2

·               Liquid Manufacturing line

         4.4.7. R.O water

·               Water town plant

·               R.O vial washing

·               R.O Bungs washing (0.2µm)

·               R.O water from ultrasonic vial washing

·               R.O water plant from

         4.4.8. W.F.I

·               Ampoule section

·               Vial washing

·               E/E drops section

·               Water town plant

·               WFI plant 

·               Washing Area (Sterile Penicillin      

4.5.   Frequency

         4.5.1. Daily

·               Potable water from Main supply line water

·               Potable water from Underground tank water

·               Potable water from Basic Department

·               Purified water from Liquid manufacturing area

·               WFI Ampoule Section

·               WFI E/E Drop  Section

·               WFI from Water town plant

         4.5.2. Weekly

·               Potable water Liquid manufacturing area

·               Potable water Purified water plant

·               Potable water Canteen

·               Process water, D.I water and purified water from Purified water plant

·               Mixed bed sample from D.I water plant

·               D.I water from a tablet, Basic, ointment manufacturing and Water town plant

·               Potable and D.I water plant  

·               R.O water samples from ceph. Area and water town plant

·               WFI samples from ceph. Area

5.0.      Procedure:

5.1.   Potable Water

S. No	Action	Responsibility
5.1.1	Aseptically Add 4 -5 drops of sterilized 10% sodium thiosulphate to 200 ml of water sample for neutralization of chlorine.	Microbiologist
5.1.2	Pour plate method: Dilutions are to be made aseptically. For this purpose transfer 1ml of the sample to the 9ml of sterile water blank with the help of a sterile pipette, this will give a dilution of 1:10.	Microbiologist
5.1.3	To make a dilution of 1:100, transfer 1ml of the sample from 1:10 dilution to the 9ml of sterile water blank.	Microbiologist
5.1.4	From the above dilutions transfer 1ml sample to the sterile Petri plates marked with respective dilutions aseptically in the replica.	Microbiologist
5.1.5	Pour cooled melted media R2A media & Standard Plate Count Agar into these plates by gentle rotation, allow the media to solidify.	Microbiologist
5.1.6	Incubate the plates of Standard Plate Count Agar at 30 to 35°c for 72 hrs and plates of R2A agar at 20 to 25°c for 5 to 7 days.	Microbiologist
5.1.7	Transfer 1 ml sample into TSB tube. In the case of weekly underground water, sample Inoculate filter of 0.45µ after passing 100ml sample into TSB medium. Incubate TSB at 30 to 35°c for 24 hrs.	Microbiologist
5.1.8	After incubation note down the results. In the case of turbidity in TSB transfer the growth by streaking with the help of sterilized wire loop on the surface of plates of Macconkey’s, EMB, and Cetrimide media plates.	Microbiologist
5.1.9	Alert Limits: NMT 250cfu/ml of total microbial count Action Limits: NMT 500cfu/ml of the total microbial count. Ecoli and Pseudomonas aeruginosa should be absent.	Microbiologist

 

5.2.   D.I Water /Purified water

S. No	Action	Responsibility
5.2.1	Pour plate method: Aseptically pipette out 1ml of the sample and transfer in sterile Petri plates in the replica.	Microbiologist
5.2.2	Pour cooled melted media R2A media and Standard Plate Count Agar into these plates by gentle rotation, allow the media to solidify and at the same time add 1ml to TSB broth also.	Microbiologist
5.2.3	Incubate the plates of Standard Plate Count Agar at 30 to 35°c for 48 to 72 hrs and R2A agar at 20 to 25°c for 5 to 7 days.	Microbiologist
5.2.4	Transfer 1 ml sample into TSB tube. In case of purified water Liquid manufacturing (weekly) Inoculate filter of 0.45µ after passing 100ml sample into TSB medium. Incubate TSB at 30 to 35°c for 24 hrs.	Microbiologist
5.2.5	After incubation note down the results. In the case of turbidity in TSB transfer the growth by streaking with the help of sterilized wire loop on the surface of plates of Macconkey’s agar, EMB, and Cetrimide media plates.	Microbiologist
5.2.6	On Daily Basis: Aseptically pipette out 1ml of the sample and transfer in sterile Petri plates in the replica.	Microbiologist
5.2.7	Pour cooled melted Standard Plate Count Agar, about 45°c into these plates by gentle rotation, allow the media to solidify.	Microbiologist
5.2.8	Incubate the plates of Standard Plate Count Agar at 30 to 35°c for 72 hrs.	Microbiologist
5.2.9	Alert Limits: Total microbial count For DI water, NMT 80 CFU/ml and for Purified water NMT 25 CFU/ml. Action Limits: Total microbial count For DI water, NMT 100 CFU/ml and for Purified water NMT 50 CFU/ml.Ecoli and Pseudomonas aeruginosa should be absent	Microbiologist

 

5.3.   R.O Water and W.F.I

S. No	Action	Responsibility
5.3.1.	Membrane Filtration method: Aseptically assemble the sterilized filter assembly under a laminar flow, a hood having 0.45 µm and aseptically transfer about 100ml of the sample through filtration assembly.	Microbiologist
5.3.2.	RO water: After passing the sample remove the filter and transfer aseptically in pre-incubated plates of R2A agar and Standard Plate Count Agar medium.	Microbiologist
5.3.3.	Incubate the plates of Standard plate count medium at 30 to 35°c for 48 to 72 hrs and R2A agar at 20 to 25°c for 5 to 7 days.	Microbiologist
5.3.4.	Transfer 1 ml sample into TSB tube. In the case of R.O water from water town plant (weekly) inoculate filter of 0.45µ after passing 100ml sample into TSB medium and Incubate TSB at 30 to 35°c for 24 hrs.	Microbiologist
5.3.5.	After incubation note down the results. In the case of turbidity in TSB transfer the growth by streaking with the help of sterilized wire loop on the surface of plates of Macconkey’s, EMB, and Cetrimide media plates.	Microbiologist
5.3.6.	W.F.I: After passing the sample remove the filter and transfer aseptically in pre-incubated plates of Standard plate count agar medium. In case of weekly WFI transfer the filter on a pre-incubated plate of both SPC and R2A.	Microbiologist
5.3.7.	Incubate the plates of Standard plate count medium at 30 to 35°c for 48 to 72 hrs and R2A agar at 20 to 25°c for 5 to 7 days.	Microbiologist
5.3.8.	Alert/Action limits: For R.O water, N.M.T 10cfu/100ml, Ecoli and Pseudomonas aeruginosa should be absent and for W.F.I ,N.M.T 01cfu/100ml.	Microbiologist

 

 6.0.      Definitions:

Term/word/statement	Meaning/Description
Cfu	Colony-forming unit
RO water	Reverse Osmosis water
SPC	Standard plate count
WFI	Water for Injection

 7.0.      References Used:

S. No	Description
1.	USP 2009.
2.	Guideline for Drinking water quality, Second Edition Volume 3, World Health Organization Geneva.

 

9.0.      Referred to Document(s):

Document No	Description
N/A	N/A

 

10.0.   Annexure:

S. No	Description of Annexure
1.	Weekly Report of Microbiological Examination of water
2.	Weekly Report of Microbiological Examination of Purified Water Plant
3.	Daily Report of Microbiological Examination of  Water
4.	Weekly Report of Microbiological Examination of R.O  Water
5.	Weekly Report of Microbiological Examination of water.