PROCEDURE FOR PREPARATION, STERILIZATION, STORAGE AND USAGE OF MICROBIOLOGICAL MEDIA

• PROCEDURE FOR PREPARATION, STERILIZATION, STORAGE AND USAGE OF MICROBIOLOGICAL MEDIA:
• Media is a culture media, or growth media. This is a specially prepared gel or liquid that provides nutrients and an environment for microorganisms, like bacteria, to grow in a lab setting.

• Media Preparation:
• Collect approximately 75% of the required volume of purified water into a suitable container.
• Weigh the quantity of dehydrated media for the preparation of agar or broth as per instructions mentioned on the manufacturers label and suspend in the container with purified water.
• Add the remaining 25 % of purified water to make up the required volume.
• Dissolve the powder by gently stirring with glass rod/stirrer, followed by boiling.
• Check the pH of medium and adjust the pH using 1.0 M NaOH or 1 M HCl solution, if necessary.
• If required, dispense the required volume of dissolved medium in desired containers.
• Close the container tightly with a suitable cap for bottles or cotton plug in case of flasks and cover with butter paper sheets and tighten them using cotton thread / rubber bands.
• Label the flask/bottle with medium name, Media reference Number, Date of preparation and use before.
• Record the details in Media preparation record as per Format –I.Assign a Media reference number to the prepared media and enter in Media Reference Number allocation register as per Format-II.

• Assigning of Reference Number to prepared media:
• All prepared media shall be identified with unique number.
• The numbering system consists of 10 characters.
• The first three characters denote the name of the Media as per the format -III.
• Next character is a “/” slash.
• Next three characters are the serial number starting with 001in every year.
• Next character is a “/” slash.
• Next two characters denote current year.

Example: The first SCD medium in the year of 2025 shall be numbered as SCD/001/25.

• Sterilization of Media:

• All the prepared media shall be subjected for sterilization by boiling or autoclaving as mentioned on the manufacturer Container label.
• Wrap the mouth of the media containers with Kraft / butter paper.
• Affix the autoclave chemical indicator strip on the media containers.
• Sterilization by boiling can be done by using water bath / Hot plate.
• Sterilization by Autoclave can be done as per the SOP.
• After completion of sterilization, ensure the colour of the indicator is changed to black
• If there is no change in colour, discard the material and prepare the media freshly.
• Check the pH of sterilized media and record in Media preparation record. (Format-I)
• All the sterilized media shall be subjected to Growth Promotion test as per SOP.
• If the media shows satisfactory growth, then the media shall be used for routine analysis, if not reject the material.

• Record the results in Media preparation record (Format-I)

• Pre Incubation and storage of Pre-Incubated Media:
• All the prepared and sterilized media shall be kept for incubation at 30-35°C up to 48 hours (For Bacterial media) and 20-25°C up to 72 hours (For fungal media).
• After completion of pre-incubation period, visually check the media tubes and plates for the absence of growth, if any contamination observed discard the plates. 5% is the acceptable range for contamination.
• All pre poured plates should be labeled with Name of Media, Media reference Number, Date of preparation and use before date.
• Enter the pre incubation details in Media preparation record. (Format-I).
• Storage and usage of Pre-Incubated Media:
• Transfer the pre-incubated media tubes and plates at 20-25°C Incubator and store up to15 days.
• Take the required pre-incubated media from 20-25°C Incubator and use for Microbiology analysis.
• Enter the details of media usage in media reconciliation record Format IV.

NOTE: Compare the current lot with the previous lot of media for recovery Factor2.

• RecoveryFactor 2 Calculation:
• For minimum value calculation – Number of inoculated cells are divided by 2 E.g.: Number of inoculated colonies – 50/2 = 25 cells. So, the minimum value is 25 cells.
• For maximum value calculation – Number of inoculated cells are multiply with 2 E.g.: Number of inoculated colonies – 50×2 = 100 cells So, maximum value is 100 cells.
• The recovery should between > 50 % to < 200 %.

• ANNEXURES: 

• ABBREVIATIONS:

Annexure-I
MEDIA PREPARATION RECORD

Annexure-II
Media Reference Number Allocation register

Annexure-III
LIST OF MEDIA CODES

Annexure-IV
MEDIA RECONCILIATION RECORD