PREPARATION, STORAGE AND USES OF CULTURE MEDIA

  1. OBJECTIVE 

To lay down a procedure for preparation, storage and use of culture media for microbiological analysis. 

 2. SCOPE

This SOP shall be applicable to preparation, storage and uses of culture media for microbiological   analysis.        

  3. RESPONSIBILITY

3.1        Execution: Officer, Executive and above –QC Department

3.2        Checking : Asst. Manager and above QC Department

  4. ACCOUNTABILITY

Head of the QC Department 

 5. PROCEDURE  

1. Media preparation:
   1. Use the pre-formulated dehydrated media for media preparation.
   2. Weigh the required quantity of the dehydrated media as per the manufacturer’s instruction and dissolve it in purified water and make up the specified volume with purified water. 
   3. Dissolve the media by shaking, tilting and / or heating with continuous stirring, if required.
   4. Distribute the required volume of media in appropriate glass containers with ensuring that the container used must have double volume capacity of the volume of substance to be sterilised. 
   5. Allot the Lot No. to each prepared media as following.                 

             Where 

1. XXXX is the short form represents the media name in capital alphabets.
2. Batch. No. is the Manufacturer Batch No..                                                                                                                                                                                                         
3. ZZZZ is denoting the Autoclave cycle numbers of particular media to be sterilised.
4. YY represents the year in which sterilization carried out. 

For example: 

Soyabean casein digests agar media of Batch No. 2J450 with Autoclave cycle No. 0004 of the year 05

1. Autoclave cycle No. will start from 0001with effect from Ist  January of every year.

1.  Write the following details and Lot. no on to the each media container. 

1. Close the media container by cotton plug and cover with butter paper or metal caps and sterilize as per specified instructions.  
2. For steam sterilization place the media container in autoclave as per SOP
3. The pH of each batch of medium should be confirmed after it has colled to room temperature  ( 25 oC) by aseptically withdrawing a sample for testing , if necessary adjust the  pH with 0.1N HCl /  0.1 n NaOH as required..
4.  After sterilization, cool down the liquid media at room temperature and use.

5.1.11      In case of solid media, cool it up to 40-45 oC with ensuring the molten form and distribute. 

   5.1.12     If any delay in distribution then to maintain its molten form keep it on to a hot plate or oven or water  bath at a temperature 45 – 50 oC up to a period NMT 6 Hrs.

1. Slant preparation: Aseptically fill the molten media up to ¼ spaces of sterile tubes and keep the tube    at approximately 20 to 30 o   angle position to form slant and allow to solidify.

1. Stab preparation: Aseptically fill the molten media up to 1/ 2 space of sterile tubes and keep in the   upright position to form the stab and allow solidifying.
2. Plate preparation: Aseptically pour the 15 to 20 ml of molten media in sterile petri plate, keep in the upright position and allow it to solidify. 

1. In case of solid media not used on the day of preparation store the media in Microbiology room / incubator at NMT 250C and before use melt it by boiling only once “Do not autoclave and over heat ” cool it up to 45 – 50 oC  with ensuring the molten form and distribute .

1. Storage
   1. Store all the prepared media e.g. plates, broth tubes, slants, stabs and broth or solid media container at  a temperature NMT 250C.
   2. Use the media with in one month from its date of preparation, if physical properties allow like no  dehydration, shrinking, drying, decolourisation and contamination.
   3. Discard all unused media as per SOP.
2. Growth Promotion Test:
   1. Ensure the growth promoting properties of the each autoclave lot of medium by   challenge with standard culture organism (Table- 2) as per given Table-1.
   2. Inoculate two containers (for liquid media) or two Plates (for solid media) with NMT 100 CFU of challenge organism.
   3. If media distributed before sterilisation in different volume in different container then use the media from container which have minimum distributed volume during autoclaving (sterilisation) for growth promotion test.
   4. Select the challenge organism specified in table -I 
   5. If the challenge organism is more than one, separately inoculate the NMT 100 CFU of each challenge organism in duplicate.
   6. Incubate at 20-25o C temperature for the growth of fungal challenge organism for NMT 5days or at 30-35oC temperature for NMT 48 hrs for the growth of bacterial challenge organism.
   7. The medium is satisfactory for use, if bacterial growth observed with in 48 hrs for bacteria and 5 days for fungi as per the specific incubation condition.
3.  Maintain the media preparation record in Annexure.

Table-1

About the Author

Author

Administrator

"A seasoned professional with a Master's in Chemistry, the author brings more than two decades of expertise in Quality Control, Analytical Method Validation, and advanced QMS investigation. Their experience is globally validated by successful management of major regulatory audits, including those conducted by the US, EU, Brazil, and Russian authorities."

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