SOP for Storage and Preparation of Microbiological Culture Media

1.0 PURPOSE

To lay down the procedure for storage, preparation and testing of microbiological culture media.

2.0 SCOPE

This SOP is applicable for the storage, preparation and testing of media being used for the various testing purpose.

3.0 RESPONSIBILITY

Trained Microbiologist is responsible to prepare the media as per the media instruction provided by the manufacturer.

4.0 ACCOUNTABILITY

Head of Department

5.0 PROCEDURE

5.1 General Instructions

5.1.1 Use clean glassware and accessories for media preparation.
5.1.2 Before initiating the media preparation, ensure that the Weighing Balance and pH meter are calibrated.
5.1.3 Use purified water or WFI – bulk for preparation of media.
5.1.4 Check the dehydrated media container for its direction for preparation and use before date. Ensure that media used is within the use before the date and no clumps in the media.
5.1.5 Do not keep the media container open for long durations as dehydrated medium are hygroscopic in nature.
5.1.6 Prepare the media as per manufacturer’s instructions, do not overheat the media and do not re-melt the solidified media for use.
5.1.7 Store the dehydrated media and prepared media at recommended storage conditions.
5.1.8 Wear appropriate gloves and mask while preparation of media, especially selective media that may contain toxic ingredients.

5.2 Procurement, Testing and Storage of Dehydrated Culture Media or Ready to Use Media

5.2.1 Procure the dehydrated culture media or ready to use culture required for microbiological testing either from HiMedia or other reputed media manufacturers.
5.2.2 Enter the media consignment details in dehydrate media consumption record.
5.2.3 On receipt of dehydrated culture media or ready to use media check the following :

• Name of Media
• Lot/Batch No.
• Code of Media
• Storage Condition
• Direction for Use
• Use Before /Expiration Date
• Certificate of Analysis

5.2.4 Read the directions on the containers and CoA and perform following tests as applicable by sampling required quantity of the media from one of the container. Enter all the details.
5.2.5 Appearance: Observe the contents of its appearance and compare with manufacturer’s CoA and appearance should comply as per manufacturer’s CoA.
5.2.6 Solubility: Prepare the medium as per manufacturer’s directions, by dissolving in purified water or WFI-bulk and heat or boil if required. Solubility should comply as per manufacturer’s CoA.
5.2.7 Appearance of Solution – Prepared Medium: Observe the above-prepared solution/medium and compare with Manufacturer’s CoA. The appearance of prepared media should comply as per manufacturer’s CoA.
5.2.8 pH of Media: Check the pH above prepared media before and after autoclaving. pH of media should comply as per manufacturer’s CoA.
5.2.9 Growth Promotion and Inhibitory Property Test: Perform the growth promotion test (including inhibitory organisms) as per 5.7 and shall comply with acceptance criteria.
5.2.10 Enter the observation and if the above parameters complies then accept the media for use, label containers as per following format and store at recommended storage conditions.

Received On: Opened On: Container No.: Sign:

5.2.11 When the media container is opened put the date of opening and sign on the label of the container.
5.2.12 When media is opened for used observe the media for its appearance if satisfactory then proceed to take the medium for use. Enter the details in dehydrate media consumption record.

5.3 Preparation of Media

5.3.1 Take required clean glassware and accessories before preparation of each type of media.
5.3.2 Take required dehydrated media containers and check for its batch number and use before dates. Enter the details in the media preparation record.
5.3.3 Read the usage directions for the media preparation and calculate the total amount of each ingredient required to formulate the required volume of media. Multiply the amount of material in g or ml by the batch size being formulated which will yield the total amount of material required for the batch.
5.3.4 Label the glassware to be used for media preparation and dispensing with following details:

Name of Media: Lot Number: Date of Preparation: Sign:

5.3.5 Using weighing boat/butter paper or appropriate glassware, weigh the amount of media ingredient required to formulate the media. The weight of the media should be within 100 % to 101 % of the required calculated weight.
5.3.6 Transfer the weighed media into a clean container and add required volume of purified water or WFI bulk from the sidewalls of the container. If required use hot WFI-bulk to facilitate proper dissolution of media.
5.3.7 With help of a glass rod or magnetic stirrer completely dissolve the media. If required boil the media using a hot plate /water bath to dissolve the media (do not overheat the medium).
5.3.8 Take a representative sample from the prepared media and measure the pH as per SOP.
5.3.9 If the pH is not within the acceptable range, adjust the pH using 1N Hydrochloric acid or 1N Sodium Hydroxide solution (not exceeding 0.1% of prepared volume) such that the pH of the medium will be within acceptable range after sterilization.
5.3.10 Dispense the medium into required glassware (test tubes, conical flasks or bottles) with the help of measuring cylinder or pipettes.
5.3.11 Close the containers with non-absorbent cotton plugs or appropriate caps. For media required for sterility testing by canisters, close the bottles with rubber stoppers and seal with aluminum seals.
5.3.12 Keep the prepared media containers in the autoclave as per the validated load pattern and sterilize the media at 121°C for 15 minutes as per SOP. For heat labile media, which does not require autoclaving, prepare as per manufacturer’s instructions by heating or boiling.
5.3.13 After completion of sterilization, unload the media and allow the liquid medium to cool. For dispensing of sterile agar plates proceed to 5.5.
5.3.14 After the sterilized medium is cooled to room temperature, take one container representing a lot of each medium and measure the pH as per SOP. The pH measurement of agar plates should be measured with a flat-probe electrode after solidification of the agar media.
5.3.15 Enter all details in media preparation record.

5.4 Dispensing of Agar Media

5.5.1 After sterilization of agar media, allow the media to cool to about 45 to 50°C. If the agar medium is required for pour plate method, then keep the flasks in water bath or incubator set between 50 to 60°C till it is to be used. Do not keep the medium for longer duration and do not re-melt the medium.
5.5.2 Prepare for dispensing of agar media by operating the Bio-Safety Cabinet (BSC) as per SOP for Laminar Air Flow (LAF). Arrange the required sterile Petri plates in the BSC or LAF.
5.5.3 If the medium requires the addition of any supplement, add the required quantity of supplement to each flask and shake gently for uniform mixing of supplement.
5.5.4 Preparation of 90 mm plates: Aseptically pour approximately 20 ml of media into sterile plates and allow the plates to solidify at room temperature.
5.5.5 Preparation of 55 mm contact plates: Aseptically and gently pour agar media into sterile contact plate such that raised convex agar surface will form. Ensure that medium does not overflow from the plate. Allow the plates to solidify at room temperature.
5.5.6 Preparation of cassettes for M air T Sampler: Aseptically pour approximately 15 ml of media into 75 mm sterile cassettes and allow the plates to solidify at room temperature.
5.5.7 After complete solidification of agar collect the plates and pack in stacks of appropriate numbers using aluminum foil.
5.5.8 Preparation of Slants: Place the test tubes containing agar media in a slanted position such that a suitable slope is obtained. Allow the slants to solidify at room temperature.
5.5.9 After solidification of media, use one of the plate/container representing the lot and measure the pH with a flat probe.
5.5.10 Enter details in media preparation record.

5.5 Pre-incubation of Media

5.5.1 Pre-incubation of agar media

5.5.1.1 Pre-incubate 100 % of all agar media plates at 30 – 35°C for 48 hours for all media with following exceptions;

• Fungal and Yeast specific media (Sabouraud Dextrose Agar), which shall be incubated at 20 – 25°C for 48 hours.
• Media, which are freshly prepared media and used, or media used for pour plates; in such case keep negative control plates.

5.5.1.2 At the end of the pre-incubation, check the plates under LAF for following:

• Breakage of plate/lids
• Less volume, dehydration/cracks and presence of particles or excessive bubbles.
• Microbial Contamination
• The raised surface in case of 55mm contact plates

5.5.1.3 Remove the plates showing breakage, less volume, dehydration/cracks and presence of particles or excessive bubbles and microbial contamination. In case of 55mm contact, plates remove plates in which there is inadequate raised surface or plates with agar overflowed to outside.
5.5.1.4 Acceptance criteria for microbial contamination: The contaminated plates should not be more than 5% of the prepared plates. If more than 5% of contaminated plates observed, discard the particular lot and an investigation should be conducted.
5.5.1.5 If pre-incubation results are satisfactory, repack the Petri plates and release the plates for use. Store the plates at recommended conditions.

5.5.2 Pre-incubation of liquid media

5.5.2.1 Pre-incubate at least 2 containers if the lot size is less than 50 containers and 4 containers if the lot size is more than 50 containers representing a prepared lot of medium at 30 to 35°C for 48 hours with the exception as in 5.5.1.1.
5.5.2.2 Acceptance criteria: No contamination should be observed in pre-incubated containers representing the lot. If contamination is observed then discard the particular lot and an investigation should be conducted.
5.5.2. If pre-incubation results are satisfactory release the medium for use. Store the medium at recommended conditions.

5.6 Sterility Check of Media

5.6.1 Sterility Check of Agar Plates: After completion of pre-incubation of media, incubate 2 plates if lot size is less than 100 plates and 5 plates if lot size is more than 100 plates at intended test incubation temperature and longest duration of the test. For example, Soybean casein digest agar plates should be incubated at 30 – 35°C for 5 days and sabouraud dextrose agar should be incubated at 20 – 25°C for 5 days. In case of bio-chemical and selective media incubate at 30 – 35°C for 3 days.
5.6.2 Sterility Check of Liquid Media: After completion of pre-incubation of media, incubate the containers used for pre-incubation at intended incubation temperature and longest duration of the test. For example, sterility test media soybean casein digest medium should be incubated at 20 – 25°C for 14 days and fluid thioglycollate mediums should be incubated at 30 – 35°C for 14 days. In case of bio-chemical and selective media incubate at 30 – 35°C for 3 days.
5.6.3 Acceptance criteria: No contamination/growth should be observed. If any growth/contamination is observed, it should be investigated.
5.6.4 Enter test details in media preparation record.

5.7 Growth Promotion Test and Inhibitory Property Tests

5.7.1 Perform growth promotion test and inhibitory property test (for selective media) on each lot of prepared medium and ready to use the medium.
5.7.2 Organisms to be used and incubation conditions and acceptance criteria for each media and perform accordingly.
5.7.3 Use standardized inoculum prepared as per SOP for preparation of culture inoculum.

5.7.4 Growth Promotion Testing of Solid Media

5.7.5.1 Inoculate 10 to 100 CFU of each specified organism in duplicate by either pour plate or spread plate and incubate at specified conditions.
5.7.5.2 After completion of incubation, count the number of colonies recovered.
5.7.5.3 Acceptance Criteria
5.7.5.3.1 General Agar Media: The growth should be obtained within the specified duration and the number of CFU recovered should not differ by a factor greater than 2 from the calculated value of the standardized inoculum (recovery with 50 to 200%).
5.7.5.3.2 Selective and Differential Media: Characteristic/indicative growth should be obtained within the specified duration.

5.7.5 Growth Promotion Testing of Liquid Media

5.7.5.1 Inoculate 10 to 100 CFU of each specified organism in duplicate and incubate at specified conditions.
5.7.5.2 After completion of incubation, observe for growth.
5.7.5.3 Acceptance Criteria: The clearly visible (turbid) growth should be obtained within the specified duration. The selective and differential media should show characteristic/indicative growth.

5.7.6 Inhibitory Property Testing of Selective / Differential Media

5.7.6.1 Inoculate at least 100 CFU of each specified organism in duplicate and incubate at specified conditions for not less than the duration specified.
5.7.6.2 After completion of incubation, observe for growth.
5.7.6.3 Acceptance Criteria: No growth of specified organism should occur.
5.7.7 Enter test details in media preparation record.

5.8 Labeling and Storage of Prepared Media

5.8.1 Allot lot number for each lot of media as per format ABCXXX, wherein ABC will indicate the unique three letter code of the media and XXX will indicate the lot number starting with 001 every calendar year.
5.8.2, For example, the first lot Bismuth Sulphite Agar prepared every year will be BSA001.
5.8.3 Label the packs or stands of prepared petri dishes or tubes with following details:

Name of Media: Lot Number: Date of Preparation: Use Before Date: Sign:

5.8.4 Store the media at specified storage condition.

6.0 ABBREVIATIONS

6.1 SOP - Standard Operating Procedure
6.2 LAF - Laminar Air Flow
6.3 % - Percent
6.4 °C - Degree centigrade
6.5 gm - gram
6.6 ml - Millilitre
6.7 CFU - Colony Forming Unit

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