SOP on Media Preparation
- Objective:
To lay down the procedure for Preparation of Media.
- Scope:
This SOP is applicable for preparation of sterile media used in Microbiology Laboratory of
(Pharmaceutical Company Name).
- Responsibility:
- Microbiologist or above of Microbiology Laboratory.
- Head – Microbiology section.
- Accountability:
- Head – Quality Control
- Head – Quality Assurance.
- Abbreviations and Definitions
QC : Quality Control
LAF : Laminar Airflow
MLT : Microbial Limit Test
RODAC : Replicate Organisms detection and Counting.
- Procedure:
- Selection and Preliminary checks to be done for media preparation (applied for every container that has to be used):
- Use the formulated commercially available dehydrated culture media or individual ingredients (if formula is available) for the preparation of medium in the microbiology laboratory.
- Read and use the directions given on the label of dehydrated medium container as per manufacturer for the preparation.
- Check the container for expiry date before using the medium.
- Ensure that the dehydrated media powder have the following physical characteristics at the date of opening.
- Free flowing
- Uniform colour
- Absence of lumps
- Reject the media, if it is not satisfied with reference to the above mentioned characteristics,
- Preparation and Sterilization of media
- Place a piece of butter paper on the pan of the weighing balance, large enough to accommodate the quantity to be weighed.
- Open the container containing the dehydrated medium to be used in a respective way.
- With the help of a clean and dried spatula / spoon, transfer the dehydrated media powder on the butter paper.
- After weighing the required amount take it slowly from the pan without spilling the dehydrated media and transfer it into a clean container supposed to be dissolved in it.
- Add small volume of purified water into the container and swirl to dissolve completely.
- Make up the volume by adding purified water through the sidewalls of the Container.
- Boil the medium if required to dissolve completely using a hot plate / water bath.
- Take care to avoid excessive heating or scorching of the medium.
- Test the media before sterilization for pH (at 25 ± 2 °C).
- If the pH doesn’t meet the specification, correct the pH by adding 0.1N Hydrochloric acid or 0.1 N Sodium hydroxide to the prepared medium.
- Dispense the dissolved medium as desired i.e. in test tubes / screw capped bottles / septum bottles/ conical flasks etc. as appropriate. Follow the directions given in the table -2 for dispensing of the dehydrated media. In medium used for environmental monitoring, add 0.5% of glycerin.
- Close the tubes and flasks with non-absorbent cotton plugs, screw capped bottles with screw caps. Septum bottles shall be closed with septum & aluminum seals.
- Wrap the tubes/ flasks with parchment paper and arrange the tubes in the test tube stands and then arrange all the prepared media containers in the autoclave.
- Sterilize the media in the autoclave by operating the autoclave as per SOP No.
- Unload the media in the sterile buffer room, place the media required for sterility testing in the SS rack and transfer the media to sterility testing room, whenever tested.
- Transfer the media to MLT section (for the tests which are supposed to be performed in the MLT section) through dynamic pass box No.:
- Perform the pH check of sterilized media by taking an aliquot from the sterile media. Discard the media if it doesn’t meet the specification.
- Addition of supplements in the media
- Some of the routinely used media shall be supplemented with few additives during certain stage of the media preparation and sterilization. Supplements such media with those particular additives as mentioned in the table-3.
- Selection and Preliminary checks to be done for media preparation (applied for every container that has to be used):
- Preparation and sterilization of heat sensitive media
- Some of the routinely used media cannot be sterilized in autoclave. Prepare such media in the conical flasks and sterilize by heating on water bath for 15 minutes. Such type of media are listed below
- Fluid Selenite-Cystine Medium
- Fluid Tetrathionate Medium
- Bismuth sulphite agar
- Xylose-Lysine-Desoxycholate Agar Medium
- After sterilization dispense the boiled media under LAF (of MLT section) into the presterilized petriplate / tubes as appropriate.
- Preparation and sterilization of Media for Sterility test
- Prepare and sterilize the media used for Sterility test, like Soya bean Casein Digest medium and Fluid Thioglycollate medium in 100 ml septum bottles.
- Prior to use, ensure that the media contained in the bottles doesn’t contain any abnormal characters like haziness,
- Ensure that the pink colour of resazurin layer (pink coloured) is not more than the upper one third of the Fluid Thioglycollate medium contained in the bottle.
- Do not use the media if any abnormalities observed in the media.
- Preparation of Agar medium plates used for Environmental and Personnel Monitoring
- Prepare the agar medium plates under LAF of MLT room.
- After sterilization and before pouring the agar into sterile Petri plates, cool the agar medium to 45 to 50 °C.
- For settling plate exposure and for swabs of surface monitoring: Pour around 20 ml of agar medium in to each of sterile 90 mm plate, close the lids and allow them to solidify.
- For volumetric air sampling: Pour in 75 mm sterile cassettes intended for specific purpose, close the lids and allow them to solidify.
- For personnel and surface monitoring: Fill the agar medium into sterile 55 mm surface contact (RODAC) plates, in such a way that the upper surface of the agar medium shall be slightly concave (bleb), close the lids and allow them to solidify. Take care to avoid touching of the lids to the upper surfaces of the agar during closing of the plates.
- Preparation of agar medium slants for sub-culturing and other purposes
- To prepare the agar media slants, follow the description of volume of media, and height on which the top of the tube is placed as specified in table.1
Table-I: Parameters for slant preparation
| Tube parameters | Volume of agar medium suspended | Height to be placed |
| 18×150 mm | 15.0 ml | 16.0 mm |
| 25×200 mm | 30.0 ml | 20.0 mm |
- Keep the upper end of the molten agar medium tube meant for slants on certain height (as per table) in such a way that the solidified medium shall form a butt-slant. For anaerobic culture, keep the molten agar medium containing the mineral oil in upright position to form butt with mineral oil on the top.
- Preparation of 0.1 N Sodium Hydroxide and 0.1 N Hydrochloric Acid
- Dissolve 40 g of Sodium Hydroxide pellets in 1000 mL of purified water to prepare 1 N sodium hydroxide solution and 18 mL of Hydrochloric acid in 1000 mL of purified water to prepare 1 N hydroxide solution.
- Make a 1:10 dilution of the above solutions to obtain 0.1N strength.
- Sterilize the solution in the autoclave by operating the autoclave as per SOP.
- Suitability Tests for Sterilized medium
- Perform the suitability tests (Growth Promotion test, Sterility check and Inhibition checks as applicable) for all sterilized lots of media following the procedure given in the SOP No.: (SOP on Growth promotion and Sterility check of Media). After suitability tests, discard the used medium as per SOP No.: (SOP on Decontamination and Disposal of Used Media).
- Pre-incubation
- Incubate all the media plates used for Environmental and Personnel monitoring, selective and differential agar media plates, agar media plates used for membrane filters (for test for water for injection, pure steam condensate and surface monitoring by swabs) and slants used for sub-culturing for 48 hrs. at 30-35 °C and check all the plates for any microbial contamination prior to usage. Do not use the media lot and discard all the plates / slants, if any of the plate / slant shows contamination. Label required no. of the plates / slants with Name of medium, sterile medium lot No. and date, before keeping the media for preincubation.
- Storage
- Store the media at 20°C to 25°C and use the media upto 7 days (including pre- incubation, if done).
- Use the freshly prepared Fluid Selenite-Cystine Medium and Fluid Tetrathionate Brilliant green Medium.
- Precautions shall be followed during preparation and sterilization of Media
- The dehydrated media container shall not be left open for too long to avoid the lump formation of the media.
- Overheating, prolonged sterilization, use of alkaline glass, prolonged storage at high temperature shall not be performed to avoid a drift in the pH of the media.
- Adequate heating of agar media shall be performed to avoid incomplete solubility.
- The medium shall not be overheated to avoid the darkening of the media.
- Repeated re-melting, excessive heating, incomplete mixing shall not be performed to avoid the loss in growth promotion properties of the media.
- Media shall be prepared in well cleaned vessel to avoid the abnormal colour of the media.
- Media shall be prepared with Proper sterilization, proper loading pattern in the sterilizer chamber, as per the validated time and temperature to avoid any contamination.
- Excessive shaking of sterilized media shall not be performed to avoid bubble or froth formation on the surface of solidified media.
- Perform the sterilization of media as soon as re-hydrated.
- Record the media preparation details in sterile media preparation record as per the Annexure No. (Format No.).
Table-2: Dispensing of routinely used media in the containers after re-hydration
| Name of Medium | Container volume | Volume of medium | Use |
| Soyabean-Casein Digest Agar Medium | 100 ml / 250 ml / 500 ml / 1000 ml screw capped or septum bottles / conical flasks. | 75 ml / 200 ml / 400 ml /
750 ml respectively |
Environmental
Monitoring. |
| Sabouraud Dextrose Agar Medium (or)
Potato Dextrose Agar Medium |
100 ml / 250 ml / 500 ml / 1000 ml screw capped or septum bottles / conical flasks. | 75 ml / 200 ml / 400 ml /
750 ml respectively |
Total combined yeasts and molds count test of non-sterile specimens |
| Plate Count Agar Medium / R2A Agar medium | 100 ml / 250 ml / 500 ml / 1000 ml screw capped or septum bottles / conical flasks. | 75 ml / 200 ml! 400 ml /
750 ml respectively |
Water analysis |
| Soyabean-Casein Digest Medium
/Soy lecithin polysorbate 20 medium |
250 ml | 90 ml | Microbial Limit Test |
| Soyabean-Casein Digest Medium | 100 ml septum bottle | 100 ml | Sterility test |
| Fluid Thioglycollate Medium | 100 ml septum bottle | 100 ml | Sterility test |
| 0.1% Peptone water (Rinse and Dissolution fluid) | 250 ml / 500 ml / 1000 ml screw capped or septum bottles. | 200 ml /400 ml / 800 ml
respectively |
Sterility test |
| Fluid Lactose Medium | 250 ml | 90 ml | Microbial Limit tests |
| MacConkey Agar and Levine Eosin-
Methylene Blue Agar Medium |
100 ml / 250 m l/ 500 ml / 1000 ml screw capped or septum bottles / conical flasks | 75 ml / 200 ml / 400 ml / 750 ml respectively | Escherichia coli test |
| Fluid Selenite-Cystine Medium / Fluid Tetrathionate Brilliant green Medium | 18 mm x 150 mm Test tubes with cotton plugs | 10 ml | Salmonellae test |
| Bismuth Sulfite Agar Medium / Brilliant Green Agar Medium / Xylose-Lysine- Desoxycholate Agar Medium | 100 ml / 250 ml / 500 ml / 1000 ml screw capped bottles / conical flasks. | 75 ml / 200 ml / 400 ml / 750 ml respectively
|
Salmonellae test |
| Triple Sugar-Iron-Agar Medium | 18 mm x 150 mm Test tubes with cotton plugs | 10 ml | Salmonellae test |
| Normal saline | 18 mm x 150 mm Test tubes with cotton plugs | 10 ml and 9 ml | Swab samples and Dilution of cultures |
| Cetrimide Agar Medium / Pseudomonas
Agar Medium for the Detection of Fluorescin / Pseudomonas Agar Medium for the Detection of Pyocyanin |
100 ml /250 ml / 500 ml / 1000 ml screw capped or septum bottles / conical flasks. | 75 ml / 200 ml / 400 ml / 750 ml respectively | Pseudomonas aeruginosa test |
| Mannitol-Salt Agar Medium/Vogel-
Johnson Agar Medium / Baired-Parker Agar Medium |
100 ml / 250 ml / 500 ml / 1000 ml screw capped or septum bottles / conical flasks. | 75 ml / 200 ml / 400 ml / 750 ml respectively | Staphylococcus aureus test |
| Agar media for butt-slants | 18 mm X 150 mm test tubes with cotton plugs | 10 ml | Sub-culturing of reference and other isolated cultures |
*For anaerobic cultures additionally add 10 ml of mineral oil.
Table-3: Addition of supplements in routinely used media
| Medium | Supplement | Stage of addition |
| Soybean Casein Digest Agar medium used for environmental monitoring | 0.5 % Glycerin | After re-hydration of dehydrated medium |
| Soyabean Casein Digest Agar medium used for environmental monitoring | 0.1% β-lactamase solution | Before pouring in to Petri plates |
| Cetrimide Agar medium | 10 ml of glycerin per 1000 ml | After re-hydration of dehydrated medium |
| Tetrathionate brilliant green medium | 20 ml of iodine solution per 1000 ml of medium | After boiling |
| Mannitol salt agar medium | 5 % Egg yolk emulsion | Before pouring in to Petri plates |
| Vogel-Johnson Agar medium | 20 ml of sterile Potassium Tellurite solution per 1000 ml | Before pouring in to Petri plates |
| Baired Parker Agar medium | 20 ml of egg yolk Tellurite emulsion per 1000 ml | Before pouring in to Petri plates |
- Forms and Records (Annexures)
- Sterile media preparation record
- Distribution
- Master copy – Quality Assurance
- Controlled copies – Quality Assurance, Production, Quality Control, Stores, Engineering and Human Resource
- History:
| Date | Revision Number |
Reason for Revision |
| 00 | NEW SOP |
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