To lay down the procedure for Sampling and analysis of Water/Pure steam for Microbiology Lab.


This SOP is applicable to provide the instructions and guidance for Sampling and analysis of Water/Pure steam for Microbiology Lab.


Microbiologist Person shall be responsible for:

To follow this procedure during sampling and analysis of water/pure steam for microbiology lab & maintain the record.

Head QC/Designee shall be responsible for:

To review new or revised SOP.

To maintain the controlled copies of SOPs at required location

Head QA/Designee shall be responsible for:

To approve new or revised SOP.

To ensure implementation of system as per defined SOP

Plant Head shall be responsible for:

To approve new or revised SOP

To ensure implementation of system as per defined SOP.



All apparatus are directly in contact with water/Pure steam i.e. condensate sample shall be sterilized.

Wear sterile gloves before sampling.

Discard the bottles/tubes which have chips, cracks and etched surfaces.

The Sample bottle/tubes shall be opened at the time of sampling.

During sampling the screw cap and the neck of the bottle should not be touched and they should be protected from contamination.

Leaking points, which allow water to flow over the outside of the point should be avoided for sampling and sampled after rectification.

Sample shall be analyzed as soon as possible i.e. within 2 hours after sampling, in case it is delayed, sample shall be stored at 2 -8°C and analyzed before 12 hours.

If the samples are not analyzed within 12 hours, sample shall be discarded and collect the sample again for analysis. Initial A.R. No shall be considered invalid and same shall be handled through incidence.

Allow the samples to reach at ambient temperature prior to analysis.

Pure Steam sampling shall be done by using Pure Steam Sampler.

Heat resistant gloves shall be used for Water for injection/Pure Steam Condensate Sampling.

Preparation of water/Pure Steam Condensate sampling bottles/Tubes

Sterilized glass bottles shall be used for sampling of Water/Pure steam condensate sample for microbiological analysis.

Sterilize and use the cleaned 250 ml glass screw cap bottles for Raw Water, Pretreated water, Purified Water & Pure Steam condensate and 500ml glass bottles shall be used for Water for injection

Note: For the collection of chlorinated water add 0.1 ml 3% of Sodium Metabisulphite solution in to the glass bottle before sterilization in autoclave to neutralize the effect of chlorine. Affix the Label on the bottle as per current version.

Affix appropriate label on the bottle before sampling as per current version.

About 10 ml WFI/Pure Steam condensate sample shall be collected for BET in depyrogenated glass tube covered with aluminum foil of at least 15 ml capacity.

Depyrogenation of glass tube shall be done as per respective procedure.

Sampling of Water/Pure Steam Condensate samples for MLT Analysis

Take required number of sterile bottle 250 ml/500 ml with label, sterile hand gloves, nose mask and 70 % filtered Isopropyl alcohol in perforated container for sampling of Raw Water, Pretreated water, Purified Water & Water for injection & pure steam condensate and sampled as per sampling schedule.

Sample shall be collected directly from sampling point, in case silicon pipe is being used for water collection in manufacturing area, sample shall be collected by considering the silicon pipe.

Follow the entry-exit procedure of respective area from where the sample is to be collected.

Follow the aseptic techniques while collecting the water sample for the prevention of any accidental contamination during the sampling.

Pure Steam Condensate sampling shall be done by Pure Steam Sampler as per current version of SOP.

Before starting the sampling, wear nose mask & sterile hand gloves and sanitizes the gloves with 70 % filtered Isopropyl alcohol.

Before collecting the sample arrange the bucket (whenever required) to prevent water from getting on the floor.

Uncap the sterilized bottle near the sampling point and immediately take the water/pure steam sampling without rinsing the bottle with water/pure steam.

Place the cap immediately and close it tightly. Mention all the information required on the label as per Attachment-II.

Keep all sampled bottles in the perforated SS container and transfer to Microbiology lab.

Follow the instructions given below during sampling of different points of water/pure steam.

Ensure the sampling point have been labeled properly.

Ensure the system/ circulating loop is under operation.

Perform the sampling from the hose pipe or any other delivery device, if it is used during routine usage of water/pure steam.

Collect the sample up to the neck to avoid the head space in sample bottle for microbial analysis to entrapment of environment air.

Avoid touching of the sample container mouth to the sampling point while sampling.

In case any sample container/ bottle fall down or compromised during sampling or analysis same sample point shall be considered for analysis same day or the next day and addressed through QMS if required.

Sampling Plan & Frequency for Analysis of Water/Pure Steam Condensate.

Sampling of water (WFI) & Pure Steam Condensate samples for BET (Bacterial Endotoxin Testing)

Sample for BET shall be collected in depyrogenated tube covered with aluminum foil.

Remove the aluminum foil from the tube immediately prior to collecting the sample.

The “sample contact area of aluminum foil” shall be avoided to contact with any surface, including the fingers or hands of the individual collecting of the sample.

The foil may be set on the top of a clean surface, topside against the surface.

Pure Steam Condensate sampling should be done by Pure Steam Sampler as per current version of SOP.

Open the sampling/user point valve until there is a steady stream of water.

Drain for approximately 30 seconds, collect water in sampling tube.

Do not allow the ‘tube’ to come in contact with the valve.

Fill the tube, without overflowing the tube.

Remove the tube from the sample stream & place the aluminum foil on the tube as quickly as possible and close the valve.

Detail mentioned on the tube for identification of the sample.

Keep all sampled tubes in the SS container and transfer to Microbiology lab.

Receiving storage and inventory of water/Pure Steam condensate sample:

After receiving the sample in microbiology lab, A. R. No. shall be generated for the sample as per SOP

Transfer all the water/Pure steam condensate sample to testing room through the pass box for MLT analysis and perform the analysis as per current version of GTP.

Transfer all the water for injection/Pure Steam condensate sample to BET testing room and perform the BET analysis as per current version of GTP.


Limits for water samples are tabulated below.


Type of water Limit for TVAC Limit for TYMC BET Limit for pathogen
Purified water 100cfu/ml NA NA Saureus, salmonella, P.aeruginosa E.coli, Shigella should be Absent/100ml
Raw water 500cfu/ml Nil cfu/ml NA
In process water (RO-Feed Water,RO-I,Softner,Sand Filter,Chlorinated, Dechlorinated water) 300cfu/ml Nil cfu/ml NA
RO Water 100cfu/ml Nil cfu/ml NA
Pure steam condensate 10cfu/100ml NA 0.25EU/ML
Water for injection 10cfu/100ml NA 0.25EU/ML


Note- Alert and Action limit shall be define.

Also computer copy shall be maintained for reference purpose wherever required. Type

In case results found out of alert limit same shall be inform to QA department as per respective procedure. Incase results found out of action limit and within the limit, same shall be informed to QA department for further course of action. In case results found out of limit same shall be handled through OOS/OOL procedure and production will be on hold till rectification.

After sanitization or adequate rectification, Maintenance department/QA shall inform to Quality Control Department for re-sampling and analysis.

After re-analysis, Quality control Department inform the re-analysis results to Quality Assurance & Maintenance department.

Water Trend shall be prepared on Monthly basis as per respective procedure.

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

Check Also