Media Preparation in Microbiology 2023

Media preparation in microbiology refers to the process of preparing culture media, which are nutrient-rich substances used to grow and cultivate microorganisms in the laboratory for various purposes, such as identification, characterization, and study of their growth patterns and metabolic activities. Proper media preparation is crucial for obtaining accurate and reliable results in microbiological experiments.

Here are the general steps involved in Media Preparation in microbiology:

  1. Selection of Media: Depending on the type of microorganisms being cultured and the specific purpose of the experiment, the appropriate type of culture media is selected. There are various types of media, such as agar plates, broth, and slants, each with different compositions to support the growth of specific microorganisms in Media Preparation.
  2. Gathering Ingredients: The ingredients for the media, such as peptones, agar, sugars, salts, and water, are gathered. It is important to use high-quality ingredients to ensure the consistency and reliability of the media in Media Preparation.
  3. Measuring and Weighing: The appropriate quantities of each ingredient are measured and weighed according to the specific recipe or formula of the media being prepared. Precision and accuracy in measuring and weighing are essential for obtaining consistent results.
  4. Mixing: The ingredients are mixed thoroughly in distilled water or deionized water to create a homogenous solution. Heat may be required to dissolve some solid ingredients, such as agar.
  5. Adjusting pH: The pH of the media is adjusted to the appropriate level using pH indicators or a pH meter. Different microorganisms have different pH requirements for optimal growth, and the pH of the media must be adjusted accordingly.
  6. Autoclaving/Sterilization: The media are then sterilized using an autoclave, which is a high-pressure steam sterilization equipment that kills all forms of microorganisms, including spores. Autoclaving is the most common method of sterilizing media, but some heat-sensitive ingredients may require alternative methods, such as filtration.
  7. Pouring or Dispensing: After sterilization, the media are poured into sterile Petri dishes or tubes, or dispensed into sterile containers, while maintaining aseptic techniques to prevent contamination.
  8. Solidification: If preparing agar plates, the media are allowed to cool and solidify before being stored in a refrigerator or used immediately. Agar solidifies at around 45-50°C, but should be cooled to around 30-40°C before pouring into plates to prevent damage to heat-sensitive microorganisms.
  9. Labeling and Storage: The prepared media are labeled with appropriate information, such as media type, date of preparation, and any special instructions, and stored properly in a designated area, such as a refrigerator or an incubator, to maintain their stability and sterility until they are ready to be used.

It is important to follow standard operating procedures (SOPs) and good laboratory practices (GLPs) during media preparation to minimize the risk of contamination and obtain reliable results in microbiological experiments. Regular monitoring and quality control of media, such as performing sterility tests, are also important to ensure the integrity and performance of the media.

Media Preparation SOP


To lay down the procedure for Media Receiving, issuance & Preparation.

2.0   SCOPE:

This SOP is applicable for Media Receiving, issuance & Preparation in microbiology Department.

3.0   Responsiblities while Media Preparation

Analyst – Microbiologist

To follow written procedure defined in SOP

To report any non compliance to the procedure

Head Microbiology QC / Designee

To implement the procedure at their site.

To investigate any non compliance to the procedure


Head QA

To ensure adherence to the procedure.

5.0 PROCEDUREof Media Preparation:

5.1 Media management in Media Preparation:

General Instructions in Media Preparation:

  1. Ensure that the COA is received with each lot. If not received, download from the manufacturer’s website and file the same. Inspect the dehydrated media & reagents for quantity ordered/received, lot number, expiry date and any special storage conditions recommended by supplier.

  1. Check the physical condition of all the media containers, if not acceptable, reject the container/s which is received in damaged condition or which does not match with the item code indented.

  1. Make relevant entries of all the media received in Annexure-I.

  1. Affix label(s) on every container as per Annexure-II. “Use Up to date” is one year from Date of Opening or the Expiry date of de-hydrated media, whichever is less.

  1. Arrange the media according to the container number (for e.g. if 4 containers are received in consignment label them as 1/4, 2/4, 3/4, 4/4) & follow FIFO system when the container is withdrawn for use.

  1. Perform initial GPT on each consignment of media before using it for routine test. .

  1. Use the dehydrated media consignment only after result of initial GPT passes, otherwise reject the media consignment.

  1. Keep the ‘under-test’ GPT consignment containers physically segregated from the media stock for effective management of media.

  1. When same lot number of culture media is received in two different consignments carry out GPT for both the consignments received.

  1. Media used for some special tests Eg. Sterility test, Antimicrobial effectiveness test, Bioassay test, Certain investigational tests etc. may need additional controls and procedures which shall be documented in respective SOPs or protocols.

  1. If media to be used after 1 year of receipt and is under safe period of expiry date, then it can be used provided a fresh initial GPT of the media and physical conditions of media are found satisfactory.

  1. Store the media as indicated on the container as per manufactures instructions. The culture media received are hygroscopic in nature hence container shall be tightly closed and kept away from bright light at specified temperature and humidity (if applicable).

  1. Use hand gloves & nose mask during handling/preparation of dehydrated media.

5.2 Physical Verification in Media Preparation:

  1. Prepared media should be checked by appropriate inspection of plates and tubes for the following:

  2. Cracked containers or lids

  3. Unequal filling of containers

  4. Dehydration resulting in cracks or dimpled surfaces on solid medium • Hemolysis

  5. Excessive darkening or color change • Crystal formation from possible freezing

  6. Excessive number of bubbles

  7. Microbial contamination

  8. Status of redox indicators (if appropriate)

  9.  Lot number and expiration date checked and recorded

  10. Sterility of the media

  11. Cleanliness of plates (lid should not stick to dish)

5.3 Media Preparation:

  1. Media shall be prepared in clean & dry glassware.

  2. Open the media container. Check the container and if lumps/ discoloration/ cake formation is observed then do not use the media. Discard the media and record in the relevant autoclave log book.

  3. Microbiological Media should be prepared as per manufacturer’s recommendations that are stated on the label of each dehydrated media container. Accidental dehydrated media spillage e.g. during weighing, transferring etc., shall be recorded on media preparation record.

  4. Add required volume of purified water using calibrated measuring cylinder and allow the media to hydrate. Boil & dissolve the medium if necessary.

  5. Adjust the pH of the media, if necessary, using 1N NaOH or 1N HCl solution to the pH specified and recorded.

  6. Dispense appropriate volume of prepared media in tubes/bottles.

  7. Stopper the reconstituted media containers (flasks/ tubes/ bottles etc.) as per that used during validation of autoclave load patterns.  Assign a lot number to each hydrated media before sterilization.

  8. Sterilize the media in autoclave as per site SOP as per validated load pattern. Affix the label on each container or rack of containers

5.4 Allotting Hydrated Media Lot Number in Media Preparation:

  1. Hydrated media lot no. will be given as per following pattern:  SCDA/01/251122

Where, SCDA – Abbreviation of the medium (Refer Table for abbreviations)

     01  – Serial number of particular medium prepared on a particular date

     02     251122        –  Date (DD-MM-YY) of preparation

      If the same medium (SCDA) is prepared for the second time on date 25/11/2022,

        then it shall be numbered as SCDA/02/251122.

  1.  Next day (i.e on 26/11/2022) when SCDA is prepared, it shall be numbered as       SCDA/01/261122.

  2. Follow instructions given by manufacturer for preparation and sterilization parameters of a medium.

  3. The molten agar medium shall be held in water bath at a temperature of 45°C to 50°C for not more than 8 hours, unless otherwise validated.

  4. Check the pH of media after sterilization of one unit of each medium as per  SOP. Use flat pH probe for agar based media. If media pH is not within the acceptance criteria then discard the media.

  5. Before usage of media check for volume, discoloration, drying and contamination. If any of the above discrepancy is observed then discard the media as per procedure.

  6. Maintain the record of each medium prepared.

  7. ‘Media preparation’ details entry in a page should be of only same supplier, new page shall be used in case using same media from another supplier.

  8. Incase pre- sterilized media is procured as ready to use media, then store it as per vendor’s recommendation and perform the growth promotion test.

  9. To prepare the plates pour sterilized media aseptically in required quantity into sterile petridishes under the LAF and allow them to solidify. If required, add the neutralizer to the media.

  10. Perform GPT of each sterilized lot of medium concurrently.

  11. Store the prepared and sterilized media at validated conditions and period.

5.5 Pre-incubation of prepared culture media:

  1. Carry out the pre-incubation of prepared and sterilized media at 30-35°C temperature for 24-48 hours for all plates prepared, unless otherwise justified. Unused plates shall be transferred to 20-25°C temperature after completion of pre-incubation.

  2. Liquid media can be used on date of preparation provided appropriate negative and positive control of medium are conducted. If any abnormality is observed in negative / positive control (i.e. no growth in positive and growth in negative) all the tests for which the media of that sterilized lot is used will be repeated. Investigation to be performed for failure of negative control through deviation.

5.6 Handling of Spilled Media in Media Preparation

If any media is spilled, collect the spilled media, in disposable polybags with the help of scoop/spoon. Such accidental loss of quantity of dehydrated medium spilled shall be recorded in formats. The surface shall be immediately cleaned with filtered 70% isopropyl alcohol. Autoclave the disposable polybags containing media at 121o C temperature for 30 minutes. After autoclaving the same shall be sent to EHS for destruction as per Destruction  SOP.

5.7 Discard of empty media containers/ dehydrated spilled media:

When the media is totally consumed or expired then deface the label of empty container and destroy the medium as per Destruction SOP.

Media Preparation in Microbiology 2023

Media Preparation in Microbiology 2023

You may also read about : Qualification/Validation Concept


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