BF Biosciences Limited Doc. No.

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

SOP Review Date:

Page:
30-06-2012
1 of 8

PREPARATION OF CULTURE MEDIA

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 preparation of culture media for

microbiological evaluation of raw materials, bulks, purified and drinking
water, intermediates and finished products.

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 : Microbiology Manual

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

SOP Review Date:

Page:
30-06-2012
2 of 8

PREPARATION OF CULTURE MEDIA

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

LIST OF CONTENTS

1. Objective

2. Scope

3. Responsibilities
4. Requirements

5. Procedure for Preparation of Media

5.1. General Requirements
5.2. Media and their Typical Concentration

6. Precautions

7. Training

8. Distribution

9. Related Document

10. History

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

SOP Review Date:

Page:
30-06-2012
3 of 8

PREPARATION OF CULTURE MEDIA

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

1. OBJECTIVE

Objective of this SOP is to define a preparation procedure for cultural media for microbiological
evaluation of raw materials, bulks, purified and drinking water, intermediates and finished
products.

2. SCOPE

This SOP describes the execution of preparation of cultural media in microbiology laboratory.

3. RESPONSIBILITIES

4.1. 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.

4.2. AM Microbiology:

- To follow the instructions laid down by this SOP.

- To keep the relevant SOPs valid and updated.

4.3. Manager Quality Operations:

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

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

SOP Review Date:

Page:
30-06-2012
4 of 8

PREPARATION OF CULTURE MEDIA

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

4. REQUIREMENTS
4.1.
4.2.
4.3.
4.4.
4.5.
4.6.
4.7.
4.8.
4.9.
4.10.
4.11.
4.12.
4.13.

5. MEDIA PREPARATION PROCEDURE

5.1. General Requirements

5.1.1. Before preparation check the expiry date and label the bottle with
name and date of preparation.
5.1.2. Complete instructions for the preparation of culture media are given
on the label of each bottle, according to it,
5.1.3. Open the culture medium bottle away from direct air flow and
moisture. Weigh the desired quantity of required medium quickly,
accurately and without creating 'clouds of dust'.
5.1.4. Reclose the medium bottle as soon as possible.
5.1.5. Always use freshly prepared purified water.

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

SOP Review Date:

Page:
30-06-2012
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PREPARATION OF CULTURE MEDIA

Written By Reviewed By Approved By
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& Date & Date & Date

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

5.1.6. Pour half of the required volume of purified water in a clean bottle
which is twice the final volume of the medium to allow the adequate
mixing.
5.1.7. Then transfer the weighed quantity of medium slowly along with
agitation as the medium can not accumulate to make clump.
5.1.8. Pour the rest of the purified water down the sides of the vessel to
wash any adherent medium back into solution. This is an important
step because dry culture media powder above the level of the water
may not be sterilized in the autoclave and may be a source of
contamination.
5.1.9. Sterilize using a validated process of autoclave (SOP #. ), then
check its pH value, if adjustment required then adjust it by using
NaOH/HCl.

NOTE: The pH of a dehydrated medium should not require adjustment

providing it has been prepared correctly using pure water and clean
equipment, and it has not been over-autoclaved. The
manufacturers instructions must be followed strictly.

5.1.10. After sterilization, pre-incubate sterilized medium at 20 25 C for

3 5 days, if medium shows any sign of turbidity then DO NOT it for
testing purpose.

5.1.11. Broth medium is kept in the same medium bottle whereas agar
medium is poured in pre-sterilized petri dishes when the medium
temperature drops at 45 50 C.

5.1.12. Mix the medium gently by rotating the flask or bottle. Avoid forming
air bubbles. Dispense aseptically about 15 20 mL medium into
petri dishes (90 100 mm diameter) under the laminar airflow
cabinet.

5.1.13. Agar plates should be of an even depth and of a firm gel.

5.1.14. When the medium has gelled and cooled, stack the plates and pre-
incubate them for 3 5 days, if plates show any sign of colony then
DO NOT use it for testing purpose.

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

SOP Review Date:

Page:
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PREPARATION OF CULTURE MEDIA

Written By Reviewed By Approved By
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& Date & Date & Date

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

5.1.15. Record the medium preparation activity in its specified log book.

5.2. Media and their Typical Preparation Concentration

* Required
Required ** Sterilization C, Time
Media Quantity
% & pH & Pressure
per 100 mL

Tryptic Soy Agar 4.0%, 7.3 4g 121 C, 15 minutes, 15 psi

Tryptic Soy Broth 3.0%, 7.3 3g 121 C, 15 minutes, 15 psi

Sabouraud Dextrose Agar 6.5%, 5.6 6.5 g 121 C, 15 minutes, 15 psi

Sabouraud Dextrose Broth 3.0%, 5.6 3g 121 C, 15 minutes, 15 psi

Enterobacteria Enrichment Mossel Broth 4.5%, 7.2 4.5 g 121 C, 5 minutes, 15 psi

Mannitol Salt Agar 10.8%, 7.4 10.8 g 121 C, 15 minutes, 15 psi

Cetrimide Agar Selective & Pseudomonas

4.53%, 7.2 4.53 g 121 C, 15 minutes, 15 psi
Cetrimide agar

Reinforced Medium for Clostridia 3.8%, 6.8 3.8 g 121 C, 15 minutes, 15 psi

MacConkeys Broth 3.5%, 7.3 3.5 g 121 C, 15 minutes, 15 psi

MacConkeys Agar 5.0%, 7.1 5g 121 C, 15 minutes, 15 psi

Rappaport Vassiliadis Salmonella

4.18%, 5.2 4.18 g 115 C, 15 minutes, 15 psi
Enrichment Broth
Heat in Water bath, boil for 2
Violet Red Bile Glucose Agar 3.95%, 7.4 3.95 g
minutes, dont autoclave
Dont autoclave, sterile on
Xylose Lysine Deoxycholate Agar 4.0%, 7.1 4g
boiling

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

SOP Review Date:

Page:
30-06-2012
7 of 8

PREPARATION OF CULTURE MEDIA

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

Columbia Agar 4.2%, 7.3 4.2 g 121 C, 15 minutes, 15 psi

Buffered Peptone Water 2.55%, 7.0 2.55 g 121 C, 15 minutes, 15 psi

Antibiotic medium 11 3.05%, 8.3 3.05 g 121 C, 15 minutes, 15 psi

* Weigh medium as your volume required. ** Sterilization time can increase up to 20 min.
Avoid inhaling the powder and prolonged skin contact.

6. PRECAUTIONS
6.1. During weighing, care must be taken and weigh accurately according to
manufacturers specifications.

6.2. Before weighing, properly label the bottles with name, %age and date.

6.3. Weigh the desired quantity of required medium quickly, accurately and
without creating 'clouds of dust'.

6.4. After weighing, reclose bottle as soon as possible because dehydrated

medium is hygroscopic.

6.5. Always wear mask and gloves for avoiding inhalation and skin contact.

6.6. After sterilization, Do Not make heavy agitation for mixing as air bubbles not
form, mix in gentle clockwise motion especially agar medium.

6.7. If shrinkage OR dryness of agar surface found, then it should be discarded

without its usage for test.

6.8. Contaminated medium either broth OR agar medium should be disposed-off

according to SOP # .

6.9. Do Not use agar plates that contain heavy moisture.

7. TRAINING

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

SOP Review Date:

Page:
30-06-2012
8 of 8

PREPARATION OF CULTURE MEDIA

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

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.

8. 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

9. RELATED DOCUMENT

NA

10. HISTORY

This is the First version.

- First Issue : JUN,2009

- Reason of composition / review new SOP.

Microbiology
BF/SOP/Mic