WATER SYSTEM VALIDATION

1.0 PURPOSE :
To evaluate bioload in purified water systems used for manufacturing.
2.0 SCOPE :
The procedure covers the relevant testing method of purified water for bio-load.
3.0 RESPONSIBILITY :
Microbiologist
4.0 D.M. WATER ANALYSIS
4.1 FREQUENCY :
As per Inhouse Norms (To be declared)
4.2 LIMITS :
Standard Limit : NMT 100 CFU/ml
Action Limit : NMT 25 CFU/ml
Alert Limit : NMT 10 CFU/ml
4.3 ACTION TO BE TAKEN :
In case of exceeding the action limits of bioburden, check up the intensity of the UV tube and give formaldehyde treatment to the resin.
4.4 SAMPLING TECHNIQUE :
Sampling quantity about 200ml of DM water for microbiological testing. Before sampling run the DM water plant and drain 250 ltr of DM water. Take the sample of aseptically in a previously sterilised bottle by rinsing 2-3 times before collection.
4.5 SAMPLING POINTS :
As per the annexure II of SOP No :xxxx
Action plan for out of limit
If the microbial count exceeds to the action limit, the information has to be sent by Q.C Manager to Production Manager, Q.A. Assistant manager and Maintenance Manager as an advance information. Mean while microbial analysis to be carried out daily to confirm the results. If it exceeds the Pharmacopoeial limit immediately inform to Production Manager not to use DM water and inform to Quality Assurance and maintenance department to arrange for resin treatment by formaldehyde.
Analyse the water for microbial count and if the results are withi in the limits then release the water for use and continue the analysis.
4.6 METHOD : By Membrane Filtration
Apparatus
i. Petridishes
ii. Membrane filter ( 0.22 Micrometer ).
iii. Filtration Unit.
iv. Measuring cylinder
Media
Soyabean casein digest agar and sabouraud dextrose agar or sabouraud chloramphenicol agar.
Procedure
Total microbial count by membrane filtration method.
Sterilise all the above mentioned apparatus and media at 121°C for 20 mins at 15 lbs pressure.
Filter 100 ml of the sample through the membrane filter by using the sterile filtration unit under aseption conditions.
Transfer one of the membrane filter’s intended for the enumeration of bacteria, to the surface of a plate of casein soyabean digest agar and the other, intended for the enumeration to fungi to the surface of a plate of saboraud dextrose agar with antibiotics or sabouraud chloramphenicol agar. Similarly filter 100 ml of sterile distilled water as control and carry out the above mentioned procedure.
Incubate the plates for 3 days(72 hrs) at 30-35°C in the test of bacteria and 20-25°C in the test of fungi for 5 days (120 hrs). Count the number of colonies that are formed in distilled water. Calculate the number of microorganisms per ml of the preparation being examined.
5.0 BORE WELL WATER
5.1 FREQUENCY :
As per Annexure II of SOP NO: xxxxx
5.2 LIMITS :
Standard Limit : NMT 500 CFU/ml
Action Limit : NMT 100 CFU/ml
Alert Limit : NMT 50 CFU/ml
5.3 ACTION TO BE TAKEN :
In case of exceeding the action limits of bioburden take up the tank for sanitation.
5.4 SAMPLING TECHNIQUE :
Sampling quantity about 200ml of bore well water for microbiological testing. Before sampling run the bore well water plant for 10 mins. Take the sample of bore well water aseptically in a previously sterilised bottle by rinsing 2-3times before collection and the mouth of the bottle is plugged tightly
5.5 SAMPLING POINT :
As per Annexure enclosed (User have to establish the same)
5.6 METHOD : By Pour Plate Method
Apparatus
i. Petridishes
ii. 1ml sterile pipettes.
Media
Soyabean casein digest agar and sabouraud dextrose agar or sabouraud chloramphenicol agar.
Procedure
Total microbial count by Plate count method.
Sterilise all the above mentioned apparatus and media at 121°C for 20 mins at 15 lbs pressure.
Pipette out 1ml of the water sample into a sterile petriplate aseptically. To the plate add 20ml of molten soya bean casein dextrose agar at 45° and immediately mix the media and inoculum by shaking the plate to and fro and clock wise and anti-clock wise. Ensure that the plate is kept flat on the bench during mixing operation and no agar is allowed on the lid of the dish. Allow the plate to set. Invert and incubate the plate at 30-35°C for the enumeration of bacterial count.for 3 days (72 hrs).
Repeat the above procedure using sabouraud dextrose agar or sabouraud chloramphenicol agar for the enumeration of fungal count.Incubate the plate at 20-25°C for 5 days (120 hrs). After 5 days count the sum of the colonies in the above plates, inoculated for bacterial and fungal count as the total colony forming units/ml of the sample water being examined.
6.0 TEST FOR PATHOGENS
ESCHERICHIA COLI :
Take 5 ml of water sample in 50ml of nutrient broth, shake well, allow to stand for one hour. Shake again and incubate for 24 hours at 30-35°C.
Primary Test :
Add 1ml of enrichment culture to a tube containing 5 ml of MacConkey broth. Incubate for 48 hours at 30°C. If the contents show acid and gas carry out the secondary test.
Secondary Test :
i) Add 0.1 ml of contents of tube showing acid & gas to each of the 2 tubes containing
a) 5 ml of MacConkey broth and
b) 5 ml of Peptone water.
Incubate for 24 hours at 43.5°C to 44.5°C and examine tube (a) for acid and gas and tube (b) for indole. To test for Indole add 0.5 ml of Kovac’s reagent, shake well and allow to stand for one minute. If red colour is produced in the reagent layer, Indole is present. The presence of Indole and acid & gas production indicates the presence of E.coli.
ii) Portions of the contents of the tube is streaked on a plate of MacConkey agar and incubated for 24 hours at 37°C. Presence of characteristic colonies appearing brick red in colour and having a surrounding zone of precipitated bile indicates the presence of E.coli.
Limit :
E. coli should be absent.
SALMONELLAE :
Take 5ml of the sample in 50ml of nutrient broth and shake well and allow to stand for one hour and incubate for 18-24 hours at 30-35°C.
Primary Test :
Add 1ml of enrichment culture to tetrathionate broth or Selenite broth and incubate at 30-35°C for 48 hours. After incubation inoculate any two plates containing a layer of any two media.
1. Brilliant green agar
2. Bismuth sulphite agar
3. Deoxycholate citrate agar
Incubate the plates at 30-35°C for 24 hours, if any colonies as per Table 2 is obtained carry out the secondary test.
Table 2
For Salmonellae identification
MEDIUM
DESCRIPTION OF COLONY
1.
Brilliant Green agar
Small, transparent & colourless or opaque, pinkish or white (frequently surrounded by a pink or red zone).
2.
Deoxycholate Citrate agar
Colourless and opaque, with or without black centres.
3.
Bismuth Sulphite agar
Black or Green
Secondary Test :
Subculture any colonies showing the characteristic growth in table 2 in triple sugar iron agar by first inoculating the surface of the slope and then making a stab culture with the time same inoculation needle and at the same time inoculate a tube of urea broth. Incubate at 30-35°C for 24 hours. The formation of acid and gas in the stab culture (with or without concomitant blackening) and absence of acidity from the surface of a red colour in urea broth, indicates the presence of salmonellae. If acid but no gas is produced in stab culture, identity of the organisms should be confirmed by agglutination tests.
Limit :
Salmonellae should be absent.
PSEUDOMONAS :
Take 5ml of the sample to 50ml fluid soyabean casein digest medium, Cetrimide Broth & incubate at 32°C for 72 hours. Subculture this on a plate containing Cetrimide Agar and incubate at 32°C for 48 hours. Examine the resulting growth by Gram's staining and apply the Oxidase test : Gram Negative bacilli giving a positive oxidase test indicates the presence of Pseudomonas.
Oxidase Test :
Place 2-3 drops of a freshly prepared 1% w/v solution of `N` -Tetra Methyl -p- Phenylene diammonium dichloride on a piece of filter paper (whatmann no 1) and smear with the suspect colony. If purple colour is produced with in 5 to 10 seconds, the test is positive.
Limit :
Pseudomonas should be absent.
STAPHYLOCOCCUS AUREUS :
Transfer 10 ml of the sample to 100ml soyabean casein digest broth. Incubate at 30-35°C for 72 hour. Portions of the enrichment broth is streaked on Vogel Johnson agar plates. Incubate the plates at 30-35°C for 48 hours. If colonies with black surrounded by yellow zones are found, proceed for a coagulase test using 0.5ml of mammalian plasma and 0.5ml of overnight broth culture of S.aureus, mix, incubate in a 30-35°C water bath, examine every half an hour for coagulation. If no coagulation is observed staphylococcus is absent.
Limit :
Staphylococcus aureus should be absent.