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BF Biosciences Limited Doc. No.

: Mic-SOP-009-00
Effective Date: 30-06-2009

SOP Review Date:
Page:
30-06-2012
1 of 17

PURIFIED & DRINKING WATER TESTING
Written By Reviewed By Approved By
Signature Signature Signature
& Date & Date & Date

Name Muhammad Adnan Khan Name Asadullah Khan Name Ejaz Ahmad
Title Microbiologist Title Assistant Manager Microbiology Title Manager Quality Operations

COVER SHEET

Issue Issue Date Originator Amendments/Justification

00 27-06-2009 Muhammad Adnan Khan New Procedure
_______________________

Valid from : 30-06-2009

Purpose : To establish a procedure for testing of water (purified water as well as
drinking water) samples chemically and microbiologically.

Responsible for Implementation : Manager Quality Operations
Manager Quality Control/Microbiology
A.M. Microbiology
Microbiologist

Total pages : 08

Issued to : 1. Manager Quality Operations
2. Quality Control Manager/Microbiology
3. AM Microbiology
4. Quality Assurance Manager

Reference : WHO
USP
BP/EP
District Laboratory Manual, Vol. 2, Monica Cheesborough

Microbiology
BF/SOP/Mic
BF Biosciences Limited Doc. No. : Mic-SOP-009-00
Effective Date: 30-06-2009

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Title Microbiologist Title Assistant Manager Microbiology Title Manager Quality Operations

LIST OF CONTENTS

1. Objective

2. Scope

3. Responsibilities

4. Requirements for Microbiological Analysis

5. Testing Procedure
5.1. Pour Plate Technique
5.2. Spread Plate Technique
5.3. Membrane Filtration Technique
5.4. Most Probable Number (MPN) Technique
5.5. Determination of Objectionable Micro-organisms by
Filtration
5.6. Determination of Objectionable Micro-organisms by MPN

6. Alert & Action Limits and Testing Schedule of Water

7. Requirements of Chemical Analysis

8. Testing Procedure

9. Training

10. Distribution

11. Related Document

12. History

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Title Microbiologist Title Assistant Manager Microbiology Title Manager Quality Operations

1. OBJECTIVE

Objective of this SOP is to define a testing procedure for water (purified water as well as drinking
water) for microbiological and chemical evaluation.

2. SCOPE

This SOP describes the execution of routine microbiological and chemical analysis of water
(purified water as well as drinking water) in microbiology laboratory and it is applicable to all water
points of water system.

This SOP also describes the policy and procedure and components that should be followed to
achieve the water quality requirements and these are following:
 Locations to be monitored
 Frequency of monitoring
 Type of monitoring
 Procedure of monitoring
 Alert and Action levels
 Procedures followed then these levels are exceeded

3. RESPONSIBILITIES

3.1. Microbiologist:

- To interface this SOP to the actual practices of flow patterns.

- To ensure that the SOP is updated & reflects the actual execution.

- To review the SOP in case of any change or whenever it is due for revision.

3.2. AM Microbiology:

- To follow the instructions laid down by this SOP.

- To keep the relevant SOPs valid and updated.

3.3. Manager Quality Operations:

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- To provide adequate resources for the implementation of this SOP as define.

4. REQUIREMENTS FOR MICROBIOLOGICAL ANALYSIS
4.1. Tryptic Soy Agar
4.2. Sterile filtration assembly
4.3. Sterile latex gloves
4.4. Filtered 70% Isopropyl Alcohol spray bottle
4.5. Sterile lint free duster
4.6. Violet Red Bile Agar
4.7. Cetrimide Agar
4.8. Peptone Water
4.9. Sterile petri dishes/plates (9 cm in diameter)
4.10. Incubator
4.11. Autoclave
4.12. Sterile forceps
4.13. Sterile membrane filters having pore size 0.45 µm
4.14. Micropipette with sterile micro tips having 1 mL capacity/1 mL sterile pipette
4.15. Vacuum pump
4.16. Sterile screw-capped glass bottles 250 mL
4.17. “L” shaped sterile glass rod

5. TESTING PROCEDURE
Take sterile screw-capped bottle from dry heat sterilizer for taking water samples from different
designated locations as per SOP #.
Prepare Tryptic Soy Agar medium as per manufacturer’s specifications SOP #, and then pre-
incubate medium containing plates at 30 – 35° C for their sterility check.
There are 4 different testing methods which are followed to evaluate the water samples.
a) Pour Plate Technique
b) Spread Plate Technique
c) Membrane Filtration Technique
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d) Most Probable Number Technique

5.1. Pour Plate Technique
5.1.1. Transfer 1 mL of sample into two petri dishes having 9 cm diameter labelled with
sample name, location and date, by micro pipette OR 1 mL sterile pipette.
5.1.2. Cool the medium approximately 45°C (feel on the dorsal side of the hand, it should
be bearable).
5.1.3. Then approximately pour 15-20 ml of Tryptic Soy Agar into each petri dish.
5.1.4. Cover the petri dishes, mix the sample with the agar by rotating the dishes in
direction of clock wise then anti-clock wise and allow the contents to solidify at room
temperature.
5.1.5. Incubate both petri dishes at 30-35°C for 48 – 72 1 hours in inverted position.
5.1.6. After incubation, examine the plates for growth, count the number of colonies and
express the average for the two plates in terms of the number of colony forming
units per mL (CFU/mL).
1
Incubation at lower temperatures for longer period of time like 20 – 25 °C for 5 – 7 days.

5.2. Spread Plate Technique
5.2.1. Transfer 0.1 – 0.5 mL of sample onto surface of agar of two petri dishes having 9 cm
diameter labelled with sample name, location and date, by micro pipette OR 1 mL
graduated sterile pipette.
5.2.2. Spread the sample over the agar surface by “L” shaped sterile glass rod completely.
5.2.3. Cover the petri dishes containing sample and let these plates to dry at room
temperature.
5.2.4. Incubate both petri dishes at 30-35°C for 48 – 72 hours in inverted position.
5.2.5. After incubation, examine the plates for growth, count the number of colonies and
express the average for the two plates in terms of the number of colony forming
units per mL (CFU/mL).

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5.3. Membrane Filtration Technique
5.3.1. Take sterile filtration assembly to Laminar Airflow Cabinet (LFC), assemble it
aseptically, and don’t make any contact with sterile surface.
5.3.2. Use membrane filters having a nominal pore size not greater than 0.45 µm.
5.3.3. Aseptically filter 100 mL of diluted water sample (1:10) through membrane filter.
5.3.4. After completion of filtration, take the filter using sterile forceps and transfer it
immediately to the previously prepared petri dish with Tryptic Soy Agar.
5.3.5. Place the membrane carefully so that the air should not be trapped under the filter,
as this will prevent nutritions from medium to entire membrane surface.
5.3.6. Cover the petri dishes containing membrane filters; incubate both petri dishes at 30-
35°C for 48 – 72 hours.
5.3.7. After incubation, examine the plates for growth, count the number of colonies and
express the average for the two plates in terms of the number of colony forming
units per mL (CFU/mL).

5.4. Most Probable Number (MPN) Technique
Prepare Tryptic Soy Broth medium as per manufacturer’s specifications SOP #, and then
pre-incubate medium containing tubes at 30 – 35° C for their sterility.
5.4.1. For this test we require 14 sterile test tubes of similar size. Into each tube place 9 ml
of sterile Tryptic Soy Broth.
5.4.2. Arrange twelve of the tubes in four sets of three tubes each and label 01st set as
(“100”), 02nd as (“10”), 03rd as (“1”), 04th as (“controls”) and remaining two tubes as
(“A”) & (“B”).
5.4.3. Into each of three tubes of one set (“100”) and into fourth tube (“A”) pipette 1 mL of
the water sample, and mix.
5.4.4. From tube (“A”), pipette 1 mL of its contents into the one remaining tube (“B”), and
mix.
5.4.5. These tubes like (“A”) & (“B”) contain 100 µL) and 10 µL) of the specimen,
respectively.

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5.4.6. Into each of the 02nd set (“10”) of three tubes pipette 1 mL from tube (“A”), and into
each tube of 03rd set (“1”) pipette 1 mL from tube (“B”), and mix. Discard both tubes
(“A”) & (“B”).
5.4.7. Close well, and incubate all of the tubes for 48 hours at 35 ºC ± 2.
5.4.8. Following the incubation period, examine the tubes for growth: the three control
tubes remain clear and the observations in the tubes containing the specimen.
5.4.9. For interpretation, a reference table 1 has given below. When interpret the result,
indicate the most probable number of microorganisms per mL of the specimen.
Table 1. Most Probable Number Count by Multiple-Tube Method

Observed Combinations of Numbers of Tubes Most Probable
Showing Growth in Each Set Number
No. of mg (or µL) of Specimen per Tube Number of
100 10 1 Microorganisms
(100 µL) (10 µL) (1 µL) per g / ml
3 3 3 > 1100
3 3 2 1100
3 3 1 500
3 3 0 200
3 2 3 290
3 2 2 210
3 2 1 150
3 2 0 90
3 1 3 160
3 1 2 120
3 1 1 70
3 1 0 40
3 0 3 95
3 0 2 60
3 0 1 40
3 0 0 23

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5.5. Determination of Objectionable Micro-organisms by Membrane
Filtration Technique
Prepare Violet Red Bile Agar (VRBA) and Cetrimide Agar (CA) media as per
manufacturer’s specifications SOP #, and then pre-incubate medium containing petri dishes
at 30 – 35° C for their sterility.
5.5.1. Take sterile filtration assembly to Laminar Airflow Cabinet (LFC), assemble it
aseptically, and don’t make any contact with sterile surface.
5.5.2. Use membrane filters having a nominal pore size not greater than 0.45 µm.
5.5.3. Aseptically filter two cycles of 100 mL of water sample through membrane filter.
5.5.4. After completion of filtration, take the filter using sterile forceps and transfer it
immediately to the previously prepared petri dish with VRBA.
5.5.5. Again after completion of second cycle of filtration, take the filter using sterile forceps
and transfer it immediately to the previously prepared petri dish with CA this time.
5.5.6. Place the both membranes carefully onto the agar surface of separate petri dishes
so that the air should not be trapped under the filter, as this will prevent nutritions
from medium to entire membrane surface.
5.5.7. Cover the petri dishes containing membrane filters; incubate both petri dishes at
30-35°C for 48 – 72 hours.
5.5.8. After incubation, examine the plates of VRBA medium for growth, if pink colored
colonies developed those indicate the presence of lactose fermenting micro-
organisms i.e. coliforms (E. coli,) and if brown colored developed those indicate the
presence of Pseudomonas, Salmonella, etc.
5.5.9. As above step, examine the plates of CA medium for growth, if yellow-green to blue-
green OR yellow-green to green colored colonies developed those indicate the
presence of Pseudomonas species.

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5.6. Determination of Objectionable Micro-organisms by Membrane
Filtration Technique
5.6.1. Depending on the type of water sample (treated or untreated), the
following are required:
Number of bottles mL of broth Strength
of broth*

Treated water samples 1 of 50 mL Double
5 of 10 mL Double

Untreated water samples 1 of 50 mL Double
5 of 10 mL Double
5 of 5 mL Single
5.6.2. MacKonkey’s broth, sterile test tubes containing 9 mL broth, and each test tube must
contain a sterile inverted Durham tube for collection of gas.

5.6.3. Label each test tube with the sample code number.
5.6.4. Mix thoroughly the sample of water by inverting the bottle several
times.
5.6.5. Remove the bottle cap and cover, and inoculate the bottles of sterile
broth as follows:
– Add 50 mL of water to the bottle containing 50 mL of broth. This is
most easily done by pouring direct into the bottle of broth up to a
100 mL graduation mark scratched previously on the bottle.
– Using a sterile pipette, add 10 mL of water to each of the five bottles
containing 10 mL of broth.
– If required (for untreated samples), pipette 1 mL of water into each
of five bottles containing 5 mL of broth.
Note: The total volume of water inoculated is 100 mL for treated
samples, and 105 mL for untreated poor quality samples.
5.6.6. Incubate the inoculated broths in a water bath or incubator at 44° C for
24 hours with the loose caps.

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5.6.7. After incubation, examine and count each bottle which has produced
both acid and gas.
Note: Acid production will be shown by a change in colour of the
MacKonkey broth from purple to yellow and gas production by the
collection of a bubble in the Durham tube.
5.6.8. Refer to the probability tables shown below to determine the most
probable number (MPN) of faecal coliform bacteria in the 100 mL or 105
mL water sample.

PROBABILITY TABLES FOR ESTIMATING THE MPN
OF FAECAL COLIFORM BACTERIA
For Treated Water Samples
Volume of sample 50 10
in each bottle: mL mL
Number of bottles 1 5
used:
MPN/100mL
0 0 0
0 1 1
0 2 2
0 3 4
Number of tubes 0 4 5
giving positive 0 5 7
reaction 1 0 2
1 1 3
1 2 6
1 3 9
1 4 16
1 5 18+

For Untreated Water Samples
Volume of sample 50 10 1
in each bottle: mL mL mL
Number of
bottles used: 1 5 5
MPN/100mL
0 0 0 0
0 0 1 1
0 0 2 2
0 1 0 1
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0 1 1 2
0 1 2 3
Number of tubes 0 2 0 2
giving positive 0 2 1 3
reaction 0 2 2 4
0 3 0 3
0 3 1 5
0 4 0 5
1 0 0 1
1 0 1 3
1 0 2 4
1 0 3 6
1 1 0 3
1 1 1 5
1 1 2 7
1 1 3 9
1 2 0 5
1 2 1 7
1 2 2 10
1 2 3 12
1 3 0 8
Number of tubes 1 3 1 11
giving positive 1 3 2 14
results 1 3 3
18
1 3 4 20
1 4 0 13
1 4 1 17
1 4 2 20
1 4 3 30
1 4 4 35
1 4 5 40
1 5 0 25
1 5 1 35
1 5 2 50
1 5 3 90
1 5 4 160
1 5 5 180+

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6. ALERT & ACTION LIMITS TAMC AND TESTING SCHEDULE OF WATER

Potable Drinking Purified
Raw Water Soft Water
Testing Water Water Water
Parameters
Total Aerobic Microbial Count

Minimum Volume 1 mL

Method Pour Plate/Filtration

Medium (Media) Tryptic Soy Agar

Incubation Temp.
30 – 35° C for 48 – 72 hours
& Time

Alert Limits 300 CFU 200 CFU 150 CFU 100 CFU 50 CFU

Action Limits 500 CFU 500 CFU 500 CFU 500 CFU 100 CFU

Specified Pathogenic Microorganisms

Escherichia coli Must be Absent

Medium (Media) MacKonkey Broth / MacKonkey Agar / Eosine Methylene Blue

Pseudomonas
Must be absent
aeruginosa

Medium Cetrimide Agar
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Salmonella sp. Must be absent

Medium Xylose-Lysine Deoxycholate Agar / Brilliant Green Agar

Volume 100 mL

Method(s) Membrane Filtration / MPN for E. coli

Incubation
30 – 35° C but 42 – 44° C for MPN Test detection of E. coli
Temperature

Incubation Time 24 – 48 hours

7. REQUIREMENTS FOR CHEMICAL ANALYSIS
7.1. Clean and dry 500 mL – 1 L screw-capped bottles
7.2. Alkaline Potassium Tetra-iodomercurate
7.3. Ammonia Standard Solution
7.4. Methyl Red Solution
7.5. Bromothymol Blue Solution
7.6. Diluted Nitric Acid Solution
7.7. Silver Nitrate Solution
7.8. Ammonium Chloride Buffer Solution pH 10.0 R
7.9. Mordant Black 11 Triturate R
7.10. 0.1M Sodium Edetate Solution
7.11. Calcium Hydroxide Solution
7.12. Potassium Chloride Solution
7.13. Diphenylamine Solution
7.14. Nitrate standard Solution
7.15. Dilute Sulphuric Acid R
7.16. 0.02M Potassium Permanganate
7.17. Dilute Hydrochloric Acid R
7.18. Barium Chloride Solution
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7.19. Saturated Potassium Chloride Solution
7.20. Test Tubes with stand
7.21. pH Meter
7.22. Water Bath
7.23. Dry Heat Oven
7.24. Analytical Balance
7.25. Total Organic Carbon (TOC) Analyzer
7.26. Conducto-meter
7.27. 1 – 10 mL Graduated Clean and dry Pipette with filler

8. TESTING PROCEDURE
8.1. Appearance
Clear, colorless & odorless liquid.

8.2. TESTS:

8.2.1. Total Organic Carbon (TOC): 500 ppm.
Analyze sample for TOC with calibrated TOC analyzer as per TOC SOP #.
8.2.2 Ammonium: Maximum 0.2 ppm.
To 20mL add 1mL of alkaline potassium tetra-iodomercurate solution R. After 5
min, examine the solution down the vertical axis of the tube. The solution is not
more intensely colored than a standard prepared at the same time by adding
1mL of alkaline potassium tetra-iodomercurate solution R to a mixture of 4mL of
ammonium standard solution (1ppm NH4) R and 16mL of ammonia-free water
R.

8.2.3. Acidity or Alkalinity:

To 10mL freshly boiled & cooled in a borosilicate glass flask, add 0.05mL of
methyl red solution, the resulting is not red. To 10mL add 0.1mL of bromothymol
blue solution, the resulting solution is not blue.

8.2.4. Chloride:

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To 10mL add 1mL of dilute nitric acid R and 0.2 mL of silver nitrate R2. The
solution shows no change in appearance for at least 15 min.

8.2.5. Calcium & Magnesium / Hardness:

To 100mL add 2mL of ammonium chloride buffer solution pH 10.0 R, 50mg of
mordant black 11 triturate R and 0.5mL of 0.1M sodium edetate. A pure blue color
is produced.
8.2.6. Carbon Dioxide:

To 25mL add 25mL of calcium Hydroxide Ts, the mixture remains clear.

8.2.7. Nitrate: Maximum 0.2 ppm.

To 5mL in a test tube immersed in ice water, add 0.4mL of a 100g/L solution of
potassium chloride 0.1mL of diphenylamine solution & dropwise with shaking,
5ml of sulfuric acid. Transfer the tube to a water bath at 50º C and allow to stand
for 15 minutes. Any blues color produce in the solution is not more intense than
that in a reference solution prepared at the same time in the same manner using
a mixture of 4.5mL of water and 0.5mL of nitrate standard solution (2ppm NO3)
R.

8.2.8. Oxidizable Substances:

Boil 100mL with 10mL add dilute sulphuric acid R. and 0.1mL of 0.02M
potassium permanganate and boil for 5min. The solution remains faintly pink.

8.2.9. Sulphates:

To 10mL add 0.1mL of dilute hydrochloric acid R and 0.1mL of barium chloride
solution R1. The solution shows no change in appearance for at least 1 hour.

8.2.10. Residue on Evaporation: Maximum 0.001%.
Evaporate 100mL on a water bath and dry in an oven at 100 – 105 ºC. The
residue weighs a maximum of 1mg.
Residue on Evaporation: W2 – W1 X 100 (mg/L)
mL of Sol. Taken
W1 = Weight of dish
W2 = Weight of dish + residue
8.2.11. Conductivity: 1.3 µS/cm at 25 °C.

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Take sample in a suitable container, and stir the test sample for maintaining the
temperature at 25 °C ± 1 °C, then measure the conductivity with calibrated
conducto-meter.

8.2.12. pH: Between 5.0 – 7.0.

To 100mL sample add 0.3ml of saturated potassium chloride solution, mix the
solution well and then measure the pH with calibrated pH meter.
Note: If results are out of specified limit in chemical analysis of water, then follow the
SOP.
9. TRAINING

Training about the contents of this SOP must be imparted to all the concerned personnel once in
a year and also to new employees before starting with the job and the records documented
according to training SOP.

10.. DISTRIBUTION
Issuance of New Copy Retrieval of Old version

Department /
Signature Copy # Retrieved from Retrieved by
Person

Original Copy

Copy # 1

Copy # 2

Copy # 3

Copy # 4

11. RELATED DOCUMENT

 NA

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12. HISTORY

This is the First version.

- First Issue : JUN,2009
- Reason of composition / review new SOP.

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