SOP for Media Preparation and Growth Promotion Test

1.0 OBJECTIVE

To lay down a procedure for media preparation and growth promotion test.

2.0 SCOPE

This procedure is applicable for media preparation and growth promotion test for microbial analysis.

3.0 RESPONSIBILITY

Microbiologist - Quality Control

4.0 ACCOUNTABILITY

Manager - Quality Control

5.0 PROCEDURE

5.1 General precautions

5.1.1 Take clean, dried glass conical flask of the desired volume as per Quantity of media.
5.1.2 Use a separate spatula for separate media to avoid cross contamination.
5.1.3 Clean the balance in between to successive weighing.
5.1.4 Use purified water for media preparation.
5.1.5 Media preparation and media discard activities will perform at different location/Time

5.2 Preparation of media

5.2.1 Take clean dried conical flask as per the requirement of media. Transfer the half of the volume of purified water of required quantity in the conical flask. Weight the quantity of the dehydrated media as per volume required, transfer in a conical flask and re-hydrate it as per manufacturer instructions. Make up the volume with remaining quantity of water.
5.2.2 Check the pH of media using calibrated pH meter, adjust the pH using 0.1M HCl/0.1M NaOH if required.
5.2.3 Follow all instructions/ precautions of the manufacturer at the time weighing and re-hydration of media.
5.2.4 Record following details:
Name of media, B.No. Exp. date, Date of preparation, Prepared by, pH before sterilization and after sterilization (Broth medium), the volume of media.etc. as per Annexure-II.
5.2.5 Dispense the medium in an individual container as per requirement plug the containers with a cotton plug or screw cap.
5.2.6 Sterilize the Media for 20 minutes at 121°C & 15 psi or for the validated time period.

5.3 Growth promotion test of sterilized media.

5.3.1 Select the quantified microbial culture as per Annexure –I, for the growth promotion test
5.3.2 Quantified culture should be 24 hours old or a validated period old microbial culture can be used for growth promotion having count 10-100 cfu/ml..
5.3.3 For Growth promotion test of the agar medium, transfer microbial culture to sterile Petri dishes in duplicate aseptically and pour 15 to 20 ml of agar medium (maintain the temperature of the medium before pouring at 40-45°C)
5.3.4 Incubate the Petri plates for 24 - 48 hours for the bacterial count at 30°C-35°C and 5 to 7 days for the fungal count at 20°C-25°C.
5.3.5 After completion of incubation period count the colonies of microorganisms. Record the average result as per Annexure II.
5.3.6 For the growth promotion test of broth medium add the prescribes culture to the tube of respective medium (10cfu –100cfu) and Incubate the tubes for 24-48 hours for the bacterial count at 30°C-35°C and 5 to 7 days for the fungal count at 20°C-25°C.
5.3.7 After completion of incubation period observe the growth of microorganisms as turbidity. Record the observation as per Annexure II.
5.3.8 Recovery of the microbial count of the agar medium should be more than 70% of the count added to the medium.
5.3.9 The medium passes in growth promotion test can be used for analysis of the sample.
5.3.10 Always run negative control of medium to check the sterility of medium.

6.0 ABBREVIATIONS

6.1 SOP - Standard Operating Procedure
6.2 WFI - Water for injection
6.3 LAF - Laminar Air Flow
6.4 HCl - Hydrochloric Acid
6.5 NaOH - Sodium Hydroxide
6.6 Ml - Milliliter
6.7 Cfu - Colony Formation Unit
6.8 °C - Degree Centigrade

ANNEXURE –1

S.No	MEDIA	Micro-organisms used for growth promotion test
1	Soya bean casein digest agar	E.coli	Bacillus subtilis	Staph.aureus	Aspergillus niger	Candida albicans
2	Soya bean casein digest Broth	E.coli	Bacillus subtilis	Staph.aureus	Aspergillus niger	Candida albicans
3	Sabouraud dextrose agar	Aspergillus niger	Candida albicans	NA	NA	NA
4	Potato dextrose agar	Aspergillus niger	Candida albicans	NA	NA	NA
5	Fluid lactose medium	E.coli	NA	NA	NA	NA
6	Eosin methylene blue agar	E.coli	NA	NA	NA	NA
7	Brilliant green agar	Salmonella.sp	NA	NA	NA	NA
8	Bismuth sulfite agar	Salmonella.sp	NA	NA	NA	NA
9	XLDA	Salmonella.sp	NA	NA	NA	NA
10	Triple sugar iron agar	Salmonella.sp	NA	NA	NA	NA
11	Cetrimide agar	P.aeruginosa	NA	NA	NA	NA
12	Pseudomonas isolation agar for florescence	P.aeruginosa	NA	NA	NA	NA
13	Baired parker agar	Staph aureus	NA	NA	NA	NA
14	Mannitol salt agar	Staph aureus	NA	NA	NA	NA
15	Vogal Johnson agar	Staph aureus	NA	NA	NA	NA
16	Fluid thio glycolate medium	Clostridium	NA	NA	NA	NA
17	Mac Conkey¢s agar	E.coli	NA	NA	NA	NA
18	Mac Conkey¢s Broth	E.coli	NA	NA	NA	NA
19	Peptone water	E.coli	NA	NA	NA	NA
20	Pseudomonas isolation agar for pyocyanin	P.aeruginosa	NA	NA	NA	NA

ANNEXURE-II 

MEDIA PREPARATION AND GROWTH PROMOTION RECORD

Media

Batch No.

Exp.Date

Date of preparation

Prepared by

Lot  No. issued

pH Before sterilization

pH after sterilization

Volume of media

Growth promotion test(use the culture 10-100 cfu) E.coli, S. aureus P.aeruginosa, Salmonella, A.niger C.albicans

Observation

Done by

Checked by

Culture added

Cfu added

Cfu recovered

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