SAMPLING, TESTING OF PURIFIED AND POTABLE WATER

OBJECTIVE:

This SOP describes the procedure to sampling and testing of potable water and purified water used in the manufacture processes and washing purposes. RECEIPT, STORAGE, USAGE, MAINTENANCE OF MICROBIOLOGICAL MEDIA.

SCOPE

Water used for the purpose of manufacturing and washings must be monitored for bioload to ensure that microbial limits are within the specified limits.

RESPONSIBILITY

Maintenance Department, Production Department, Quality Control Department & Quality Assurance.

TESTS

Purified water and potable water are tested for total microbial count and for pathogens like E.Coli, Salmonella, Pseudomonoas and Staphylococcus.

1). SAMPLING PROCEDURE

1. Sampling container: Take a 100ml Sterile bottles

b) Flush out water from the sampling point for 1-2 minutes.

c) Collect about 100 ml of water in the sterile bottles.

d) After sampling analyze the sample with in 2 hrs if not possible within 2 hours from the time of sampling. Store the sample in refrigerator2-8° C upto 8 hrs.

2). EQUIPMENT AND GLASSWARES

a) Sterilize  the manifold for filtration of water sample.

b) use 45 micron membrane filter for sample filtration

3). PROCEDURE

A) TOTAL AEROBIC MICROBIAL COUNT BY FILTRATION METHOD

Set the filtration assembly as shown in the figure.

Membrane Filter

To Vacuum

Water Collecting Vessel

1. Transfer the sterilized equipment to LAF.
2. Remove the stopper from the candle, tighten the candle with holder and connect the vacuum line.
3. Open the cap of the candles and carefully transfer 10 ml of buffered sodium chloride peptone for pre wetting membrane. Apply vacuum and filter the sample.
4. Rinse the membrane with 100 ml of Sterile Buffered Sodium Chloride Peptone

Solution pH 7.0.

1. Remove the membrane from the candles with the sterile forceps and transfer to R2A Plate.

6. Incubate the plates at 30-35° c for five days,

1. After incubation period count the number of CFU per plate and record. Confirm that the counts.
2. Carry out the negative control.

B) DETECTION OF PATHOGENS

Add 100 ml of the test sample to 100 ml of soybean casein digest broth medium and incubate at 35° C for 24 hours. This is the Enriched Sample for detection of pathogens.

I. TEST FOR E.COLI

1 ml of the enriched Sample is added to 100 ml of MacConkey broth and incubated at 40-45 °C for 48 hours. After incubation completed take loopful from MCB and streak on MacConkey agar and incubate the plates at 30-35° C for 24-72 hrs. serve the results if brick red colonies found on MCA plate its indicats positive of E.Coli and perform the indole test for confirmation.

INDOLE  TEST

Add 0.5ml of the Kovac’s reagent shake well and allow to stand for 1 minute. If a red colour is produced in the reagent layer Indole is present. Carry out a positive control test by repeating the primary and secondary  tests  using a positive control  and a negative control. The test is not valid unless the results indicate that the positive control contains the organism under test and the negative control giving the absence of the organism.

LIMIT: E.Coli should be absent.

II. TEST FOR SALMONELLA

a). Transfer 1 ml of the enriched sample to 10 ml of RVSEB .  Incubate at 35 -35 ° C for 24 hours. After incubation period completed take loop full and streak on XLD Plate. And incubate plates 24-78 hrs. If any colonies found with Red With or Without Black Centers. Indicates present of Salmonella.

b) .SECONDARY TEST

Any of the suspected colonies observed are subcultured on triple sugar iron agar by first inoculating the surface  of the slope and then making a stab culture with the same inoculating needle. Incubate at 35 degree centigrade for 24 hours.  If no evidence of tubes having alkaline (red) slants and acid (yellow) butts (with or without concomitant blackening of the butt from the hydrogen sulphide production) the sample meets the requirements of the test for the absence of Salmonella. Carry out a positive control test by repeating the primary and secondary  tests using a positive control  and a negative control. The test is not valid unless the results indicate that the positive control contains the organism under test and the negative control giving the absence of the organism.

LIMIT : Salmonella should be absent.

III. TEST FOR PSEUDOMONAS AERUGINOSA

Sub-culture the enriched sample on cetrimide agar.  Incubate at 30-35 degree centigrade for 24-72 hours. If upon examination none of the plates contain colonies having the characteristics as listed in table below for the media used, the sample meets the requirement for freedom from Pseudomonas aeruginosa.  If any colonies confirming to the table are found carryout the oxidase and pigment tests. Streak the representative suspected colonies from the agar surface of cetrimide agar on the surfaces of  Pseudomonas agar  medium for detection of fluorescein and pseudomonas agar medium for detection of Pyocyanin contained in petridishes. Invert the petri dishes and incubate at 30 to 35 deg centigrade for three days. Examine the streaked surfaces  under  UV light.  Examine the petridishes to determine whether colonies confirming to description in table- are present.  If growth of respective colonies  are found, place two or three drops of freshly prepared 1% w/v solution of N,N,N,N-tetramethyl-4-pheny­lenediamine dihydrochloride on filter paper and smear the colony. If there is no development of pink colour changing to purple the sample meets the requirement of the test for the absence of Pseudomonas aeruginosa.

TABLE-II

Carryout  the control test by repeating the test adding the prescribed quantity and a volume of broth containing 10  to  50 Pseudomonas aeruginosa NCTC 6750 prepared from a 24 hour  culture in nutrient broth to a sterile screw capped bottle containing 100 ml of soyabean casein medium. The test is invalid if the results do not indicate that the positive control contains Pseudomonas and the negative control giving the absence of the organism.

LIMIT : Pseudomonas should be absent.

IV). TEST FOR STAPHYLOCOCCUS AUREUS

Subculture the enriched sample on Mannitol Salt agar. Incubate the plates for 24-72 hours at 30-35 degree centigrade. Examine for any suspected colonies as described in the table below:

MORPHOLOGIC CHARACTERISTIC OF STAPHYLOCOCCUS AUREUS ON SELECTIVE AGAR MEDIA.

TABLE-III

If growth occurs, carry out the  coagulase test.  Transfer representative suspect colonies from the agar surface of any of the media listed in Table to individual tubes, each containing 0.5 ml of Mammalian, preferably Rabbit or Horse, plasma with or without additives. Incubate in water-bath at 37 degrees examining the tubes at 3 hours and subsequently at suitable intervals up to 24 hours. If no coagulation in any degree is observed, the sample meets the requirements of the test for the absence of Staphylococcus aureus. Carryout the control test by repeating the test adding the prescribed quantity and a volume of broth containing 10 to 50 Staphyloccus aureus NCTC 10788 prepared from a 24 hour culture in nutrient broth to a sterile screw capped bottle containing 100 ml of Soyabean casein medium. The test is invalid if the results donot indicate that the positive control contains pseudomonas and the negative control giving the absence of the organism.

LIMIT : STAPHYLOCOCCUS AUREUS should be absent.

TABLE- IV LIMITS FOR POTABLE WATER AND PURIFIED WATER

4). ACTION TO BE TAKEN IN CASE OF EXCEEDED ALERT LIMITS;

If the result exceeds the alert limit the following corrective action to be taken:

A) FOR POTABLE WATER :

1. Inform QA/Production Departments.

1. Add water purifier to the over head tank after water is pumped apart from adding the water purifier to the water tank directly after receipt of water.
2. Check the cleaning log of water tank.
3. After adding the purifier to the tank, carry out the analysis. If the count obtained is not less than the alert limit inform, house keeping and initiate for thorough cleaning and disinfection of water tank. At the immediate available opportunity repeat the above procedure.

B) FOR PURIFIED WATER

1. Inform QA/Maintenance/Production/ Departments.

2. Take up necessary action in maintenance of water tanks cleaning and sanitation of pipe lines, at immediate available opportunity.

3. Carry out the analysis and check for the counts.

4. If the counts obtained are not below the alert limits, Inform Maintenance

Department to sanitize again and repeat.

5). ACTION TO BE TAKEN IN CASE OF EXCEEDED ACTION LIMITS:

If the result exceeds the Action limit the following corrective action to be taken:

A) FOR POTABLE WATER

1. Microbiologist and QC Manager should communicate to QA/Production/Maintenance Managers and Senior Manager QA/QC and black spot report should be initiated by QC Manager in consultation with Senior Manager QA/QC.

1. Instruct and stop procuring the water immediately.

3. . Get the water tank cleaned thoroughly followed by disinfection.

4. On receipt of the water, before pumping to the over head tank, carry

out microbial test (Preferably the water to be collected from the source

of the supplier).

1. If the results are out of limits inform management about the same and advice to get the water from the second approved supplier.

B) FOR PURIFIED WATER:

1. Inform to all section Heads about the results and instruct them through Senior Manager- QA/QC to stop all manufacturing and Primary Packing activity.
2. Prepare Black spot report.
3. Review all the D.M water logs thoroughly.
4. Inform the maintenance Department to carryout sanitization of complete loopline.
5. After sanitization, run the loop line for one hour and discard the water completely.
6. Restart the loopline. Collect water from all User points including RO outlet and subject for microbial tests.
7. If the counts are found to be within alert limits, Inform Production Department to use the water.
8. However monitor the water from all user points for further three continuous days.
9. If the counts is not below alert limits, Inform Maintenance Department to sanitize the loopline completely.
10. Simultaneously the supplier of the RO Plant to be contacted by the Maintenance Department to sanitize the RO Plant.
11. Repeat the analysis at all user points and RO outlet as described above.

ABBREVATIONS:

MB -MICROBIOLOGY

SOP -Standard operating procedure

REFERENCE:

USP<1231>

ANNEXURES:

Annuxure-1 (water sample report)-FMT/MB/115