SOP Title: Procedure for Media Preparation and Sterilisation for various Microbiological Testing

SOP Title: Procedure for Media Preparation and Sterilisation for various Microbiological Testing

1. Objective:

To lay down a procedure for the Media Preparation and Sterilisation for various Microbiological

Testing.

2. Scope:

The scope of this SOP is applicable for the Preparation of Media, Sterilisation, Dispensing of sterile Culture Media, Storage and Reuse of Sterilized Culture Media, Assigning Batch Number to the Media and the related documentation in Microbiology Section of Quality Control Department.

3. Responsibility:
   • Quality Control Department: To prepare and review the SOP. To follow the procedures laid down for the Procedure for Media Preparation and Sterilisation as per this SOP.
   • Quality Assurance Department: To review and approve the SOP and Annexure.
4. Accountability:

Head Quality Control Department, Head Quality Assurance Department.

 

5. Procedure:

5.1		Procedure for Media  Preparation:
5.1.1		Refer to MSDS of the respective media before handling it for the first time.
5.1.2		Verify that the physical characteristics of the media are as per the Certificate of  Analysis of individual culture media.
5.1.3		Always wear gloves and nose mask while weighing the dehydrated media.
5.1.4		Use a dry spatula for withdrawing media from the container and close the bottle lid tightly after use.
5.1.5		When using a new bottle of dehydrated media enter the date of opening on Media Receipt Label as per SOP ‘Procedure for Receipt and Storage of Dehydrated Culture Media used for Microbiological Analysis’.
5.1.6		Weigh accurately the required quantity of dehydrated culture medium as per Annexure on a butter paper.
5.1.7		Transfer the weighed quantity of media to a suitable flask and reconstitute it with required volume of purified water.
5.1.8		Enter all the details of preparation on “Media Preparation Record” as per Annexure.
5.1.9		Enter the consumption record of Dehydrated Media in usage record as per Annexure.
5.1.10		Update the “Instrument/Equipment Usage Log Book” for balance as per SOP “Calibration of Instruments and Equipments”.
5.1.11		The pH values of the reconstituted media as prepared above should be in compliance with the values indicated on the container label of the dehydrated culture medium or Certificate of analysis.
5.1.12		Check the pH and if necessary, adjust with 0.1 N Hydrochloric acid to decrease the pH value or 0.1 M Sodium Hydroxide solution to increase pH.
5.1.13		Record the pH of reconstituted media in Annexure and update the “Instrument/Equipment Usage Log Book” for pH as per SOP “Calibration of Instruments and Equipments”.
5.1.14		Agitate briskly for a few minutes to dissolve or suspend the adherent powder to form a uniform slurry or solution. Avoid spillage.
5.1.15		Place the flask on boiling water bath and occasionally shake up to complete dissolution of media or a suspension.
5.1.16		Transfer the media in flasks or tubes as per the requirement with appropriate plugging either with non absorbent cotton or other suitable material such as plastic caps.
5.1.17		Broth media should be dispensed before sterilisation directly in the test tubes to be used for analysis.
5.2		Procedure for Sterilisation of Media:
5.2.1		Check the water level of autoclave. Ensure that the electric supply is off during media loading and unloading.
5.2.2		For operation procedure of autoclave, refer SOP ‘Procedure for Operation and Cleaning of Autoclave’.
5.2.3		Place the sterilisation indicator strip with media container and affix it as a reference after sterilisation on Annexure, ‘Autoclave Sterilization Record’ of SOP, ‘Procedure for Operation and Cleaning of Autoclave’.
5.2.4		Transfer the flasks/tubes containing reconstituted media to the autoclave with proper plugging. Avoid overloading.
5.2.5		Tightly close the lid of autoclave and sterilise the media at 121°C +2°C at 15 psi for 15 minutes or as specified in Annexure.
5.2.6		Record the autoclave cycle number on Annexure as well as on Annexure of ‘Autoclave Sterilization Record’ of SOP, ‘Procedure for Operation and Cleaning of Autoclave’ as a reference after sterilisation.
5.2.7		After sterilisation unload the media using the heat resistant gloves (asbestos free) to prevent burns.
5.2.8		Transfer the reconstituted media to the inoculation room through the pass box for dispensing and solidification.
5.2.9		Check the pH of sterile media by taking a small quantity of media in a test tube or in suitable glassware under aseptic conditions and make the entry in Annexure.
5.2.10	Record the pH of reconstituted media in Annexure and update the “Instrument/Equipment Usage Log Book” for pH as per SOP “Calibration of Instruments and Equipments”.
5.3	Procedure for Dispensing of Sterile Culture Media:
5.3.1	Molten agar media should be poured around 15-20 ml in sterile Petri plates and 5-10 ml if preparing the slants under the Laminar Air Flow, when cooled to about 45 °C.
5.3.2	Allow the media to solidify at room temperature.
5.3.3	Record the balance quantity of prepared media along with its validity period in Annexure.
5.3.4	Label the flasks and test tubes of 100 ml volume or above as per Annexure ‘Reconstituted Media Label’.
5.3.5	In case of petri plates and test tubes of 30 ml volume, mark the Batch No. and the Date of preparation manually with a marker pen.
5.4	Procedure for Storage and Reuse of Sterilized Culture Media:
5.4.1	1.   The petri plates/slants shall be stored in the refrigerator or at room temperature.
5.4.2	2.   Prepared media shall be used for a maximum period of 15 days from the date of preparation or as per the SOP ‘Procedure to Determine Shelf Life of Reconstituted Media.’
5.4.3	3.   Remove the stored agar media from refrigerator before use and allow it to stand to attain the room temperature.
5.4.4	4.   Re-melt the solid agar media on the day of use in the original container on the water bath at 100° C.
5.4.5	5.   The media usage record shall be entered in Annexure.
5.4.5	6.   Stored culture media showing any change in appearance or any signs of visible microbial contamination or cracks or dehydration shall be discarded as per SOP ‘Procedure for destruction of media, cultures and microbial waste’.
5.4.6	7.   All used glassware’s shall then be cleaned as per SOP ‘Procedure for Cleaning of Used Glassware in Microbiology Section’.
5.5	Assigning Batch Number to the Media
5.5.1	Allocate each reconstituted media with an individual batch number.
5.5.2	Batch numbering shall be done as Media code/Serial number/Year; wherein
5.5.3	Media code specifies about the unique code defined for each dehydrated media on its receipt.  Refer Annexure, In-house Code of Dehydrated Media, Bases and Reagents of SOP, ‘Procedure for Receipt and Storage of Dehydrated Culture Media used for Microbiological Analysis’
5.5.4	Serial number is the batch number allotted to each reconstituted media.
5.5.5	Year specifies the last two digits of calendar year.
5.5.6	For example; M1/001/24 stands for the batch number of reconstituted media of MacConkey agar, where; M1 : Media code of MacConkey Agar. 001: First batch of reconstituted MacConkey Agar media. 24  : Last two digits of calendar year.
5.5.7	Record the batch number of reconstituted media in Annexure of Media Preparation and Usage Record of Agar/Broth Medium.
5.5.8	Unused solid media to be discarded by melting in water bath after its expiry.
5.5.9	Decontaminate all the used Media by referring procedure given in SOP, Decontamination of culture media and microbial waste.

 

6. Definitions / Abbreviations:

• Definitions :

 

• Abbreviations :

Abbreviation	Expansion
°C	Degree Centigrade
0.1 N	0.1 Normal
0.1 M	0.1 Molar
ml	millilitre
psi	Per square inch
+	Plus
MSDS	Material Safety Data Sheet

  

*Note – Ready to use SOP available in “DOWNLOAD” Section.