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Pharmaceuticals

Bayer Pharmaceuticals Pvt. Ltd., Kolshet Road, Thane

400607.Purified Water :Microbiological test procedure , limits and test frequency

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Valid Since: 28.09.2006

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Bayer Pharmaceuticals Private Limited, Kolshet Road,Thane

Purified Water :Microbiological test procedure , limits and test frequency

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Method

The total microbial is determined by the membrane filtration method using filters withpore size of 0.45 m (e.g. Sortorius membrane filter SM 11306) with a diameter of 47mm.

Sampling: Allow the water to pass through the sampling point outlet for 2 - 3 minutesto rinse the valve & then collect the sample in sterilized flask. If water samplescannot be tested within two hours of discharge, they must be kept cool by storing inthe refrigerator. The samples must be tested on the day of discharge.

Quantity of sample required: Not less than 300 ml per sampling point.

1. Total Aerobic Microbial count:The total microbial count is determined by the membrane filter method.

Microbial count of Purified water:The amount of water to be filtered depends on the expectedMicrobial count. For high Microbial counts dilution series should be prepared.Filter each sample at least twice.E.g. Purified water generally 2 X 1 ml or if required dilution series

When filtration is completed remove the membrane filters from the apparatus and,with the filtration side upper most, place them on the Agar culture medium(CSA/SBA) in such a way that no air bubbles form between the filter and culturemedium. The Agar plates are incubated at 30 2 C. They are evaluated at theearliest on the fifth day after the start incubation unless a shorter incubation periodpermits a more reliable result.

2. Test for E. coli,Salmonella and Coliform Organisms: (Purified water only)Agar (Alternative method): Filter 100 ml water, remove the membrane filters fromthe apparatus and, with the filtration side upper most, place them on MacConkey orEMB agar in such a way that no air bubbles form between the filter and the culturemedium. Incubate at 36 1 C for not less than 24 4 hours. If the result isnegative, increase incubation to not less than 40 hours.MacConkey agar : Red or Pink colour clonies in case of Coliform organism:

MacConkey agar: Large red or pink ,not slimy colonies in case of E.coli.MacConkey agar : Colourless,Translucent in case of SalmonellaEMB agar: Dark blue- violet,metallic lustre in case of E. coli are present.EMB agar : Brown,slimy in case of coliform organismsEMB agar: Translucent, amber coloured in case of Salmonella present

3. Test for the absence of Pseudomonas aeruginosa:(Purified water only.)Agar (Alternative method): Filter 100 ml water, remove the membrane filters fromthe apparatus and, with the filtration side upper most, place it on Cetrimide agar insuch a way that no air bubbles form between the filter and the culture medium.Incubate at 36 1 C for not less than 24 4 hours. If the result is negative,

increase incubation to not less than 40 hours. A positive result (yellowish greenpigmentation,partially fluorescing) indicates formation of pyocyanin.

Coliform organisms:

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Bayer Pharmaceuticals Private Limited, Kolshet Road,Thane

Purified Water :Microbiological test procedure , limits and test frequency

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1. If gram negative and oxidase-negative rods are present, prepare a dilution streakon MacConkey agar. After isolating individual colonies, differentiate further, e.g.with Indole and MR.VP test.

2. Pseudomonas aeruginosa:If gram negative and oxidase-positive rods are present, perform a further

test, e.g. with proteolytic activity on skim milk agar.

Sampling points : As per ACME Plan

frequency of testing: Weekly1

Limits:Microbial countAlert level >10 cfu / ml and 100 cfu / mlBiological indicators E.coli,Salmonella & coliform bacteria,

Pseudomonas aeruginosa mustnot bedetected in 100 ml.

Alert level exceededMeasures to be taken if limits are exceeded .Plant informed immediately by the note.

Test repeated immediately.

Evaluation of microbiological spectrum in terms of cause of

contamination.

If repeated test confirms that alert level has been exceeded and thetrend is reconizable.Q.C.Micor. Lab. Documents the final evaluationand sends a copy of this to Mfg. Plants.

Action level exceeded

Plant inform immediately by noteTest repeated immediately on three successive working days.Evaluation of microbiological spectrum in terms of cause of contamination.

Measures which has taken by manufacturing are documented and passedon to Q.C.D. for informationDocumentation Q.C. Microbiology Lab. documents the results.

List of Media used.

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Bayer Pharmaceuticals Private Limited, Kolshet Road,Thane

Purified Water :Microbiological test procedure , limits and test frequency

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CodeNo.

Abbreviation Name Compositionweight in 1 litdist. water

pH ofready tousemedium

Sterilization

201 CSA or SBA Casein-peptone Soyameal Peptone-agar/Soyabeancasein digestagar

40 gm Hi-mediaM 290

7.3 0.2

15 min at121 C

231 EMBA EosinMethylene blueLactose-Sucrose agar.

36 gm Merck 1347or37.5 gm Hi-mediaM 022

6.9 - 7.3 15 min at121 C

232 MCA MacConkeyagar

50 gm Merck 5465or51.5 gm Hi-mediaM 081 or M 008

6.9 - 7.3 15 min at121 C

244 CA Cetrimide agar 44.5 gm Merck5284 + 10 mlglycerolor45.3 gm Hi-mediaMM 024

7.0 - 7.4 15 min at121 C

SMA Skim milk agar 51.5 gm Hi-mediaM 763

6.8 - 7.2 15 min at121 C

HISTORY OF CHANGES

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Bayer Pharmaceuticals Private Limited, Kolshet Road,Thane

Purified Water :Microbiological test procedure , limits and test frequency

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Issue number Reason for Change Valid from

01 New inclusion 28.09.2006