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Standard Operating Procedure (SOP) and Guideline for the Receipt, Storage, Preparation, Growth Promotion Test, use, and Disposal of microbiological media.

Microbiological Media Guideline

1.0   Purpose:

• • To define the procedure for the receipt, storage, preparation, growth promotion test, use, and disposal of Microbiological Media used during microbiological analysis.

2.0   Scope:

• • This guideline is applicable to microbiological media used by the microbiology laboratory at drug substance manufacturing and formulation plants.

3.0   Reference,  and Attachments:

• • References :

• • IP, general chapter- 2.9 — Microbial contamination in non-sterile products.

• • Guideline on Aseptic Technique

• • USP, general chapter <61> Microbiological examination of non-sterile products: microbial enumeration

• • Ph Eur, general chapter 2.6.12-Microbiological examination of nonsterile products: Microbial enumeration

• • TGA guidance on sterility

• • Attachments :

• • Microbiological Media stock (Attachment-1)

• • List of Microbiological Media with storage condition and pH (Attachment-2)

• • Microbiological Media preparation, sterilization and GPT Record (Attachment-3)

• • List of Microbiological Media, organisms, and incubation conditions for GTP (Attachment-4)

• • Microbiological Media disposal (Attachment-5)

4.0   Definition of terms & Abbreviations:

• • Growth promotion test (GPT): Also referred to as fertility or nutritive properties test, which is performed on the Microbiological Media used during different tests like sterility test, microbial limit test, preservative efficacy test, etc to demonstrate that it is capable of supporting the growth of micro-organisms

• • Colony-forming unit (CFU): Visible outcome of the growth of micro-organisms arising from a single or multiple

• • GPT: Growth Promotion Test

• • ATCC: American type culture collection

• • R2A: Reasoner’s 2A

• • RODAC: Replicate organism detection and counting

• • lbs: The traditional symbol stands for libra, the Latin word for the unit-

• • CFU: Colony-forming unit

• • NTCC: National collection type culture

5.0   Procedure – Microbiological Media Management :

• • Receipt of Microbiological Media:

• • On the receipt of the Microbiological Media, check for the expiry date of the media and mention the date of receipt on the label of the media container.

• • The analyst shall mention the date of opening with a sign-on label while opening the container.

• • After receipt of the Microbiological Media, make necessary entries in media stock record as per Attachment-1.

• • After opening the Microbiological Media container, the media shall be used up to shelf life defined on the media container.

• • Use culture media as per the supplier’s recommendation.

• • Storage of dehydrated Microbiological Media:

• • The dehydrated media are highly hygroscopic and must be stored as per the supplier’s storage condition mentioned on the label.

• • The temperature of the microbiological media storage room/facility shall be monitored through a min/max thermometer and the limit shall be followed as per the media storage requirement.

• • List of media having storage condition and pH limit shall be prepared as per Attachment-2 and shall be displayed near media storage facilities.

• • Microbiological Media preparation:

• • Dissolve the specified amount as mentioned on the label of dehydrated Microbiological Media in the required volume of distilled water (water for injection) or purified water as mentioned on the label of dehydrated media container.

• • Add 1% w/v of glycerol AR grade in all the agar Microbiological Media before sterilization to avoid drying of media during exposure/incubation.

• • Plug the mouth of the conical flask containing dissolved media in case of agar medium with non-absorbent cotton plugs or steri-caps and wrap with butter paper.

• • After adding the dehydrated Microbiological Media in the purified water/water for injection/distilled water, it should be sterilized within 1 hr.

• • For slant preparation, distribute about 7 ml of molten media in each tube (20 mm x 150 mm) and plug it. Note: Before sterilization, ensure that agar is molten /disso1ved and mixed properly which otherwise shall affect the solidification process.

• • For the liquid Microbiological Media preparation, appropriate volume shall be dispensed either in the test tube or in the conical flask as required.

• • Tube/flask mouth shall be closed using sterile-caps or plastic caps / stainless steel caps/cotton.

• • • pH Check of Microbiological Media:

    • Check the pH of the medium before sterilization with a calibrated pH meter.

• • • If required adjust the pH with the help of 1N or 0.1N HCI or NaOH solutions.

• • • Record pH check/adjustment details in the Microbiological Media preparation record.

• • • Transfer 10-15 ml of dissolved media in an autoclavable test tube & label with media lot number and purpose (for pH check).

• • • This shall be subjected to sterilization and shall be used for pH check after sterilization.

• • Microbiological Media sterilization & Pouring:

• • Sterilize the media by autoclaving at 121°C, 15 pressure for 20 mins, or as per the media preparation method described by the media supplier on the media container.

• • For example, where the instruction is given “DO NOT AUTOCLAVE”, keep in free-flowing steam or in boiling water bath for a time as specified on individual media container.

• • After sterilization, check the pH of the medium (contained in a separate test tube) with calibrated pH While pH check.

• • Ensure that the pH meter electrode dips into the medium.

• • Make entries in the Microbiological Media preparation record as per attachment-3.

• • For agar plates, mix the sterile medium ensuring the absence of bubble When it reaches to about 45-50°C temperature (to avoid the formation of water droplets on the lids of Petri plates) pour approximately 15-20 ml sterile agar media in sterile Petri plates under LAF wearing a mask & sterilized or sanitized hand gloves. Allow solidifying.

• • The molten agar medium should be held in a monitored water bath at a temperature of 45°C to 50° C & a standard time shall be derived to know that approx what time one has to keep the media in a water bath after transferring the same from autoclave to water bath.

• • For slants, keep the tubes in a slanting position to cool in the dedicated test tube stand.

• • Follow the below procedure for the RODAC plate Preparation:

    • Wear sterile gloves and a nose mask before working under the laminar airflow unit.

• • • Disinfect the external surface of the plastic bag of the pre-sterilized empty sterile RODAC plates in laminar airflow. Allow the bag to air dry.

• • • Open the plastic bag and takeout the empty sterile RODAC plate avoiding contact with the inner side of the plates.

• • • Place them in not more than two rows with a distance of approx two inches between each plate and in each row under laminar airflow.

• • • Pour the prepared medium gently into each RODAC plate till the formation of a raised convex surface.

• • • Approx 14-15m1 of medium shall be required for a RODAC plate having a 55 mm diameter.

• • • Keep the lid of the RODAC plate open till the Microbiological Media gets solidified.

• • • After solidification, cover the lid on to the RODAC plate and label.

• • Microbiological Media lot numbering and labeling:

• • ln the media preparation,steri1ization & GPT record ( attachment -3), allot Microbiological Media lot number against provision given for entry of lot no, using prefix M (stands for media), serial number (001) and year (20 for 2020).

• • This means the first lot number of the media prepared in the year 2020 shall be M/001/20. Continuous, unique lot number shall be given to all media irrespective of the type/name of media.

• • Media label shall contain media lot number as well as the date of media

• • Each and every unit (tube/Petri plate) of the medium shall be labeled with media lot no.

• • Preincubation of Microbiological Media:

• • After the Microbiological Media is solidified/cooled container containing media plates/tubes shall be identified with the status label as “Under Pre-incubation”.

• • Preincubation for the media to be used for environmental monitoring shall be at 30-35°C for a minimum of 24 hrs. incubation time.

• • After completion of pre-incubation, and examination of Microbiological Media (Plates shall be observed visually for any microbial growth, if detected such plates shall be removed ) the media shall be labeled as “Ready for use”.

• • To ensure 24 hrs preincubation time, while usage of microbiological media, preincubation start date & time as well as date & time of use of the media shall be recorded in the respective test template.

• • The time interval between pre-incubation start and usage shall not be less than 24 hrs.

• • Storage of prepared Microbiological Media:

• • Prepared media shall be stored at room temperature after pre-incubation.

• • Growth promotion test (GPT) of Microbiological Media:

• • The growth indicative and inhibitory properties of any media shall be established on three different lots of the same vendor.

• • The growth promotion test shall be performed for each lot of prepared (sterilized) medium.

• • This test is applicable to the media listed in attachment-4.

• • Cultures to be used and incubation conditions to be followed are also given in Attachment-4.

• • Incubate at the specified temperature for not more than the shortest period specified in the test.

• • Prepare the medium as per the manufacturer’s instruction and separately inoculate <100viable microorganisms in the test container of the medium under test.

• • For liquid media inoculate the broth with less than 100 CFU of selected dilution of the respective Microbial Organism

• • For agar media, inoculate the media with less than 100 CFU of selected dilution of the respective organism by pour plate method.

• • If more than one organism is required for GPT of a particular medium, one plate/tube shall be inoculated for each of the organisms.

• • In the case of ready-made prepared Microbiological Media are used, a certificate required to be taken from the supplier to ensure that growth promotion test is done at the manufacturer’s end as per the pharmacopoeial requirement for a lot of media.

• • Growth promotion tests for each lot shall be performed at the user end also.

• • House organisms (environment and water isolates) should also be used for GPT of media used in water testing, environment monitoring, swab testing, etc.

• • Incubate the plates or tubes as per conditions given in attachment-4.

The growth promotion test of the solid medium complies if,

• • • Visible growth is observed after the incubation period and,

• • • Recovery of growth obtained must not differ by a factor greater than 2 from the calculated value of standardized inoculum used.

• • • For media that is used for the preparation of RODAC, Recovery of growth shall be performed on normal 9 cm diameter Petri dishes.

• • The growth promotion test of the liquid medium complies if, clearly visible growth of the microorganisms comparable to that previously obtained with a previously tested and approved batch data of medium occurs

• • Instruction for Usage of Microbiological Media:

• • Before use carefully examine the plates for contamination, uneven filling of bubbles on the surface of the agar, color changes, and cracks due to loss of Discard such defective plates or tubes.

• • Discard such defective plates or tubes.

• • Recommendation of BP/USP for use of sterilized media is,

• • • “If prepared media are stored in unsealed containers, they can be used for l month, provided that they are tested for GPT within two weeks of the time of use and that color indicator requirements are met”.

• • • Means media of the particular lot shall be used within 15 days of Remaining media shall be discarded if not used within 15 days.

• • • If required to be used beyond 15 days, GPT shall be performed again.

• • • In case GPT found satisfactory & color indicator requirement of media is met, it shall be used for further 15 days.

• • • If the remaining media not used within 30 days, shall be destroyed.

• • It is mentioned in USP and BP that Microbiological Media used to comply with the sterility and GPT carried out before or in parallel with the test on the product to be examined.

• • Hence plated can be used after pre-incubation.

• • Any failure in GPT shall be investigated and (if required) the tests shall be repeated in which media failing in GPT is used.

• • Disposal of used Microbiological Media:

• • Mention the items to be disposed of (used media/biological indicator/culture suspension etc) in the media disposal record.

• • Take out the media for disposal to the washing room.

• • Prepare l liter of 5 % v/v Dettol in SS bucket with lid.

• • Wear a nose mask and hand gloves.

• • Transfer all the used media and cultures into the SS bucket of sanitization solution and cover the bucket with a lid.

• • If Microbiological Media is in test tubes as agar slants, then add the above sanitization solution to cover the surface of media and keep it in the test tube stand.

• • Keep the test tube stand in the autoclave.

• • Drop a deodorizing pearl into the bucket, close the lid, and place it in the autoclave.

• • Run a sterilization cycle at 121°C for 30 minutes as per the instrument operating procedure.

• • Transfer the content of the tubes into the SS bucket having autoclaved

• • • Allow the medium to cool and dispose of the medium in the effluent treatment plant.

• • • Wash the empty container under running tap water.

• • • Record the details in the Microbiological Media disposal record as per Attachment-5.

• • Microbiological Media reconciliation:

• • Media reconciliation shall be done for each lot of the prepared dehydrated medium.

• • For a reconciliation of the dehydrated medium, the limit shall be 98-101% (Refer attachment -l).

Attachments  – Microbiological Media:

Attachment-1: Microbiological Media stock

Name of Media:	Code no :
Lot no :	Expiry date :
Quantity & container received :	Date of receipt :
Manufacturer’s name :	Reconciliation (98-101%)

 

Date	Quantity Withdrawn	Balance Quantity	Sign

Attachment-2: List of Microbiological Media with storage condition and pH 

Medium	Storage condition recommended by the supplier	pH Limit

Attachment-3: Microbiological Media preparation, sterilization and GPT Record 

Activity						Prepared by & date
Name of the media:____________________
Manufacturer’s lot/batch no: and expiry/use before date:________
In House Lot No. _________________________
Weighed _________g of dehydrated media. Balance no . _______Calibration due on .__________ Suspended in _______ml distilled water (water for injection) or purified water, A. R. No. __________. Heated to dissolve.
pH of the medium before sterilization: ______________ Qty of 1N/O.IN NaOH /HC1 used for pH adjustment. : _________ Lot no. of NaOH/ HC1: : ______________ pH after pH adjustment: : ______________ pH meter no: : _______Calibration valid upto :__________ Add 1% w/v glycerol AR grade in all agar media before sterilization Glycerol AR lot no: ___________ validity: ___________ Qty of glycerol added ; : _________gm. in________ ml of media.
Sterilized by autoclaving / boiling at______ °c for ______mins
Sterilization run no: _________. Autoclave no:____________
pH of the medium after sterilization:_____________
Final distribution _________Plates, __________Conical flask, _________Tubes /Slants, ________Rodac plates.
Growth promotion test:
Culture used	Dilution of culture used	The concentration of culture suspension (Acceptance criteria-<100cfu/m0	Culture suspension preparation record no.	Result	Acceptance criteria** ( cfii/m1 to cfuhnl)
Acceptance criteria-Recovery for solid media: Growth must not differ by a factor greater than 2 from the inoculum used. Acceptance criteria for GPT of liquid media: Clearly visible growth of the microorganisms occurs comparable to that previously obtained with a previously tested and approved batch data of medium.
Media preparation date :
Media use before date :
Leftover /expired media destroyed on date________ by ._______

 Attachment-4: List of Microbiological Media, organisms, and incubation conditions for GTP

Media	Test in which used	Organism	Incubation condition		Observe for
Fluid Thioglycoll ate Medium	Sterility	Cl. sporogenous ATCC 11437 or 19404 Ps. aeruginosa ATCC 9027, ATCC6633 S. aureus ATCC d538	30-35°C for NMT 3 days.		Clearly visible growth of the microorganisms comparable to that previously obtained with a previously tested and approved batch of medium occurs.
Soybean Casein Digest Medium (broth)	Sterility	Asp.niger ATCC 16404, B.subtilis ATCC 6633, C.albicans ATCC 10231.	20-25 °C, NMT 3days for bacteria, and 5 days for fungi.		Same FTGM.	as	mentioned	under
Soybean Casein Digest broth	MLT	B.s«BFI/is ATCC 6633 S. aureus ATCC 6538, Ps. aeruginosa ATCC 9027	30-35 NMT days bacteria.	°C, 3 for	Same FTGM.	as	mentioned	under
Fluid Lactose Medium	MLT(RM/FP/water)	E. coli ATCC 8739	36-38°C for 18  to     24 hrs.		Same FTGM.	as	mentioned	under
Tetrathionate Bile Brilliant Green broth	MLT (RM/FP/water)	S. ‹t6ony NCTC 6017	36-38°C for 48lx		Further confirmation shall be done using BSA/BGA/XLDA/DCA

Attachment-5: Microbiological Media disposal 

Date	Autoclave run no.	Sterilization Cycle hold time Start time	Sterilization Cycle hold time End time	Items destroyed	Disposed of by	Checked by

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