Water Sampling Procedure for Microbiological analysis

SOP for Sampling and Microbiological analysis of Water | Microbiological analysis of Water in Pharmaceutical Industry |

SOP Covers below points:

Sampling Procedure of Raw water/Purified water/DM water For Microbiological Analysis
Sampling Procedure of Water for Injection For Microbiological Analysis
Sampling procedure of Pure steam for Total aerobic microbial count
Sampling procedure of Water for Injection For Bacterial Endotoxin Test
Sampling procedure of Pure steam for Bacterial Endotoxin Test
Microbiological Analysis – Method of Analysis
- Total Viable Count (TVC) Test for Pure steam/DM Water/Purified water/water for injection
- Specified Microorganisms test for Pure steam/DM Water/Purified water/water for injection
Water Sampling points and frequency
Water analysis Trend
Water analysis report format

For sampling of water for chemical testing and release of water and pure steam. click link – https://pharmablog.in/sampling-testing-and-release-of-water

1.0 Sampling Procedure (Microbiological analysis of water):

1.1 Sampling Procedure of Raw water/Purified water/DM water For Microbiological Analysis :

Collect the sample in sterile sampling bottle as per the sampling schedule.
Clean the surface of the sampling points which are outside the core manufacturing area with 70% filtered IPA. Sampling points i.e., user points inside the core manufacturing area shall not be sanitized with 70% filtered IPA before sampling. In raw water sampling bottle pour 0.3 mL of 10% thio sulphate before sterilization of sampling bottle.
Drain out water for not less than 5 minute
During sampling, handle the sampling bottle near to the nozzle of the sampling point taking care that the nozzle of the sampling point shall not touch the surface of the sampling bottle.
Collect about 2 x 100 ml sample in a sterile glass bottle taking care not to touch the inside surface of cap/stopper or neck of the bottle.
Leave ample air space in the bottle to facilitate mixing by shaking before examination.
Collect the sample and immediately close stopper/ cap.
Mark the identity of sample, sampling point, Date of sampling, time of sampling, sampled for and sampled by on the container as per the label given below.
Transfer the sample to Microbiology laboratory for analysis.
Analyze the sample within 2 hours from collection, alternatively keep the sample in refrigerator at 2° – 8°C and analyze within 4 hours. (For Purified water or WFI allow the sample cool to room temperature before analyzing).

1.2 Sampling Procedure of Water for Injection For Microbiological Analysis :

Collect the sample in the sterile sampling bottle as per the sampling plan.
Clean the surface of the sampling points which are outside the core Manufacturing area with 70% filtered IPA. Sampling points i.e., user points inside the core manufacturing area shall not be sanitized with 70% filtered IPA before sampling.
Allow the water to drain for not less than 1 minute
During sampling, handle the sampling bottle near to the nozzle of the sampling point taking care that the nozzle of the sampling point shall not touch the surface of the sampling bottle.
Collect about 1 x 300 ml sample in sterile glass sampling bottle taking care not to touch the inside surface of cap/stopper or neck of the bottle. Immediately close the stopper/ cap of the bottle and seal the top of the sample bottle. Collect 1 x 100 ml for pathogen testing.
Mark the identity of sample, sampling point, Date of sampling, time of sampling, sampled for and sampled by on the container as per the label given above.
Transfer the sample to Microbiology laboratory.
Analyze the sample within 2 hours from collection, alternatively keep the sample in refrigerator at 2° – 8°C and analyze within 4 hours. (For Water for Injection allow the sample to come to ambient temperature before analyzing).

1.3 Sampling procedure of Pure steam for Total aerobic microbial count :

Attach the steam condensate accessories to the sampling point.
Allow the steam condensate to drain for about 1 minute.
Remove the stopper/ cap taking care not to touch the surface of the cap of neck of the bottle.
Rinse the sampling container and stopper / cap with the sample water to be sampled 2-3 times.
During sampling, handle the sampling bottle near to the nozzle of the sampling condensate accessory drain point taking care that the nozzle of the drain point shall not touch the surface of the sampling bottle.
Collect about 300 ml of sample in a sterile glass sampling bottle taking care not to touch the inside surface of cap/stopper or neck of the bottle in duplicate.
Leave sample air space in the bottle to facilitate mixing by shaking before examination.
Collect the sample and immediately close stopper/ cap.
Mark the identity of sample, sampling point, sampled for, sampled by on the sampling bottle.
Transfer the sample to MLT room for analysis.
Analyze the sample within 2 hours from collection, alternatively keep the sample in refrigerator at 2°-8ºC and analyze within 4 hours.

1.4 Sampling procedure of Water for Injection For Bacterial Endotoxin Test :

Depyrogenate the glass Test Tube at 250ºC for 2 hour.
Use depyrogenated aluminum foil sealed test tubes for sample collection.
Collect the sample in the depyrogenated test tubes as per the sampling schedule.
Clean the surface of the sampling points which are outside the core manufacturing area with 70% filtered IPA. Sampling points i.e., user points inside the core manufacturing area shall not be sanitized with 70% filtered IPA before sampling.
Allow the water to drain for not less than 1 minute.
During sampling, handle the test tube near to the nozzle of the sampling point. Taking care that the nozzle of the sampling point shall not touch the surface of the sampling test tube.
Collect 1 x 10 ml of sample in depyrogenated test tubes close and seal with aluminum foil.
Mark the identity of sample, sampling point, and date of sampling, time of sampling, sampled for and sampled by on the container as per the label given above.
BET of all user point for Water for Injection is performed twice in a year.
Allow the sample to cool at room temperature and analyze immediately

1.5 Sampling procedure of Pure steam for Bacterial Endotoxin Test :

Use depyrogenated test tube for sample collection.
Use depyrogenated aluminum foil sealed test tubes for sample collection.
Collect the sample in the depyrogenated test tubes as per the sampling schedule.
Clean the surface of the sampling points which are outside the core manufacturing area with 70% filtered IPA. Sampling points i.e., user points inside the core manufacturing area shall not be sanitized with 70% filtered IPA before sampling.
Allow the water to drain for not less than 1 minute.
During sampling, handle the test tube near to the nozzle of the sampling point. Taking care that the nozzle of the sampling point shall not touch the surface of the sampling test tube.
Collect 1 x 10 ml of sample in depyrogenated test tubes close and seal with aluminum foil.
Mark the identity of sample, sampling point, and date of sampling, time of sampling, sampled for and sampled by on the container as per the label given above.
Transfer the sample to BET room for analysis.
Analyze the sample immediately.
Quantity of sample required for the analysis is given below.

Type of water	Type of Analysis	Sample Quantity	Sample Bottle
Raw Water	Microbiology [Pour Plate ]	1 x 100 ml	Sterile
DM Water	Microbiology [Pour Plate]	1x 100ml (TVAC) 1×100 ml (Pathogen)	Sterile
Purified Water	Microbiology [Membrane Filtration]	1 x 100 ml (TVAC) 1 x 100 ml (Pathogen)	Sterile
Water for Injection	Microbiology [Membrane Filtration]	1 x 300 ml (TVAC) 1 x 100 ml (Pathogen)	Sterile
WFI / Pure steam	Bacterial Endotoxin Test	1 x 10 ml	Depyrogenated

2.0 Microbiological Analysis : Method of Analysis :

For Microbiological analyses of different grades of water carry out the analysis on different grades of water as per the procedure given below.
Pathogen testing of Storage tank of Purified Water and Water for Injection (Selected points) performed daily basis and all user point of (purified water and water for injection) pathogen testing is carried out on quarterly.

GRADE OF WATER	TESTS
Raw Water	TVC
DM Water	TVC, E. coli, Salmonella, P. aeruginosa & S. aureus
Purified Water	TVC, E. coli, Salmonella, P. aeruginosa &S. aureus
Water for Injection	TVC, Endotoxin, E. coli, Salmonella, P. aeruginosa & S. aureus & Endotoxin.
Pure Steam	TVC & Endotoxin & E. coli, Salmonella, P. aeruginosa & S. aureus.

TVC = Total Viable Count. (Total Bacterial Count + Total fungal Count) Note: For Raw water/Dm Water dilute the sample 10 times i.e. 1:10 and take 1 ml of diluted sample for analysis.

2.1 Total Viable Count (TVC) Test for Pure steam/DM Water/Purified water/water for injection :

For Raw Water / DM Water: (Pour plate method)
- Aseptically pipette out 1 ml of water sample to 9 ml of sterile saline to prepare 1:10 dilution.
- Shake the 1:10 dilution of the sample to homogenize the water sample.
- Take 2 pre-sterilized plates and aseptically add 1 ml of sample to each sterile Petri plate.
- Check the temperature of media by using non contact Infrared temperature gun before pouring into plates.
- Promptly add about 15-20 ml of sterilized R-2A agar previously cooled to 40° to 45º C into 2 petriplates.
- Cover the Petri plates, mix the sample with the agar by tilting or rotating the Petri plates and allow the contents to solidify at room temperature.
- After solidification of media, incubate the R-2A plates at 30 to 35ºC for 5 days.
- After incubation, observe the CFU/plate and calculate the CFU/ml by multiplying with the dilution factor.
- Report the average of two plates as CFU/ml of sample.

For Purified Water : (Membrane filtration method)
- Place the filtration cup on to the filtration assembly.
- Place sterile 0.45 µ, 47 mm diameter membrane filter in the filtration cup.
- Add approx. 50 ml sterile 0.1 % peptone in filtration cup
- Aseptically pipette out 1 ml of water sample and add it to filtration cup.
- Filter the water sample through the membrane filter.
- Rinse the filtration cup and membrane filter with approx. 50 ml of 0.1 % sterile peptone water.
- Aseptically place the filter membrane on the R-2A agar media plate.
- Incubate the R-2A plate at 30° to 35ºC for 5 days.
- After incubation, observe the CFU/plate.

For water for injection: (Membrane filtration method)
- Place the filtration assembly
- Place sterile 0.45 µ, 47 mm diameter membrane filter in the filtration cup.
- Filter 1 x 300 ml of water sample through the membrane filter.
- Aseptically place the filter membrane on the R-2A agar media plate.
- Incubate the R-2A plate at 30° to 35ºC for 5 days.
- After incubation, observe the count (CFU) per plate. Divide the counts observed by three to get the counts as CFU/100ml.
- Perform the Bacterial Endotoxin Test as per SOP

For Pure steam: (Membrane filtration method)
- Carry out the total microbial count of pure steam as per analysis procedure for sterile water for injection.
- Before analysis come down the pure steam temperature to room temperature.
- Generation point of pure steam tested only TVC/Chemical monthly and Pathogen testing on quarterly basis.

2.2 Specified Microorganisms test for Pure steam/DM Water/Purified water/water for injection

Inoculate 10 ml of sample to 90 ml of Soyabean casein digest medium.
Incubate the soyabean casein digest medium tube at 30-35°C for 24 hours (solution A).

Test for Escherichia coli:
- Shake the tube of solution A gently and inoculated 1.0 ml of solution A to 100 ml of MacConkey broth.
- Incubate MacConkey tubes at 42°C-44°C for 48 hours.
- After 48 hours of incubation, by means of an inoculating loop, streak a portion from the MacConkey broth tubes on the surface of MacConkey agar plate. Cover and invert the petriplates and incubate at 30ºC to 35ºC for 24-48 hours. Up on examination if none of the colonies conforms to the description as given in table I the specimen meets the requirement of the test for absence of Escherichia coli.
- If colonies conform to description as given in table I than carry out further confirmation by transferring the suspect colonies individually by means of an inoculating loop to the surface of Levine Eosin-Methylene Blue Agar plated on petriplates. If numerous colonies are to be transferred, divide the surface of each plate into quadrants, each of which is seeded from a separate colony. Cover and invert the petriplates and incubate at 30°C to 35ºC for 24-48 hours.
- Upon examination, if none of the colonies exhibit both a characteristic metallic sheen under reflected light and blue-black appearance under the transmitted light, the sample meets the requirement of the test for the absence of Escherichia coli.

Selective media	Characteristics colonial morphology	Gram stain
MacConkey’s agar medium	Brick red non-mucoid colonies surrounded by zone of bile precipitates	Gram-negative rods
Levin Eosin Methylene blue agar medium	Characteristics metallic sheen under reflected light and blue-black appearance under transmitted light	Gram-negative rods

Table: I

Indole test
- If upon observation colonies confirm to description as given in table-I carry out the confirmatory test by transferring the suspect colonies individually by means of an inoculating loop and add to tube containing 5 ml of 0.1 % peptone water. Incubate at 30°C to 35°C for 24-48 hours. After incubation add 0.5 ml of Kovac’s reagent, shake well and allow to stand for 1 minute, if a red colour is produced in the reagent layer, Iodole is present. The presence of acid, gas and Iodole indicate the presence of Escherichia coli.

Test for Salmonellae species:
- After 24 hours, shake the solution A container and transfer 0.1 ml of solution A to tube containing 10 ml of Rappaport vassiliadis salmonella enrichment broth mix and incubate at 30° to 35°C for 24 hours.
- After 24 hours, By means of an inoculating loop streak portions from Rappaport vassiliadis salmonella enrichment broth on the surface of Xylose Lysine Deoxycholate Agar (XLDA) Cover and invert the Petri plates and incubate at 30°C to 35ºC for 24-48 hours.
- Upon examination if none of the colonies conforms to the description given in Table – II, the specimen meets the requirements for the test for absence of Salmonella species.
- If colonies of Gram negative rods matching the description in Table – II are observed, proceed with further identification by transferring representative suspect colonies individually by means of an inoculating wire, to a butt slant tube of Triple Sugar Iron Agar slant by first streaking the surface of the slant and then stabbing the wire well beneath the surface at the same time inoculated by means of an inoculating wire to a tube of urea broth and incubate at 35°C to 37°C for 48 hours.
- If upon examination discloses no evidence of tubes having alkaline (red) slants and (yellow) butts (with or without concomitant blackening of the butt) and none of the tubes of the urea broth medium discloses no evidence of tubes having red colour, the specimen meets the requirements of the test for the absence of the Salmonella species.

Medium	Description of colony	Gram stain
Xylose Lysine Deoxycholate Agar (XLDA)	Red colonies with or without black centers	Negative, short rods (in cluster)

Table – II

Test for Pseudomonas aeruginosa:
- Shake the tubes of solution A gently and by means of an inoculating loop, streak a portion from the solution A on the surface of Cetrimide agar. Cover and invert the petriplate and incubate at 30°C to 35°C for 72 hours.
- After 48 hours, if none of the colonies having characteristics listed in Table – III for the media used the sample meets the requirement for the absence of P.aeruginosa.
- If colonies matching the description in table-III for cetrimide agar are found proceed with further identification by transferring representative suspect colonies individually by help of inoculating loop on agar surface of pseudomonas agar medium for detection of pyocyanin contained in petri plates. Cover and invert the Petri plates.
- Inoculated pseudomonas agar medium for detection of fluorescin and pseudomonas agar medium for detection of pyocyanin Petri plates at 33°C – 37°C for not less than 3 days.
- If none of the colonies having characteristics listed in Table – III the specimen meets the requirements of the test for absence of genus Pseudomonas aeruginosa.
- If colonies of gram negative rods matching the description given in table-III are found proceed with further identification by transferring representative suspect colonies individually by means of an inoculating wire on to oxidase test (N-N-diethyl-p-phenylene diamine oxalate). The test is positive if purple colour is produced within 5 to 10 second. Positive oxidase test and gram negative bacilli indicates the presence of Pseudomonas aeruginosa.

Medium	Characteristic colonial morphology	Fluorescence in UV light	Oxidase test	Gram stain
Cetrimide Agar	Generally greenish	Greenish	Positive	Negative Rods
Pseudomonas Agar Medium for detection of fluorescin	Generally colourless to yellowish	Yellowish	Positive	Negative rods
Pseudomonas Agar Medium for detection of Pyocyanin	Generally greenish	Blue	Positive	Negative rods

Table – III

Test for Staphylococcus aureus:
- Shake the tubes of solution A gently by means of an inoculating loop, streak a portion from the solution A on the surface of Mannitol Salt Agar. Cover and invert the petriplate and incubate at 30°C to 35°C for 72 hours.
- Observe the plates for colony characteristics as per the Table – IV given below. If none of the colonies having characteristics listed in Table – IV for the media used the sample meets the requirement for the absence of genus Staphylococcus aureus.
- If colonies of gram positive cocci matching the description in table–IV carry out the Coagulase test.
- Coagulase Test: With the help of inoculating loop, transfer the suspected colony to tube containing 0.5 ml of mammalian, preferably rabbit or horse plasma. Incubate in water bath at 37°C examining the tubes at 3 hours and subsequently at suitable interval up to 24 hours. If no coagulation in any degree is observed the specimen meets the requirements of the test for absence of Staphylococcus aureus.

Medium	Characteristic Colonial Morphology	Gram Stain
Mannitol Salt Agar	Yellow colonies with yellow zones	Positive cocci (in clusters)

Table – IV

Test Negative Control:
- Run the negative control of each media and incubate it simultaneously with the test sample at same incubation temperature and period. The test is valid if negative control shows no growth at the end of test.
Test Positive Control:
- Run the positive control of each test simultaneously with the test sample, taking 1.0 ml of 10-100 cfu/ml of inoculum of specific organism for specific media. The test is not valid unless the positive control indicates the positive result for respective organism.
Acceptance criteria:
Alert and Action Limit (Total Viable Count)
- Raw Water / Water Point Of DM water:
  - Alert limit : 200 CFU/ml
  - Action limit : 300 CFU/ml
  - Limit : Not more than 500 CFU/ml
- Purified water
  - Alert limit : 60 CFU / ml
  - Action limit : 80 CFU / ml
  - Limit : Not more than 100 CFU / ml
- Water For Injection / Pure steam
  - Alert limit : NA
  - Action limit : NA
  - Limit : 1 CFU /100 ml

3.0 Water Sampling points and frequency:

Sample from Storage Tank of RO water, storage tank of Purified water, and Storage Tank of Water for Injection and return loop for WFI shall be sampled and analyzed daily and that for User’s points shall be analyzed on rotation basis.
The sampling of water shall not be carried out on weekly off and on holidays. However the sampling points of water to be sampled falling on weekly off and Holidays shall be sampled and analyzed on previous day.

4.0 Water analysis Trends shall be prepared for the following activities:

Water analysis data- (Purified water, Water for injection, DM Water)

4.1 Frequency for trend charts preparation:

Prepare monthly trends for water analysis data for all the locations.
Prepare trends before 10^th of subsequent month.

4.2 Colors Coding:

Follow the colors code for different series in a trend chart as below:

Parameters	Colors code
Maximum counts	Green
Minimum counts	Blue
Alert limits	Pink
Action limits	Red
Recommended Limit	Brown
Other Contents	Black/colour

Note: – Parameter for location where it is difficult to calculate the minimum and maximum counts, only maximum count shall be considered.

4.3 Interpretations:

Trends charts shall be interpreted as below:
- Whether there is any specific / adverse trend or out of trend, out of specifications of trend data generated. Whether the data is well within the acceptance criteria.

5.0 ABBREVIATIONS:

QA	Quality Assurance
SOP	Standard operating Procedure
DM	De mineralized
IPA	Isopropyl alcohol
CFU	Colony forming unit
TVC	Total viable count
TAMC	Total aerobic microbial count
NMT	Not more than
SCDM	Soyabean casein digest medium
R-2A	R-2A agar
RVSE	Rappaport vassiliadis salmonella enrichment broth
CA	Cetrimide agar
XLDA	Xylose lysine Deoxycholate agar
MCA	MacConkey agar
MSA	Mannitol salt agar
VRBGA	Violet red bile agar
WFI	Water for injection
PW	Purified Water
PIC/S	Pharmaceutical Inspection Convention and Pharmaceutical Inspection Co-operation Scheme.
MHRA	Medicines and Healthcare products Regulatory Agency
USP	United State Pharmacopeia
BP	British Pharmacopeia
IP	Indian Pharmacopeia
BET	Bacterial Endotoxin Test
NA	Not Applicable

6.0 ANNEXURES:

Annex. No.	Title
01	Water report Total viable Microbial count
02	Total Viable Count for Pure Steam/ Manufacturing Tank
03	Microbial Limit Test of DM Water / Raw Water
04	Protocol for purified water
05	Protocol for water for injection

7.0 SOP REFERENCES

MHRA – Rules and Guidance for Pharmaceutical Manufacturers and Distributors – 2017.
WHO – Good Manufacturing Practices: Water for Pharmaceutical use – Annex 3, 2011.
USP 36, NF 31 – Water for Pharmaceutical purposes <1231>
BP 2018 – Monograph of Purified Water and Water for Injection
IP 2014 – Monograph of Purified Water and Water for Injection

END OF THE SOP

ANNEXURES :

Annex. No. 01 Water report Total viable Microbial count