Beruflich Dokumente
Kultur Dokumente
: Mic-SOP-009-00
Effective Date: 30-06-2009
Name Muhammad Adnan Khan Name Asadullah Khan Name Ejaz Ahmad
Title Microbiologist Title Assistant Manager Microbiology Title Manager Quality Operations
COVER SHEET
Total pages : 08
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
Name Muhammad Adnan Khan Name Asadullah Khan Name Ejaz Ahmad
Title Microbiologist Title Assistant Manager Microbiology Title Manager Quality Operations
LIST OF CONTENTS
1. Objective
2. Scope
3. Responsibilities
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
8. Testing Procedure
9. Training
10. Distribution
12. History
Microbiology
BF/SOP/Mic
BF Biosciences Limited Doc. No. : Mic-SOP-009-00
Effective Date: 30-06-2009
Name Muhammad Adnan Khan Name Asadullah Khan Name Ejaz Ahmad
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 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:
Microbiology
BF/SOP/Mic
BF Biosciences Limited Doc. No. : Mic-SOP-009-00
Effective Date: 30-06-2009
Name Muhammad Adnan Khan Name Asadullah Khan Name Ejaz Ahmad
Title Microbiologist Title Assistant Manager Microbiology Title Manager Quality Operations
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 manufacturers 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
Microbiology
BF/SOP/Mic
BF Biosciences Limited Doc. No. : Mic-SOP-009-00
Effective Date: 30-06-2009
Name Muhammad Adnan Khan Name Asadullah Khan Name Ejaz Ahmad
Title Microbiologist Title Assistant Manager Microbiology Title Manager Quality Operations
Microbiology
BF/SOP/Mic
BF Biosciences Limited Doc. No. : Mic-SOP-009-00
Effective Date: 30-06-2009
Name Muhammad Adnan Khan Name Asadullah Khan Name Ejaz Ahmad
Title Microbiologist Title Assistant Manager Microbiology Title Manager Quality Operations
Microbiology
BF/SOP/Mic
BF Biosciences Limited Doc. No. : Mic-SOP-009-00
Effective Date: 30-06-2009
Name Muhammad Adnan Khan Name Asadullah Khan Name Ejaz Ahmad
Title Microbiologist Title Assistant Manager Microbiology Title Manager Quality Operations
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 C 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
Microbiology
BF/SOP/Mic
BF Biosciences Limited Doc. No. : Mic-SOP-009-00
Effective Date: 30-06-2009
Name Muhammad Adnan Khan Name Asadullah Khan Name Ejaz Ahmad
Title Microbiologist Title Assistant Manager Microbiology Title Manager Quality Operations
Microbiology
BF/SOP/Mic
BF Biosciences Limited Doc. No. : Mic-SOP-009-00
Effective Date: 30-06-2009
Name Muhammad Adnan Khan Name Asadullah Khan Name Ejaz Ahmad
Title Microbiologist Title Assistant Manager Microbiology Title Manager Quality Operations
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.
Microbiology
BF/SOP/Mic
BF Biosciences Limited Doc. No. : Mic-SOP-009-00
Effective Date: 30-06-2009
Name Muhammad Adnan Khan Name Asadullah Khan Name Ejaz Ahmad
Title Microbiologist Title Assistant Manager Microbiology Title Manager Quality Operations
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.
Name Muhammad Adnan Khan Name Asadullah Khan Name Ejaz Ahmad
Title Microbiologist Title Assistant Manager Microbiology Title Manager Quality Operations
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+
Microbiology
BF/SOP/Mic
BF Biosciences Limited Doc. No. : Mic-SOP-009-00
Effective Date: 30-06-2009
Name Muhammad Adnan Khan Name Asadullah Khan Name Ejaz Ahmad
Title Microbiologist Title Assistant Manager Microbiology Title Manager Quality Operations
Minimum Volume 1 mL
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
Pseudomonas
Must be absent
aeruginosa
Name Muhammad Adnan Khan Name Asadullah Khan Name Ejaz Ahmad
Title Microbiologist Title Assistant Manager Microbiology Title Manager Quality Operations
Volume 100 mL
Incubation
30 35 C but 42 44 C for MPN Test detection of E. coli
Temperature
Name Muhammad Adnan Khan Name Asadullah Khan Name Ejaz Ahmad
Title Microbiologist Title Assistant Manager Microbiology Title Manager Quality Operations
8. TESTING PROCEDURE
8.1. Appearance
Clear, colorless & odorless liquid.
8.2. TESTS:
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:
Microbiology
BF/SOP/Mic
BF Biosciences Limited Doc. No. : Mic-SOP-009-00
Effective Date: 30-06-2009
Name Muhammad Adnan Khan Name Asadullah Khan Name Ejaz Ahmad
Title Microbiologist Title Assistant Manager Microbiology Title Manager Quality Operations
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.
To 25mL add 25mL of calcium Hydroxide Ts, the mixture remains clear.
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 50 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.
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.
Microbiology
BF/SOP/Mic
BF Biosciences Limited Doc. No. : Mic-SOP-009-00
Effective Date: 30-06-2009
Name Muhammad Adnan Khan Name Asadullah Khan Name Ejaz Ahmad
Title Microbiologist Title Assistant Manager Microbiology Title Manager Quality Operations
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.
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
NA
Microbiology
BF/SOP/Mic
BF Biosciences Limited Doc. No. : Mic-SOP-009-00
Effective Date: 30-06-2009
Name Muhammad Adnan Khan Name Asadullah Khan Name Ejaz Ahmad
Title Microbiologist Title Assistant Manager Microbiology Title Manager Quality Operations
12. HISTORY
Microbiology
BF/SOP/Mic