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SOP for Media Preparation and Growth Promotion Test


Standard operating procedure of media preparation for microbial analysis and growth promotion test to verify the fertility of the culture media.

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 desired volume as per Quantity of media. 
5.1.2  Use 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 conical flask and re-hydrate it as per manufacturer instructions. Makeup 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 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) ,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 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 use 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 petridishes in duplicate aseptically and pour 15 to 20 ml of agar medium (maintain the temperature of medium before pouring at 40-45°C) 
5.3.4  Incubate the petriplates for 24 - 48 hours for bacterial count at 30°C-35°C and 5 to 7 days for fungal count at 20°C-25°C. 
5.3.5  After completion of incubation period count the colonies of micro-organisms. Record the average result as per annexure II. 
5.3.6  For the growth promotion test of broth medium add the prescribes culture to tube of respective medium (10cfu –100cfu) and Incubate the tubes for 24-48 hours for bacterial count at 30°C-35°C and 5 to 7 days for fungal count at 20°C-25°C. 
5.3.7  After completion of incubation period observe the growth of micro-organisms as turbidity. Record the observation as per annexure II. 
5.3.8  Recovery of 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 use for analysis of 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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