SOP for Preparation and destruction of Culture Media
It describes the (Standard Operating procedure) SOP for Receipt, Storage, Preparation and destruction of Culture Media.
Sop for Receipt, Storage, Preparation and destruction of Culture Media
Purpose and Scope :
- The purpose of this SOP is to lay down the procedure for Media Receipt, Storage, Preparation, Sterilization and Destruction of Culture media
- This Standard operating procedure shall be applicable for All Microbial culture media in the Microbiology section of Quality Control Department
Responsibilities:
- All Microbiologist, Quality control personnel shall be responsible to follow and implement this SOP.
Introduction and Procedure :
Receipt of culture Media:
- All Required dehydrated culture Media shall be procured from reliable source.
- On the receipt of consignment, check each media container for any kind of damage during transit. If any physical damage is observed, reject the container.
- Check the media container against the COA for following details e. batch number, manufacturing date, expiry date & code number
- Without opening the container, physically check the media for any agglomeration, hardening or lumps. This is done by visually observing the container against light & by gentle tilting.
- If any abnormal sound is heard while tilting the media container, reject it and return the consignment to supplier.
- Enter the quantity of media received with the batch no, received date and use before date in media stock record. Refer Annexure-VI.
- Affix a label on container as per Annexure-III
- Follow the ‘First in First out (FIFO)’ system while inventory & usage of the respective media containers.
- In case for particular media, if 5 containers of same batch no. are received, Numbering of these 5 containers should be done as 1/5, 2/5 …5/5.
- In case, for a particular media 5 containers received are of two different batches, for e.g. 3 containers of batch no. A & 2 containers of batch no. B. Then containers of batch no. A should be numbered as 1/3, 2/3 & 3/3. Similarly the containers of batch no. B should be numbered as 1/2 & 2/2.
- The numbering system mentioned above is applicable to each media container received.
Storage of culture Media :
- The dehydrated culture media as well as their ingredients are highly hygroscopic and must be stored in a cool and dry place, away from bright light or as per manufacturer Instructions.
- Stock record for all culture media’s shall be prepared and maintained for reference. On receipt of the dehydrated media, a label with the relevant details like received on, opened on, container number and use before date shall be affixed on each container of dehydrated media.
- Growth promotion test should be done before taking media for ‘In use’ or simultaneously when taking media for ‘In use’.
Preparation of Media:
- Carefully read the instructions printed on the label of dehydrated culture media containers. Check the composition of the medium and the direction for its use.
- Weigh accurately the required quantity of dehydrated culture medium on a butter paper using weighing balance; transfer the powder to a suitable container (flask/tube/bottle).
- Add a part of the required quantity of purified water and mix the contents. Most of the culture media are highly soluble in water and shall dissolve immediately on mixing. Make up the desired volume with further quantity of purified water. Mix the contents thoroughly.
- Transfer the required quantity (20ml) of the media in test tube for checking the pH (before sterilization), if necessary, adjust the desired pH using either 0.1N hydrochloric acid or 0.1N sodium hydroxide for individual culture media or as per the manufacturer’s instruction.
- Affix the Media Preparation Label to the flask/tube/bottle as per Annexure-V.
- After sterilization, the pH of the broth media shall be measured at a temperature upto 25°C and for solid Media up to 40°C.
- Dispense the reconstituted medium in suitable containers like test tubes, bottles or flasks. The volume of these containers shall be sufficiently large to facilitate proper aeration and avoid overflow of medium during sterilization.
- All culture media containers shall be plugged with non adsorbent cotton wool and covered with a butter paper/aluminium foil securely tied in place with a rubber band. If screw cap bottles are used, close their caps properly. Sufficient air space shall be available inside the containers to prevent blowing of cotton plugs or breaking of the containers during sterilization.
- Some media does not require sterilization by autoclaving. Hence they shall be heated only to dissolve the dehydrated medium completely in the water. Over heating or over boiling shall be avoided while preparing these media e.g.: XLD Agar.
- In house Lot Number shall be generated to every prepared media like the abbreviation of media with load number of Media sterilization cycle and serial Alphabates start from A.
- For example, the media sterilization load number, in which Soybean casein digest medium is prepared, is 001,then In house lot number for it shall be as ‘SCDM 001X’. Here SCDM stands for Abbreviation of the media . Here 001 represents the serial number of Autoclave sterilization cycle in a year, starts from 001and shall be continued as 002,003 and so on. The sequence shall be restart from 001 at the start of every calendar year. ‘A’ stands for first prepared media of that load. If second prepared media of that load is R2A, then In house lot number for R2A shall be as ‘R2A 001B’.
- The broth medium shall be cooled to room temperature & to be used after Pre- incubation sterility check. Store the Prepared media below 25˚C. This stored media can be used upto 7 days.
- Media plates prepared, should be used after Pre-incubation sterility check. Store the plates at below 25˚C.This plates can be used upto 7 days.
- Record of Details of media preparation in Annexure-I.
- The Media Reconciliation details shall be recorded in Annexure-IV.
Sterilization of culture media:
- Culture media after preparation and dispensing as above shall be sterilized as soon as possible and shall not be kept in unsterilized conditions for more than two hours after their preparation.
- Culture media prepared and dispensed as above shall be sterilized in an autoclave by a validated cycle at 121.6°C for 20 minutes or at a temperature prescribed for individual culture media.
- Autoclave sterilization details of media shall be recorded in Annexure-II.
- Avoid excessive heating up and cooling down time. Which can results in breakdown of sugars and lowering of pH, and it can harm the nutritive value of the media. Unload the containers of culture media from the autoclave immediately on completion of sterilization cycle to prevent over exposure to heat.
Disposal/Destruction of culture Media :
- Labels of the empty containers of dehydrated media shall be defaced and the containers shall be discarded after washing.
- Since the dehydrated media are in the form of micro fined powder, dissolve the unused quantity of the powdered medium in water and sterilize it in autoclave at 121.6°C for 30 minutes. After autoclaving, shall be emptied out in the drain of washing area, under running water.
- Unused broths and agar media contained in tubes or flasks shall be directly decontaminated by autoclaving in Destruction Autoclave. After autoclaving, contents shall be poured in the drain of washing area under running water.
- The containers/flasks/ bottles then shall be rinsed with Potable water and cleaning shall be done in washing area as per procedure given.
- All the contaminated and Washed Glasswares /Accessories should be kept in dedicated/different places.
- Microbiologically contaminated media containers / Glasswares e.g. Petri dishes used for environment monitoring, water analysis, pathogen testing, positive controls, agar slants used for cultivation of microbial cultures etc. shall be first segregated and stored separately in a specified area with a prominent identification label as “CONTAMINATED GLASSWARES – TO BE AUTOCLAVED BEFORE WASHING” in a tray/container.
- Remove the marking done on petri plates by 70% IPA or methanol; remove any label stickers on flasks and bottles.
- Add 70% IPA or 2.5% Dettol or 2.5% Savlon in every contaminated petri plate, tube and flask. Give minimum 15 minutes of contact time.
- After Contact time, these containers of contaminated culture media shall be loaded in the SS Container/autoclave basket and decontaminate it by autoclaving in Destruction autoclave at 121.6°C for 30 minutes.
- After Destruction Cycle, the media shall be poured in the drain while it is molten. The drain shall be flushed continuously with water to prevent choking with agar. Containers shall be rinsed with water and the glasswares shall be cleaned as per following procedure;
- All Glasswares shall be collected in wash tub.
- During cleaning must wear protective hand gloves, nose mask and goggle.
- Glasswares shall be washed as quickly as possible after use /destruction cycle.
- Drain the contents of glassware (if present) in running water.
- Initially glasswares shall be rinsed with the running water. Soak the glasswares in 2% Soap solution (Teepol).
- During washing of the glassware’s, all the glassware’s shall be thoroughly scrubbed with nylon brush. Rinse glass wares thoroughly with the running water. Finally rinse the glassware with purified water.
- Dry the glasswares in hot air oven at 60ºC and take out after complete drying.
- For cleaning of accessories, follow the procedure as per given above.
- For destruction of disposable Petri dishes, after destruction cycle the decontaminated media shall be poured in the drain while it is molten. The drain shall be flushed continuously with water to prevent choking with agar. Then transfer molded plates to scrap yard by covered in double polythene bag.
- Record the details of Destruction cycle in Annexure-III.
IV: Annexure :
Annexure I: Microbiological Media Preparation Report
Annexure II: Media Sterilization Record of Autoclave
Annexure III: Media Destruction Record of Autoclave
Annexure IV: Media Reconciliation Record
Annexure V: Media Preparation Label
Annexure VI: Microbiological Media Stock Record
Annexure- I
Microbiological Media Preparation Report
Date of Media Preparation:——————— Analytical Balance ID: ——————–
Media valid Up to : ——————– pH Meter ID : ————————–
- A) Media Preparation details:
| Sterilization
Load No. |
Name of Media | In house Lot Number | Batch number
of media |
Volume prepared (ml) | Qty.
Weighed (gm) |
pH after
Sterilization |
Supplement |
Media Prepared By: Checked By:
Date : Date :
- B) Media Fertility Check:
| In house Media Lot Number | Incubation
Temp & Time |
Incubator
ID. |
Positive
control |
Negative
Control |
Observed By | Checked By |
Annexure-II
Media Sterilization Record of Autoclave
Equipment I.D.: _______________
| Date | Load No. | Cycle started time
|
Temp. achieved
at time (121.6ºC) |
Cycle completed at time | Hold Time
|
Item | Done By | Checked By | Sterilization
Indicator |
Annexure- III
Media Destruction Record of Autoclave
Equipment I.D.: _______________
| Date | Load No. | Cycle started time
|
Temp. achieved
at time (121.6ºC) |
Cycle completed at time | Hold Time
(30min) |
Item | Done By | Checked By | Sterilization
Indicator |
Annexure- IV
Media Reconciliation Record
Name of Media :——————————————– Code No.: ——————–
Batch No. : ————————— Date of Received :——————-
Expiry Date : —————————– Media Quantity: —————–
| Media Used on
|
Container number
|
Taken Quantity
(gm) |
Balance Quantity
(gm) |
Done By |
Annexure V
Media Preparation Label
| MEDIA PREPARATION LABLE
Name of Media :————————————- In house Lot No.:——————————— Quantity :——————————— Date of Preparation:—————————— Media Valid Up to:——————————- Media Prepared By: —————————- |
Annexure VI
Microbiological Media Stock Record
Name of Media: ——————————————————- Code No.:——————-
| Date of Received | Quantity Received | Batch Number | Expiry Date | GPT Status | Quantity
taken |
Bottle Number | Balance Stock
(Closing Stock) |
Taken By/Date | Checked By |
