procedure for Handling of Medias to be followed for Receiving Preparation Growth promotion test Storage Destruction of media

1.0       Purpose          :                       

To lay down the procedure for Handling of Medias to be followed for Receiving, Preparation, Growth promotion test, Storage, Destruction of media.

 Scope:

This procedure is applicable for receiving, preparation, growth promotion test, storage and destruction of every lot / batch of media and also for destruction and disposal of positive controls, media, viable culture suspensions, non-contaminated media, and containers used to handle the media (includes all glass ware).

3.0       Responsibility:

The responsibility of receiving, preparation, growth promotion test, storage, destruction and disposal of media and positive controls, viable culture suspensions, non-contaminated media, and containers lies with Microbiologist / Analyst.

4.0       Definitions / Abbreviations:

Media: A substance used to provide nutrients for the growth and multiplication of microbes.

5.0       Procedure:

5.1       Receiving of media:

5.1.1    Dehydrated media shall be procured as per requirement from the suitable vendors.

5.1.2 On receipt of media, check for use before date, Batch No. / Lot No. of each and every bottle,and confirm the usage period for its validity.

5.1.3 Enter the received quantity of media in the inventory log book and fix the label on each bottle.

5.2   Preparation:

5.2.1       Ensure that the media is not deteriorated.

5.2.2       Growth promotion test passed media shall be used in regular analysis.

5.2.3       Media should be prepared as per manufacturer instructions.

5.2.4       All the media should be prepared in clean and dry container.

5.2.5       Weigh the media using a calibrated balance as per requirement / manufacturer instructions and add part of the required amount of purified water and swirl to dissolve. Gradually add the remaining water from the sides of the container.

5.2.6       If the media is not dissolving, heat the same by using hot plate or water bath; take care to avoid excessive heating.

5.2.7       Check the pH of the prepared media and it should comply with label requirements. If not, adjust the pH of the media by adding 0.1N Hydrochloric acid or 0.1N Sodium Hydroxide before sterilization.

5.2.8       Distribute the prepared media into suitable flasks / tubes / vessels and close with cotton/aluminum foil / metal caps. Load the flasks/tubes/vessels into the autoclave along with one Sterilabel / Signaloc indicator. Sterilize at 121 ºc at 15 lbs pressure for 15 minutes or  115 ºc at 15 lbs pressure for 15 minutes or as per manufacturer instructions.

5.2.9    After sterilization, unload the contents of the autoclave and agar media shall be placed on the water bath until it used.

5.2.10  Check the pH of the sterilized prepared media and it should comply with label requirements.If not complies prepare fresh media.

5.2.11     For preparation of agar plates, take sterile petriplates which have been sterilized previously by dry heat at 181ºC for 2 hours or use disposable sterile petri dishes, add 15-20mL or sufficient amount of media in to sterile Petri dishes previously has been melted and cool to approximately 45°C under LAF.

5.2.12     Preparation of pre-incubated agar plates by above mentioned step 5.2.11 and kept for incubation at 30 to 35 °C or 20 to 25°C for 18 to 48 hours in incubator as per the requirement.  These pre-incubated plates can be used for membrane filter method / spread plate / bioload.

5.2.13     Media should be prepared according to number of samples, on that day and should be consume within the same day and if incase any left over, should be discarded according to destruction procedure of media.

5.2.14  The above details shall be recorded in a Media preparation log book Form xxxxx   PRE001 for each media separately.  Media Reference number shall be assigned as below:                                   

            Example: Media abbreviation XXX                                   

            Where as,

Media abbreviation stands for the abbreviation of particular media mentioned in Mediapreparation method sheet.

XXX stands - Sequential number (001, 002,003….)

                                    For Example: xxxxx (for Soyabean casein digest medium)

5.2.15     Update the details in a Media preparation method sheet form xxxxx, when ever medias procured from new manufacturer or if any new media procured.

5.3       Sterilization of media:

5.3.1    All media shall be prepared in clean and dry container by reconstituting the dehydrated mediawith purified water.

Distribute the prepared media into suitable conical flasks or tubes or containers as per requirements and closewith cotton / metal caps. Load the conical flasks or tubes or container into the horizontal autoclave along with one Sterilabel / Signaloc indicator label.

5.3.3    Operate the Horizontal Autoclave as per the operating procedure.

5.3.4    Sterilize at 121 ºc at 15 lbs pressure for 15 minutes or 115 ºc at 15 lbs pressure for 15 minutesor as per manufacturers instructions.

5.3.5    fter sterilization, unload the contents of the autoclave.

5.3.6    Failure of horizontal autoclave could be due to either non-attainment of required temperatureor failure of the total functional system. Sterilize the media in the vertical autoclave at 121°c/15 lbs for 15 minutes or 115 ºc at 15 lbs pressure for 15 minutes or as per manufacturers

instructions. Before media sterilization in vertical autoclave it shall be sterilized by running empty chamber cycle at 121 ºc at 15 lbs pressure for 30 minutes.

5.4       Growth Promotion Test:

5.4.1    All prepared media shall be checked for its growth promotion characters before using for             

microbiological purpose. GPT shall be performed for one bottle from each batch and same

results can be considered for next same batch.

5.4.2    If same batch media received in two consignments, initial consignment GPT results can be consider.

5.4.3    Prepare and sterilize the media as per the procedure of preparation and sterilization of media.

5.4.4    Use viable culture suspensions  or Certified Bioball cultures or Ready made cultures which having 10-100 CFU of  S.aureus, E.coli, P.aeruginosa, S.abony, B.Subtilis, C.albicans

and Cl.sporogenes or other new cultures.

5.4.5       For broth (liquid) media, add 1or as per requirement mL of particular viable culture suspension or Certified Bioball cultures or Ready made cultures of 10-100 CFU (S. aureus/ E. coli / P. aeruginosa / B. subtilis / S. abony / C.albicans / Cl.  Sporogenes or other new cultures) in 100mL sterile medium and incubate as per table in step No.5.4.8. The medium shall be turbid then the broth is suitable for in regular use. Simultaneously keep one tube negative without any suspension added it should not show any turbidity.

5.4.6       For solid media, add 1or as per requirement mL of particular viable culture suspension or Certified Bioball cultures or Ready made cultures of 10-100 CFU (S. aureus / E. coli / P. aeruginosa / B. subtilis / S. abony / C.albicans / Cl.  Sporogenes or other new cultures) in sterile petri -plates then add 15-20 mL of melted media and allowed to solidify. Incubate the plates as per table in step No.5.4.8. The media shows growth of colonies which shall be + / - 2 factor of the particular suspension (dilution) used. Then the media is suitable for in regular use. Simultaneously keep one negative plate without adding any suspension, it should not show any growth of colonies.

5.4.7    Use viable suspension culture or Certified Bioball cultures or Ready made cultures for growth promotion test as per annexure-I. Annexure –I can be updated / modified if there is a change in ATCC No. or If new media added or any other changes.

5.4.8    Incubate them accordingly as shown below against each organism.

5.4.9    Acceptance criteria:

For Broth: It should show turbidity.

For Agar: It shows growth of colonies of about +/- 2 factor of the particular suspension used.

5.4.10     A logbook shall be maintained for Growth promotion test, Form No. FM004/MBLPRE001 and enter the details in the columns Name of the media, Batch No.,Date of test, Date of report, Growth promotion test reference No., sign.  Assigning the growth promotion test reference No. ALL/GPT/XXX/MMYY.

Where as ALL stands for Astrix Laboratories Ltd.   

GPT stands for Growth promotion test. 

XXX stands for Serial No.starts from 001,002.upto end of the December each                                                                      year 

 MM stands for Month, Ex.01…..12

YY stands for Year, Ex.2011 write last two digits ‘11’

5.4.11  Record the results in the report format xxxxx.

5.4.12     Opened container which is GPT passed should be used with in six months and if any left over is there discard the left over.

5.4.13     Unopened GPT passed bottles can be used for 1 year.

5.4.14     After 1 year of unopened GPT passed bottles can be used after Re conform growth Promotional test. For re conformation of GPT follow growth promotion test procedure.

5.4.15     When ever media used in analysis purpose perform the positive control test of the particular sterilized media with specific microorganism as per GPT procedure from 5.4.4 to 5.4.6. Positive control is nothing but Growth promotion test. For positive control test use any one of the specific microorganism for that specific media.

5.5 Storage:

5.5.1    The media, which has been passed for the Growth Promotion Test (GPT), shall be stored as per the manufacturer’s instructions, i.e. cool, dry place and protect from light, put the label

 indicating ‘Growth Promotion Test Passed’  Form xxxxx on every bottle

 of a lot / batch.

5.5.2 In case growth promotion test fails, all the containers of the lot / batch shall be rejected and

returned to supplier or destructed as per destruction procedure. If the same batch received in next consignment, the batch shall be rejected without performing GPT.

5.5.3    Any media showing signs of deterioration (caking/discoloration) shall be discarded.                  

5.5.4    Use the media within the expiry date indicated on the bottle label / manufacture instructions.

5.6       Destruction:

5.6.1    Wear nose mask and gloves in both the hands.

5.6.2    Ensure that destruction and disposal is done carefully and avoid the cross contamination of area and other containers.

5.6.3    Collect the agar plates, broth tubes, positive controls, culture tubes, viable culture suspensions and media bottles. Place them in a separate tub provided near the washing area and put the label ‘Items to be destructed’ on the tub.

5.6.4       Destruct the above collected items by placing them in vertical autoclave at 121 ºC/15 lbs for 30 minutes, allow the load to cool and transfer solid media into a disposable bag and liquid media into a vessel containing disinfectant solution (4%Dettol/ 4%Savlon/ Other disinfectant) and close the respective bag and vessel, send them to ETP / out side agency.     

5.6.5    Record the details in the Destruction and disposal of media log book, Form xxxxxx

5.6.6    All the used glassware shall be put into a tray containing disinfectant solution (4%Dettol / 4%Savlon / other disinfectant), leave them for 10 minutes, take out and wash thoroughly with soap solution followed by water.

5.6.7    If there is any spillage of used media, decontaminate with 70% IPA solution and wipe with tissue paper followed by disinfectant solution and discard the tissue paper safely.

5.6.8    Frequency: Destruction of used media shall be done within 48 hours after usage / as per requirement.

6.0       Related Documents:

6.1       Procedure for Sub culturing and preparation of Viable culture suspension from Pure cultures(SOPxxxxx)

6.2       Procedure for operation, calibration and temperature mapping of horizontal and vertical Autoclave.( SOPxxxxx)

Hydrochloric acid, 1n sodium hydroxide, microbiology,