| Media preparation Consumption and Sterilization in Microbiology Laboratory |
| 1.0 OBJECTIVE To lay down the Procedure for media preparation consumption and sterilization in Microbiology Department. 2.0 SCOPE This procedure is applicable for media preparation consumption and sterilization in the Quality Control Department of Microbiology Area 3.0 RESPONSIBILITY • Microbiologist : is responsible for preparation and sterilization and maintain record as per SOP. 4.0 ACCOUNTABILITY • Department Head 5.0 PROCEDURE 5.1 Safety considerations: 5.1.1 The dehydrated culture media as well as their ingredients are highly hygroscopic and must be store recommended storage condition. 5.1.2 Examine the physical appearance nature of the dehydrated medium. 5.1.3 Spillage of large or small quantities of dehydrated powder or prepared medium shall be taken care by wearing protective overalls, gloves, eye protection and nose mask. 5.1.4 The weighing operation shall be completed as quickly as possible to prevent absorption of moisture by the hygroscopic content. 5.1.5 Wear hand gloves and facemask while weighing the dehydrated media to avoid inhalation of fine powder particle of media. 5.1.6 Do not prepare media in poorly cleaned glassware always take clean, dried glassware as per required quantity of media volume and visually check there shall be no crack or breakage if any crack or breakage found then does not use that glassware. 5.1.7 Always use clean dried weighing tools (Spoon or spatula) to prevent foreign particles and moisture to enter during preparation of media. 5.1.8 Always use separate spatula for individual media or clean by dipping in purified water beaker in between of the consecutive weighing of two media. 5.1.9 Check and always take media for routine use after approval or qualification. 5.1.10 Before start of the weighing, ensure the colour of dehydrated media powder, any lumps formation and free flawless. 5.1.11 The operation shall complete as quickly as possible to prevent absorption of moisture by the hygroscopic contents of media. 5.1.12 Use Purified water/water for injection for media preparation. 5.1.13 Follow all the instructions/precautions as directed by manufacturer at the time of weighing and re-hydration of media. 5.1.14 Clean the balance in between two successive weighing operations if media spillage is occurred. 5.1.15 Prepare the media as per vender instructions given on the container or leaflet because different media types may have different preparation requirements (e.g., heating, additives). 5.1.16 Dehydrated media shall be thoroughly dissolved in water before dispensing and sterilization in microbiology department. If adding required supplement to media, adequate mixing of the medium after adding the supplement shall be performed. 5.1.17 After completion of sterilization cycle media shall not be stored in Autoclave, as it may damage the media properties. Improper heating or sterilization and conditions may result in a difference in colour change, loss of clarity, altered gel strength and pH drift from the manufacturer recommendation range as well as reduce growth promotion activity or selectivity in microbiology department. 5.1.18 If required, the prepared media Petri plate shall be incubated in an inverted position to prevent collection of condensation of the plate surface. 5.2 Requirements 5.2.1 Glassware (Screw Cap Bottle, Conical Flask, Test tube/ Petri plates/Beaker/measuring cylinder) 5.2.2 Weighing Balance 5.2.3 Dehydrated culture media 5.2.4 pH meter 5.2.5 Water bath 5.2.6 Purified water/Water for injection 5.2.7 Autoclave 5.2.8 Laminar Air Flow 5.2.9 Incubator 5.2.10 IR thermometer 5.2.11 Hand gloves, facemask 5.2.12 Spatula/Spoon 5.2.13 Disinfectant Solution/Purified water 5.3 Microbiology Media Preparation. 5.3.1 Take suitable volume of Purified water /water for injection in the cleaned dried bottle/flask as per requirement for media to be prepared and mark or paste identification label on each article (bottle/flask, tubes, vials) with details such as name of the medium (In-House short forms of media), prepared/sterilized lot No. as per procedure given in section 5.3.6 and quantity prepared or dispensed and Sign & date. 5.3.2 Use the calibrated weighing balance and weigh the required quantity of media by calculating the weight as mentioned on the media bottle/ container, after weighing immediately close the media bottle/container. For example, suppose 30 gm media is required for 1000mL media preparation and if 400mL media is to be prepared then calculation shall be as below 1Required dehydrated media quantity for 400mL media preparation = 30gmX400mL = 12 gm. 5.3.3 After weighing, add the dehydrated media powder in bottle/conical flask containing Purified water /water for injection. Shake the media bottle/flask and make up the volume with Purified water /water for injection. Add the additives if required as per manufacturer recommendation. After preparation dispensed the liquid media in clean dry container as per required quantity. Add 1% Glycerol in each SCDA media container to be prepared for Environmental monitoring plates. 5.3.4 If heating is necessary as per manufacturer instruction to dissolve the media, care shall be taken not to overheat the media. 5.3.5 Media which are not sterilized in autoclave shall be prepared and sterilized by boiling in water bath or on heating mental/Hot plate as per manufacturer instructions or 10 minutes. 5.3.6 Each lot of medium prepared and sterilized shall be assigned a specific in-house lot number as AA/YY/MM/ZZZ. 5.3.7 Allot lot number for each lot of media as per format AA/YY/MM/ZZZ, where AA will indicate abbreviation of media and YY will indicate last two digest of year and MM will indicate of month and ZZZ will indicate the lot number of starting with 001 every month. 5.3.8 Where, AA abbreviation of media SCDA, YY year 23 and MM month 07 and ZZZ 001, SCDA/23/07/001. 5.3.9 Labelled the plate’s packets or test tubes stands. 5.3.10 Sterilized the media as per validated cycle and after completion of sterilization unload the media as soon as possible. 5.3.11 After sterilization the pH of each prepared and sterilized lot of medium shall be confirmed after it has cooled to room temperature (20-25°C). A flat pH probe shall be used for agar surfaces and an immersion probe shall be used for liquid medium. 5.3.12 The pH of media shall be in a range of ±0.2 of the value indicated by the manufacturer on the media COA or on the media Box/container. The pH of liquid media shall be checked direct from the liquid media container and for solid media direct from the Petri plate. 5.3.13 If pH after sterilization does not meet the acceptance criteria, then media shall not be used and shall be discarded as per current SOP. To Read this SOP Click Here SOP for Entry and Exit in Microbiology Testing Area. 5.4 Preparation of Petri plates [90/100 mm diameter] 5.4.1 After preparation and sterilization cool the media up to approx. 45-50ºC and if required before pouring gently shake the media container and pour 20-25ml medium in sterile Petri plates (90 mm diameter) under aseptic conditions. Allow the Petri Plates to solidify and after solidification invert the Petri Plates. 5.4.2 The Petri plates shall be marked on the bottom position with media reference No. 5.5 Preparation of slants and Swab 5.5.1 After rehydrating the medium boil it to dissolve the agar and then dispense around 10-13 ml of media into culture tube close the tube with screw cap/ cotton plug properly. 5.5.2 Sterilize the Media tube as per validated load pattern cycle. 5.5.3 Unload the media tube from the autoclave and place them in a slanting position so as to achieve a butt and slant. 5.5.4 Mark the tubes appropriately with the media reference number. 5.5.5 Ensure that the media in the tubes do not touch the screw cap/ cotton plug and allow to solidifying stage, 5.5.6 Prepare 0.9% of NaCl solution or BSCP as per manufacturer recommendation for the swab preparation and pour the 09 ml solution into pre-sterilized cotton swab stick. 5.5.7 Preparation of RODAC (contact) plates: 5.5.8 After sterilization cool the media and pour 12-15 ml in sterile RODAC plates (55 mm) under aseptic conditions. The RODAC plates shall be marked on the bottom position with the media reference number. 5.6 Preparation of tubes for sterility, pathogen testing 5.6.1 After preparation of broth media pour the media an appropriately sized glass test tube with the of measuring cylinder 5.6.2 Pour required quantity in glass test tube e.g. 100ml SCDM for water and sterility testing and 90ml SCDM for pathogen and dilution testing of finished product, Raw material and Packaging materiel testing. 5.6.3 Pour 100ml Fluid Thiogylcollate Medium for sterility testing and before ensure that the pink colour of resazurin layar (pink colour) is not more than upper one third of the FTGM in the test tubes. 5.6.4 After taking the dehydrated media from the daily use container of culture media, enter the all details in the media preparation and plates/tubes preparation and consumption record in format No. XXX. 5.7 Storage of media 5.7.1 If media is not used immediate after sterilization, then it shall be kept in a water bath at 45-50°c for not more than 8hours /. Wipe the exterior of the media container with clean and dry mop before use. 5.7.2 After sterilization cool the Fluid thioglycollate medium at room temperature and the ring in Fluid thioglycollate medium shall not be more than upper one third of the medium if found more than upper one third than restore it only once by heating in water bath until the pink colour disappear. 5.7.3 Re-melting of an original container of the solid media shall be performed only once in water bath or on hot plate. 5.7.4 Pre-incubated media shall be used as per below table |
| S. No. | Name of the Media Petri plates (90 mm X 15 mm) | Hold time period | Storage Conditions |
| 01 | Violet Red Bile Glucose Agar | As per validation | 20-25°C |
| 02 | Xylose Lysine Deoxycholate Agar | ||
| 03 | Sabouraud Dextrose Agar | ||
| 04 | Soybean Casein Digest Agar | ||
| 05 | R2A Agar | ||
| 06 | Columbia Agar | ||
| 07 | Mac Conkey Agar | ||
| 08 | Sabouraud Chloramphenicol Agar | ||
| 09 | Cetrimide Agar Base | ||
| 10 | Mannitol Salt Agar Medium | ||
| 11 | De-engley’s Neutralizing Agar | ||
| S. No. | Name of the Media Petri plates (12 mm X 55 mm) | Hold time period | |
| 01 | De-engley’s Neutralizing Agar RODAC Plates 55 mm | As per validation | |
| 02 | Soybean Casein Digest Agar | ||
| S. No. | Name of the media in Slants | Hold time period | |
| 01 | Soybean Casein Digest Agar slant | As per validation | |
| 02 | Nutrient Agar slant | ||
| 03 | Triple Sugar Iron Agar slant | ||
| 04 | Sabouraud Chloramphenicol Agar | ||
| 05 | Triple Sugar Iron Agar slant | ||
| S. No. | Name of the media in tubes and bottles | Hold time period | |
| 01 | Sterilized Liquid Media in tubes | As per validation | |
| 02 | Sterilized Solid Media in Bottles | As per validation |
5.8 Growth promotion test & laboratory controls: 5.8.1 Each autoclave lot/boiled media shall be tested for growth promotion capability of media using cultures mentioned respective SOP“ Growth promotion of media” 5.8.2 Portion of each lot of the medium (or one container of the prepared lot) used for the regular microbiological testing shall be incubate as “positive control” using one or two microorganisms as per current Sop of growth promotion test of media. 5.8.3 Positive control of liquid medium and solid agar medium shall be performed using quantified suspension of < 100 CFU/ml. 5.8.4 Sterility of prepared plates and liquid medium (Negative control of medium) shall be performed by incubating a portion of prepared lot or one prepared plate along with the test sample. |