Receipt, Preparation, Storage, Quality Testing, Usage and Disposal of Microbiological Media

Procurement, Receipt and Storage of Dehydrated Microbiological Media:

Procure commercially available dehydrated media or ready to use media from qualified manufacturers. If the required dehydrated media or ready to use media is not available from first supplier, then procured from secondary qualified supplier. If media is not available with any of these manufacturers, prepare media using individual ingredients as per the composition given in respective pharmacopeia.

Upon receipt of the dehydrated media or ready to use media, check the physical conditions of each media container. If any abnormality observed in any container inform to team leads and take appropriate action.

Verify the COA (Certificate of analysis) of received media lot for its quality confirmation. If COA is not available with received media lot then download the COA from manufacturer’s website. Sign and date shall be done after verification of COA,

Record the details of received dehydrated media or ready to use media in respective log.

Label each received dehydrated media container or ready to use media container in suitable format i.e.

Media Receipt Label

Date of Receipt

Lot No of Media

Sr. No. of Container

Received By

Fill the details in label date of receipt, lot number of media, serial number of container and received by sign and date. Where serial number of the container apply the container number as X/Y, where X is serial no. of container and Y is total number of containers received. For example if 3 containers are received, the individual container shall be labelled as 1/3, 2/3 and 3/3.

Store the dehydrated media or ready to use media as per manufacturer’s storage instructions.

Approval of Dehydrated Microbiological Media and Ready to Use Media:

Perform the quality testing such as Growth promotion, inhibitory test and test for indicative properties on the first container of same lot as per point QUALITY TESTING to confirm the quality of received media.

If received media is tested first time for quality testing then compare the results with COA. Subsequence quality testing shall be compared with previously tested and approved batch/lot  of media. Record the result in respective log.

If received media meet the acceptance criteria mentioned in the point QUALITY TESTING, affix respective  for approval

Use the media on First Expire First Out or First In First Out (FEFO/FIFO) basis whichever applicable.

Expiry and Retest of Dehydrated Microbiological Media and Ready to Use Media:

After the satisfactory results of quality testing of media lot/batch, affix an “Approval Label” and assign the retesting date and expiry date.

If the manufacturer's expiry date is below one year, then consider manufacturer’s expiry date as an expiry date of media lot/batch and no resting is required. If the manufacturer's Expiry is more than one year,

Assign manufacturer’s Expiry date as an expiry date of media lot/batch.

Assign retesting date as one year from the date of approval of particular lot/batch of media.

Retesting of media lot/batch shall be performed at a frequency of once in year from the date of approval of particular lot/batch of media.

Retesting shall be performed as per point number QUALITY TESTING and record the result in respective log.

Preparation of Media:

Issue the approved dehydrated media container or ready to use media and check the expiry date and retesting date on the container. Do not use the media, if media found expired or not re-tested. Make the issuance entry of media on respective log.  

If a new bottle of the medium is taken for use, enter the date of opening on the container. Record the details of issued dehydrated media in respective log for further consumption of issued dehydrated media.

Open the bottle and check the physical appearance of the medium..

Calculate the required quantity of the dehydrated media as per manufacturer’s instructions and weigh the required quantity of dehydrated media on weighing balance as per SOP. Record the issued quantity in respective log.  

Use appropriate container for media weighing depending upon media quantity. Mark the container with media name, lot number and date of preparation.

After use, immediately close the dehydrated media container and tighten the lid. If the media is exhausted in the container than the cross mark the container with marker and discard the container in waste bin.

Slowly transfer weighed media in container containing Water for Injection (WFI) / purified water and mix thoroughly. Make up the final volume by adding purified water from the sidewalls of the container.

Heat the media if required to dissolve completely using a hot plate or water bath with agitation.

Take care to avoid excessive heating of the media.

Media for settle plate exposure and Cetrimide Agar shall be supplemented with 10 mL glycerol per litter of media before sterilization to avoid dehydration during exposure.

Preparation of thermo labile media using boiling method. DO NOT sterilize them.

Take portion of media in clean and dry glass beaker; allow media to cool down up to room temperature and check the pH as per SOP. pH shall comply as per manufacturer instruction. If require adjust the pH with 0.1 N NaOH or 0.1 N HCl.

Distribution of Media:

Dispense the dissolved media as desired i.e., in tubes (plastic cap or screw cap), screw cap flasks, screw cap bottles, filled volume NMT 75 % (3/4) volume of container capacity. If sterility testing media prepared in vial fill volume is 100 ml in 100 ml vial.

Close the bottles and test tubes with plastic caps or screw caps according to the containers used and seal the vials.

Label the each container and stand as per below label

Prepared Media Status Label¹

PREPARED MEDIA STATUS
Name of the Media
Lot No.
Date of Preparation
Use Before
Sign and Date

Individual labelling shall be done as per below label for Plates, Test tube and Vials.

Prepared Media Status Label²

Media
Lot number
Use Before

Lot Numbering of Prepared Media and Ready to use media:

Lot numbering of Ready to use media shall be followed as per assign by manufacturer.

For prepared media assign the lot number as per following:

ZZ-DDMMYY-XX

Where, ZZ is media code/abbreviation i.e. define inhouse (media code digits may fall between 2 to 5 digits). DD stand for date in two digits,

MM stands for month in two digits,

YY stand for last two digits of year, and

XX stand for sequence number of same media prepared in same day.

For example: Lot number of first lot of Mac Conkey Agar prepared on 01/04/16 shall be assign as MA010416-01, if same media prepared second time in same day the lot number shall be assign as MA010416-02 and so on. 

Record details of media preparation in respective log.

Sterilization of Media:

Affix a sterilization indicator on each container of media.

Load the prepared media containers in the autoclave as per validated load pattern.

Sterilize the media as per SOP on sterilization of media using validated cycle.

Unload the media from unloading zone after sterilization.

Transfer the media in incubator room through mobile trolley for pre-incubation through dynamic pass box.

Transfer sterilized media for plate preparation in media plate preparation room. Maintain the temperature of agar media at 45 to 50^°C by keeping in water bath to avoid solidification of agar-based media prior to plate preparation. The molten agar medium should be held at 45° to 50°C for not more than 08 hours.

Check the pH of the sterilized media after sterilization at 20°C to 25°C temperature.

If the pH of the sterile media is not within the specified limits, discard the media and record in respective log.

If an ingredient has to be added after sterilization of the media, allow the media to cool at appropriate temperature recommended by manufacturer instruction and add the ingredient to the sterilized media under aseptic conditions.

Re-melting of an original container of solid media should be performed only once to avoid media whose quality is compromised by overheating or potential contamination.

Expiration of Media:

Expiration of media shall be assigned as per media Expiration qualification protocol.

Preparation of Plates (90 mm):

Take pre-sterilized disposable 90 mm petri-dishes and remove outer polythene cover outside the LAF and discard in the waste bin and remove inner cover under LAF and arrange the plates on the LAF bench. Aseptically pour the approximately 20 mL to 25 mL of sterile media in petri-dishes.

Allow the media to solidify under LAF. Keep all the solidified plates in a tray or in appropriate container and label individual plates (as above¹).

Preparation of Contact Plates:

Take pre-sterilized disposable 55 mm petri plate and remove outer polythene cover outside the LAF and discard in the waste bin and remove inner cover under LAF and arrange the plates on the LAF bench. Aseptically pour approximately 15mL or quantity sufficient in RODAC plate to yield raised surface or proper convex surface of media.

Allow the media to solidify under LAF. Keep all the solidified plates in a tray and label the tray (as above¹) and label individual plates (as above²).

Preparation of Slants

For slant preparation dispense approximately 10 ml media in 20ml glass test tube.

After sterilization of the slant tubes, keep the tube in slanted position for slant preparation.

After solidifying, keep all slants in a container and label individual Slant (as above²).

After completion of PRE INCUBATION Store the media slants at 2° to 8°C and use within expiry period.

Media quality Testing:

Physical observation: Dehydrated media, Ready to use media and Prepared media should be checked by appropriate inspection of plates and tubes for the following;

Table I

Physical properties of different types of media

Media Type	Dehydrated Media	Ready to Use Media	Prepared Media
Criteria	Cracked containers or lids.	Cracked containers or lids.	Cracked containers or lids
	Clump formation.	Unequal filling of containers.	Unequal filling of containers.
	Lot number and expiration date checked and recorded.	Dehydration resulting in cracks or dimpled surfaces on solid medium.	Dehydration resulting in cracks or dimpled surfaces on solid medium.
	Excessive darkening or colour change.	Crystal formation from possible freezing.	Excessive darkening or colour change.
	-	Microbial contamination/ sterility of media	Excessive number of bubbles.
	-	Lot number and expiration date checked and recorded.	Cleanliness of plates (lid should not stick to dish).
	-	Bubbles formation in solid media plates	Lot number and expiration date checked and recorded.
	-	Unequal distribution of media in plates	Microbial contamination/ sterility of media
			Unequal distribution of media in plates

Pre-incubate all the media at 30°C to 35°C for not less than 48 hours.

Perform Growth promotion, Inhibitory test and Indicative properties of media after completion of pre-incubation period.

For Growth Promoting and Indicative Properties of Solid Media and Liquid Media:

For solid media perform surface spread method by inoculating not more than 100 cfu/0.1ml in duplicate plate as per required test strain given in Table II.

For liquid media inoculate not more than 100 cfu/0.1 ml in portion /container of media in duplicate as per required test strain given in Table II.

Incubate all the test media at specified temperature for not more than the shortest period of time as per Table II.

For Inhibitory Properties of Liquid and Solid Media:

Inoculate a portion of appropriate medium/container with at least 100 cfu of test strain in duplicate given as per Table II.

Incubate at the specified temperature for not less than the longest period of time as per Table II.

Negative Control:

A negative control should be performed in duplicate for each medium during growth promotion test and it should be incubate for minimum 5 days.

Table II

Growth Promoting, Indicative and Inhibitory Property of Media and Incubation Condition

Test/Medium	Property	Test Strain	Incubation Condition
Soyabean Casein Digest Agar (SCDA) / Tryptone Soya Agar with Lecithin and Tween 80 (TSA)	Growth Promoting	S. aureus ATCC 6538	30 to 35ºC for ≤ 3 days
		B. subtilis ATCC 6633
		P. aeruginosa ATCC 9027
		Environment Isolates
		C. albicans ATCC 10231	30 to 35ºC for ≤ 5 days
		A. brasiliensis ATCC 16404
Soyabean Casein Digest Medium (SCDM) For Microbial enumeration Test	Growth Promoting	S. aureus ATCC 6538	30 to 35ºC for ≤ 3 days
		B. subtilis ATCC 6633
		P. aeruginosa ATCC 9027
		Environment Isolates
Soyabean Casein Digest Medium (SCDM) For Sterility Testing	Growth Promoting	B. subtilis ATCC 6633	20 to 25ºC for < 3 days
		Environment Isolates
		A. brasiliensis ATCC 16404	20 to 25ºC for < 5 days
		C. albicans ATCC 10231
Fluid Thioglycollate Medium (FTM)	Growth Promoting	Cl.sporogens ATCC 19404	30 to 35ºC for < 3 days
		S. aureus ATCC 6538
		P. aeruginosa ATCC 9027
		Environment Isolates
R2A	Growth Promoting	B. subtilis ATCC 6633	30 to 35ºC for ≤ 3 days
		P. aeruginosa ATCC 9027
		Water Isolates, if applicable
Enterobacteria Enrichment Medium	Growth Promoting	E. coli ATCC 8739	30 to 35ºC for 24 - 48 hrs
		P. aeruginosa ATCC 9027
	Inhibitory	S. aureus ATCC 6538
Violet Red Bile Glucose Agar (VRBGA)	Growth promoting + indicative	E. coli ATCC 8739	30 to 35ºC for 18 - 24 hrs
		P. aeruginosa ATCC 9027
MacConkey Broth	Growth Promoting	E. coli ATCC 8739	42 to 44°C for 24 - 48 hrs
	Inhibitory	S. aureus ATCC 6538
MacConkey Agar	Growth promoting + indicative	E. coli ATCC 8739	30 to 35°C for 18 - 72 hrs
Rappaport Vassiliadis Salmonella Enrichment Broth (RVSEB)	Growth promoting	S. abony NCTC 6017	30 to 35ºC for 18 - 24 hrs
	Inhibitory	S. aureus ATCC 6538
Xylose Lysine Deoxycholate Agar (XLDA)	Growth promoting + indicative	S. abony NCTC 6017	30 to 35°C for 18 - 48 hrs
Cetrimide Agar (CA)	Growth promoting	P. aeruginosa ATCC 9027	30 to 35°C for 18 - 72 hrs
	Inhibitory	E. coli ATCC 8739
Mannitol Salt Agar (MSA)	Growth promoting + indicative	S. aureus ATCC 6538	30 to 35°C for 18 - 72 hrs
	Inhibitory	E. coli ATCC 8739
Reinforced Clostridium Medium (RCM)	Growth promoting	Cl.sporogens ATCC19404	30 to 35°C for 48 hrs
Columbia Agar (CLA)	Growth promoting	Cl.sporogens ATCC19404	30 to 35°C for 18 - 72 hrs (Anaerobic Condition )
Sabouraud Dextrose Broth (SDB)	Growth promoting	C. albicans ATCC 10231	30 to 35°C for 24 - 48 hrs
		A. brasiliensis ATCC 16404
Sabouraud Dextrose Agar (SDA)	Growth promoting + indicative	C. albicans ATCC 10231	20 to 25°C for ≤ 5 days and 30 to 35°C for 3 - 5 days.
		A. brasiliensis ATCC 16404
Sabouraud Chloramphenicol Agar(SCA)	Growth promoting + indicative	C. albicans ATCC 10231	20 to25°C for ≤ 5 days and 30 to 35°C for 3 - 5 days
		A. brasiliensis ATCC 16404

Table III

Indicative Reactions on Media

Test/Medium	Test Strain	Indicative property
Violet Red Bile Glucose Agar (VRBGA)	E. coli ATCC 8739	Pinkish red colour colonies should be observed.
	P. aeruginosa ATCC 9027	Colourless colony
MacConkey Agar	E. coli ATCC 8739	Brick red colonies; may have surrounding zone of precipitated bile
Xylose Lysine Deoxycholate Agar (XLDA)	S. abony NCTC 6017	Well developed, red colonies with or without black centers
Mannitol Salt Agar (MSA)	S. aureus ATCC 6538	Yellow or white colony surrounded by yellow zone
Sabouraud Dextrose Agar (SDA)	C. albicans ATCC 10231	White color colonies should be observed.
Sabouraud Chloramphenicol Agar (SCA)	C. albicans ATCC 10231	White color colonies should be observed.

Record the results of Growth promotion, inhibitory test and indicative properties of media in respective log.

If ready to use media are used perform physical verification (as per Table I), pH measurement, and growth promotion test on each lot. Record the details in respective log.

Validity of The Test and Acceptance Criteria:

There must no growth in the negative controls.

For solid media, growth obtained must not differ by a factor greater than 2 from the calculated value for a standardized inoculum for growth promotion test (i.e. 0.5 to 2).

In case of liquid media, the media passes growth promotion test, if visible growth appears within the specified incubation time and temperature.

There should no growth in the media in case of inhibitory test.

In case of indicative property, results should be complies as per Table III.

Record the result in respective log.

Rationale for Use of In-house Isolates for GPT (If applicable):

Use In-house isolates from the list of predominantly found organisms.

Minimum of two defined in-house isolates shall be used in growth promotion test and validation activities on rotation basis.

Decontamination and Disposal

Collect all the expire media, Rejected media and used media in autoclavable disposal bag and tie it with cable tie. Decontaminate autoclavable bags containing media by autoclaving it as per respective validated procedure ,

Actions To Be Taken In Case of Any Abnormalities:

If any criteria mentioned above does not meet, then investigate as per respective procedure and decide the acceptance of particular impacted media lot.

If abnormality as shown in table IV but not limited to, is observed during procurement, receipt, preparation, storage, growth promotion, Usage and disposal of microbiological media take appropriate action mentioned in the below table.

Table IV

Abnormality	What to do / what not to do
Spillage of media	-          Mop the area with wet mop and collect in disposal bag. -          Spray filtered 70% IPA/ validated disinfectant and mop with dry mop. -          Discard the media as per Disposal SOP.
Breakage of glassware	-          Wear appropriate personnel protective equipment (PPE’s). -          Collect all the pieces in a plastic bag. -          Handover plastic bag to the site scrap yard.
Injury due to breakage of Glassware	-          Exit the area if working in controlled area. -          Wash the wound with sufficient clean water. Clean the wound with antiseptic solution. Apply antibiotic ointment / powder and bandage. If the wound is severe, report to the first aid centre immediately.

Recommended Media code:

Media Name	Media Code
Cetrimide Agar	CA
Columbia Agar	CLA
Enterobacteriaceae Enrichment Broth	EEB
Fluid Thioglycollate Medium	FTM
MacConkey Agar	MA
MacConkey Broth	MB
Mannitol Salt Agar	MSA
Rappaport Vassiliadis Salmonella Enrichment Broth	RVSEB
R2A agar	R2A
Sabouraud Chloramphenicol Agar	SCA
Sabouraud Dextrose Agar	SDA
Sabouraud Dextrose Broth	SDB
Soyabean Casein Digest Agar	SCDA
Soyabean Casein Digest Agar with  polysorbate 80	SCDAT
Soyabean Casein Digest Medium	SCDM
Soyabean Casein Digest Medium with polysorbate 80	SCDMT
Tryptone Soya Agar with Lecithin and polysorbate 80	TSA
Violet Red Bile Glucose Agar	VRBGA
Xylose Lysine Deoxycholate Agar	XLDA
Reinforced Clostridium Agar	RCA
Reinforced Clostridium Medium	RCM
0.1% w/v Peptone water ( Fluid A)	PW
0.1% w/v peptone water with 0.1%v/v polysorbate 80 ( Fluid D)	PWT
Phosphate Buffer	PB
0.9%w/v Normal Saline	NS
0.9%w/v Normal Saline with 0.1%v/v polysorbate 80	NST
Sterile water	SW

Log Recommended:

Media Receipt Details

Media Receipt and Issuance Record

Media Approved Label

Media in-house Code

Media Consumption Record

Media Quality Test Report

Media Preparation Record

REFERENCE:

Reference

<61> Microbiological examination of non-sterile products: Microbial Enumeration Test

<62> Microbiological examination of non-sterile products: Test for Specified Microorganisms

<71> Sterility Test

<1117> Best Microbiological Laboratory Practices

Vol. 8 European Pharmacopeia.

ABBREVIATIONS:

Abbreviation Used	Full Form of Abbreviation
ATCC	American Type Culture Center
cfu	Colony Forming Unit
°C	Degree Celsius
COA	Certificate of Analysis
EP	European Pharmacopoeia
GPT	Growth promotion test
HCl	Hydrochloric Acid
hrs	Hours
LAF	Laminar Air Flow
mL	Millilitre
NaOH	Sodium Hydroxide
NCTC	National Collection Type Culture
No.	Number
RODAC	Replicate Organism for detection and Count
SOP	Standard Operating Procedure
QA	Quality Assurance
QC	Quality Control
WFI	Water For Injection

DEFINITATION:

Microbiological media:

Microbiological media is a nutritive substance in form of liquid or gel designed to support the growth of microorganisms.

Growth promotion test:

Growth Promotion Test determines the suitability of culture media to ensure the quality, functionality and nutritive properties of the media before using it for analysis. The media is challenged with a small number of microorganisms to ensure the nutritive properties.

Sterilization:

             Sterilization is a process to eliminate (removes) all forms of live microorganisms from an                        object or surface by treating with high heat, radiation or chemical.

Precautions:

To the extent possible, procure the dehydrated media based on regular consumption in such a way that the media will not last for more than one year.

Wear nose mask and hand gloves during media preparation.

Care shall be taken while handling pathogenic microorganisms.