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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

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 with
pore size of 0.45 µm (e.g. Sortorius membrane filter SM 11306) with a diameter of 47
mm.

Sampling: Allow the water to pass through the sampling point outlet for 2 - 3 minutes
to rinse the valve & then collect the sample in sterilized flask. If water samples
cannot be tested within two hours of discharge, they must be kept cool by storing in
the 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 expected
Microbial 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 culture
medium. The Agar plates are incubated at 30 ± 2° C. They are evaluated at the
earliest on the fifth day after the start incubation unless a shorter incubation period
permits 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 from
the apparatus and, with the filtration side upper most, place them on MacConkey or
EMB agar in such 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.
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 Salmonella
EMB agar: Dark blue- violet,metallic lustre in case of E. coli are present.
EMB agar : Brown,slimy in case of coliform organisms
EMB 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 from
the apparatus and, with the filtration side upper most, place it on Cetrimide agar in
such 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 green
pigmentation,partially fluorescing) indicates formation of pyocyanin.

Coliform organisms:
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 streak
on 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: Weekly

1
Limits:
Microbial count
Alert level >10 cfu / ml and <100 cfu / ml
Action level >100 cfu / ml
Biological indicators E.coli,Salmonella & coliform bacteria,
Pseudomonas aeruginosa must not be
detected in 100 ml.
Alert level exceeded
Measures 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 the
trend is reconizable.Q.C.Micor. Lab. Documents the final evaluation
and sends a copy of this to Mfg. Plants.

Action level exceeded

Plant inform immediately by note

Test 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 passed

on to Q.C.D. for information
Documentation Q.C. Microbiology Lab. documents the results.

List of Media used.

Bayer Pharmaceuticals Private Limited, Kolshet Road,Thane
Purified Water :Microbiological test procedure , limits and test frequency

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Code Abbreviation Name Composition pH of Sterilization

No. weight in 1 lit ready to
dist. water use
medium

201 CSA or SBA Casein- 15 min at

peptone Soya 40 gm Hi-media 7.3 ± 0.2 121° C
meal Peptone- M 290
agar/
Soyabean
casein digest
agar

231 EMBA Eosin 36 gm Merck 1347 6.9 - 7.3 15 min at

Methylene blue or 121° C
Lactose- 37.5 gm Hi-media
Sucrose agar. M 022

232 MCA MacConkey 50 gm Merck 5465 6.9 - 7.3 15 min at

agar or 121° C
51.5 gm Hi-media
M 081 or M 008

244 CA Cetrimide agar 44.5 gm Merck 7.0 - 7.4 15 min at

5284 + 10 ml 121° C
glycerol
or
45.3 gm Hi-media
MM 024

SMA Skim milk agar 51.5 gm Hi-media 6.8 - 7.2 15 min at

M 763 121° C

HISTORY OF CHANGES
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