Preparation of agar media

Preparation of agar media
 1.     Purpose:-                         

                       To describe the method of preparing media plates for carrying out different
  microbiological tests.
2.     Scope:-

 This procedure applies to preparation of Microbiological media in aseptic conditions in Microbiological Lab.
     3. HSE Statement:-
                            Face mask and gloves should be used.

4. Responsibilities:-

                               i)      Manager Quality Control is responsible to ensure that procedure & formats are followed entirely as approved.
                             ii)      Microbiologist is responsible to perform the test.
5. Materials:-

5.1 Tryptic Soya agar (prepared and sterilized) in 500 ml conical flask with cotton plug covered with  butter paper kept in water bath at 48-50°C.       
5.2  Other media such as Sabouraud Dextrose Agar, Eosin Methylene Blue, MacConkey Agar, Antibiotic Media etc. prepared and sterilized, kept in water bath at 48-50°C.
5.3  Petri-dishes sterilized at 200°C for at least two hours.
5.4  LFC kept ‘on’ for 30 minutes prior to prepare plates.
5.5  Disinfectant.
5.6  Sterilized cotton duster.
5.7  Incubator for pre-incubation.   

6.0 Definitions:
  6.1 Media:
 Media is the nutritive source for the cultivation of microorganisms. The media usually prepare from protein by acid or enzymatic digestion. Medias are usually available in dehydrated form. We have only to mix required measured quantity in distilled water. Follow instructions for preparation as mentioned on the label of media container.
  6.2 Sterilization:
                It is the process of killing or removing microorganisms. Media is sterilized by autoclaving at  
 1210C for 15 minutes at a pressure of 15 PSI.
  6.3 Agar:-
               Complex sulfated polysaccharide derived from certain marine algae(normally red algae) that is a
               gelling agent for solid or semisolid microbiological media. Agar consists of about 70% agarose and 30% agaropectin. Agars can be melted at temperature above 100ºC; gelling temperature is 40-50ºC. Agar is used for performing viable counts

7. Flow Chart:-

8.0 Description:-
      8.1 Procedure:-

     8.1.1 Prepare and sterilize agar media as per SOP.
     8.1.2 Transfer all the required materials to LFC bench taking all necessary microbiological precautions.
     8.1.3 Remove plates from SS box on to the bench and let them cool.
     8.1.4 After cooling of the plates obtain Media from water bath and mop outer surface with duster wet
              with disinfectant.
8.1.5    Carefully and slowly remove the cotton plug first uncovering the butter paper on it.
8.1.6    Gently heat the mouth of the flask or bottle containing media for decontamination of outer
8.1.7        Now carefully take one plate and half open it by one hand and pour approximately 20 ml of agar media and close the plate. Care should be taken to not to lean over the open plate or media nor take your hand or arm over opened plate.
8.1.8    Similarly pour all the plates as soon as possible as agar media starts solidifying.
8.1.9    Pour media in such a way to inhibit the bubble formation in the agar media.
  8.1.10 Let plates as such for at least 20 minutes for solidification.
  8.1.11 After solidification label each plate with abbreviation of media name, date of preparation.
  8.1.12 Gather all the plates and transfer in a container into the appropriate incubator for pre-incubation
                in inverted position for at least 24-48 hours.
     8.1.13 Before conducting any test observe the plates carefully for the evidence of any growth
     8.2 Precautions:-
     8.2.1 Store the plate in refrigerator if testing is delayed.
     8.2.2 Plates should be used as early as possible as media starts drying.
     8.2.3 See that media is not overheated.
     8.2.4 Check that the sterilization cycles are satisfactory.
     8.2.5 Check the pH of the media after sterilization of the media.
     8.2.6 Check the expiry date of dehydrated media.
     8.2.7 Check the washing process of the glassware.  
     8.3 Growth Promotion Test of Medias (Positive Control):
     Growth promotion test of each lot of media prepared should be performed as per SOP.

   9. Forms and Records:-
     9.1 Sterilization and Preincubation record of Medias.
     9.2 Log of Sterilization Cycle.
     9.3 Growth Promotion test.
10. References:-
      USP 35
  11. Distributions:-

This SOP has to be distributed in below mentioned Departments:-

Sr. NO
Distributed to
Quality Control Department

Quality Management Department

12. Revision History:-

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