SOP for Preparation of Microbial Culture Media

1.0 OBJECTIVE

To lay down a procedure for preparation of microbiological culture media and plates & slants.

2.0 SCOPE

This SOP is applicable to the Quality Control Dept.

3.0 RESPONSIBILITY

Microbiologist

4.0 ACCOUNTABILITY

Head Quality Control Department.

5.0 PROCEDURE

5.1 Requirements

5.1.1 Balance
5.1.2 Dehydrated media
5.1.3 Autoclave
5.1.4 Laminar air flow
5.1.5 Glassware

5.2 Preparation of Culture Media (using dehydrated media)

5.2.1 Store the dehydrated media in tightly closed packs in dark or as directed by the manufacturer.
5.2.2 Weigh a required amount of dehydrated media and add it to the flask containing purified water.
5.2.3 Make-up the required volume with purified water.
5.2.4 Check the pH of the media before sterilization and after sterilization and if required adjust pH as mentioned on respective media container with HCl or NaOH solution as mentioned in the respective media container.
5.2.5 Distribute the media in the flasks or tubes as required.
5.2.6 Cover the flasks and tubes with autoclavable plastic caps/ cotton plugs and wrap with butter paper/ aluminum foil. Load the materials as per the procedure mentioned in SOP for operation of autoclave.
5.2.7 Sterilize the media by autoclaving at 121°C for 15 minutes at 15 lbs pressure unless otherwise specified.
5.2.8 Enter details of prepared media in the ‘Media Preparation Register’.
5.2.9 Also make relevant entries in the Autoclave sterilization Record in SOP (Operation of Autoclave).
5.2.10 Pre-incubate the media at 30 to 35° C for 48 hrs.
5.2.11 Check visually for any growth in the form of turbidity in case of liquid media and colony forming units in case of solid agar. Discard the media if any growth is observed.
5.2.12 Perform positive control test of each load of sterilized media.
5.2.13 Store the prepared sterile solid agar and liquid media after pre-incubation below 25°C for not more than 15 days.

5.3 Preparation of Pour Plates

5.3.1 Rehydrate the medium and sterilize as per the media preparation instructions.
5.3.2 Cool the media below 45° and pour 15 –20 ml in sterile Petri plates (9 to 10 cm diameter) under aseptic conditions. The Petri plates shall be marked on the bottom position with the details of the name of the medium, lot no. and date of preparation.
5.3.2 Allow to solidify and invert the Petri plates. Pre Incubate the plates at 30° C to 35° C for 48 hours.
5.3.4 Check visually for any contamination. Discard, if necessary.

5.4 Preparation of Slants and Butts

5.4.1 After rehydrating the medium, dissolve the agar media.
5.4.2 Dispense about 10 ml in 18 x 150 mm test tubes. Plug the tubes with non-absorbent cotton and cover with a butter paper.
5.4.3 Sterilize as per media preparation instructions are given on the label.
5.4.4 Unload the tubes from the autoclave and place them in a slanting position so as to achieve a slant and put the tubes as such in a test tube stand for Butts.
5.4.5 Ensure that the top of the slant does not touch the cotton plug and allow to solidify.
5.4.6 Pre-incubate for 48 hours at the appropriate temperature.
5.4.7 Check visually for any contamination. Discard if necessary.
5.4.8 Mark the tubes appropriately with the Name of the medium, Lot no., and Preparation date.
5.4.9 Store the slants, after pre-incubation below 25°C.

5.5 Preparation of RODAC Plates

5.5.1 Rehydrate the medium and sterilize as per the media preparation instructions.
5.5.2 Cool the media and pour in sterile RODAC plates under aseptic conditions. The RODAC plates shall be marked on the bottom position with the details of the name of the medium, lot no., date of preparation.

5.6 Traceability of Media/ Sterilization Load Numbers

5.6.1 Each lot of medium prepared and sterilized is assigned a specific lot number as 8 characters as given below.
XX-YYZZM
XX: indicates Serial no. of prepared media
YY: indicates Month
ZZ: indicates Year
M: indicates the abbreviation of the media.
5.6.2 All media lots prepared/ sterilized are to be serially numbered month wise.
Record the name of the medium and specific lot number of all media used for microbiological testing in the Analytical Data Sheet.
5.6.3 Organisms for Positive Control
Bacillus subtilis NCIM 2063 (or equivalent)
Candida albicans NCIM 3471 (or equivalent)
For differential and selective media inoculate the medium with respective microorganisms as mentioned on the container having a concentration of less than 100 cfu / ml.

6.0 ABBREVIATIONS

6.1 HCl: Hydrochloric acid
6.2 NaOH: Sodium hydroxide
6.3 QC: Quality Control
6.4 IPA: Isopropyl alcohol
6.5 NCIM: National Collection of Industrial Microorganism
6.6 Dept.: Department
6.7 SOP: Standard Operating Procedure

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