To lay down a procedure for preparation of Microbial culture Media.
1.0 OBJECTIVE:
To lay down a procedure for preparation of Microbial culture Media.
2.0 SCOPE:
This procedure is applicable to preparation of Microbial culture Media in Microbiology laboratory.
3.0 RESPONSIBILITY:
Microbiologist : To perform the procedure as per SOP.
Head Quality Control : To ensure the compliance of the SOP.
4.0 PROCEDURE :
4.1 Requirements:
• Balance
• PH meter
• Dehydrated media
• Autoclave
• Laminar air flow
4.2 Preparation of culture media (using readymade dehydrated media)
4.2.1 Store dehydrated media in tightly closed packs in the dark or as directed by the manufacturer.
4.2.2 Make-up the required volume with purified water.
4.2.3 Check the pH of the media before sterilization and if required adjust pH as per manufacturer recommendation with 0.2N HCl or 0.1N NaOH solution.
4.2.4 Distribute the media in the bottles/flasks or tubes as required.
4.2.5 Cover the flasks and tubes with autoclavable plastic caps / cotton plugs and wrap with butter paper / aluminum foil. Load the materials as per the Load pattern in Appendix-I.
4.2.6 Media are sterilized in the autoclave by gravity cycle as per Preparation of Microbial Culture Media otherwise specified. Check the pH of the media after sterilization. Some medias are sensitive to autoclaving. Prepare those medias as per manufacturer instructions.
4.2.7 Enter details of prepared media in the ‘Media Preparation Record’ given in
4.2.8 Also make relevant entries in the autoclave sterilization record in format xxxxx
4.2.9 Pre-incubate the media for 48 hours at 30 to 35° C.
4.2.10 Check visually for any growth in the form of Turbidity. Discard the media if Turbidity observed.
4.2.11 Perform positive control test of each load of sterilized media.
4.2.12 Store the media below 25°C.
4.3 Preparation of plates:
4.3.1 Re-hydrate the medium and sterilize as per the media preparation instructions.
4.3.2 Cool the media and pour in sterile petriplates (9 to 10 cm diameter) under aseptic conditions.The petriplates shall be marked on the bottom position with the details of the name of the medium, load no., date of preparation,
4.3.3 Allow solidifying and inverting the petriplates. Pre-incubate the plates for 48 hours at 300 C to350 C.
4.3.4 Check visually for any contamination. Discard, if necessary.
4.3.5 Store the prepared media plates after pre-incubation below 25°C for not more than 28 days, and prepared liquid media tubes for not more than 14days.
4.4 Preparation of slants:
4.4.1 After re-hydrating the medium, dissolve the agar media.
4.4.2 Dispense about 10 ml in 18 x 150 mm test tubes. Plug the tubes with non-absorbent cotton and cover with a butter paper.
4.4.3 Sterilize as per the instructions by gravity cycle as per Preparation of Microbial Culture Media
4.4.4 Unload the tubes from the autoclave and place them in a slanting position so as to achieve a butt (minimum 1 inch).
4.4.5 Ensure that the top of the slant does not touch the cotton plug and allow solidifying.
4.4.6 Incubate for 48 hours at the appropriate temperature.
4.4.7 Check visually for any contamination. Discard if necessary.
4.4.8 Mark the tubes appropriately with the name of medium and preparation date.
4.4.9 Store the slants, after pre-incubation below 25°C.
4.5.2 Record the name of medium and specific lot number of all media used for microbiological testing in the Analytical Data Sheet.
4.6 Organisms For Positive Control :
Bacillus subtilis
Candida albicans
Hcl, naoh, sterilization, microbiology
