To lay down a procedure for media preparation and growth promotion test.
This procedure is applicable for media preparation and growth promotion test for microbial analysis.
Microbiologist - Quality Control
Manager - Quality Control
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.
6.1 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
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