Validation Protocol to Determine the Shelf Life of Prepared Microbiological Media

1.0 INTRODUCTION

The most important thing is to ensure that various media used during any test, support microbial growth to consider the test results as valid. The ability of the nutritive media to support the microbial growth is mainly influenced by pH, physical description and water content. Thus it is essential to check that, at the time of usage these parameters are unaffected, which can be done by checking the pH and carrying out growth promotion test.

This protocol provides the procedure to determine the shelf life and consistency in pH of prepared media on storage at 20-25°C and 2-8°C. All media shall be prepared as per the SOP for media preparation and the prepared media shall be tested for growth promotion test per container on opening or when required and pH after sterilization  Representative volumes of all these media shall be then taken out at different storage intervals and tested for growth promotion capability and change in pH.

1) Initial

2) After 1 day

3) After 3 days

4) After 7 days

5) After 14 days

6) After 21 days

7) After 28 days

8) After 31 days

The maximum storage period or shelf life of all these various media shall then be determined based on the results of growth promotion test and physical appearance. Check the maximum storage period over which a medium is well capable of supporting growth of test organism and also shows no variation with respect to pH (at the end of study) shall be taken into consideration for deciding the shelf life of that particular medium.

2.0 OBJECTIVE

The objective of this study is to determine the shelf life of prepared microbiological media on storage with respect to change in pH and growth promotion test to ensure that at the time of usage the media has capability to support the microbial growth and are free from any contamination and deformation after storage in defined conditions.

3.0 SCOPE

This protocol is applicable for microbiology laboratory in quality control.

4.0 REFERENCE DOCUMENT

SOP for media preparation

5.0 RESPONSIBILITY

Microbiologist

6.0 PROCEDURE

6.1 PREPARATION OF MEDIA

6.1.1  Media shall be prepared as per the SOP for media preparation.

6.1.2  Liquid media shall be distributed in parts of 100 ml in 250 ml conical flasks / bottles.

6.1.3  Solid media shall be poured in to sterilized petridishes after sterilization.

6.1.4  All the media shall be labelled for name of the medium, date of preparation and signature.

6.1.5  Media shall be stored in an incubator maintained at 20-25 °C and 2-8°C

6.2 RECORDING OF pH

6.2.1  After sterilization of media, pH shall be recorded in the datasheet as ‘INITIAL pH’

6.2.2  On storage, at different time intervals mentioned in section 1.0, individual liquid media shall be checked for pH.

6.2.3  For each medium, pH observed at different time intervals shall be recorded in the data sheet.

6.2.4  Solid media should be checked for pH initially only. 

6.3 GROWTH PROMOTION TEST

6.3.1  After preparation and sterilization of all the media, immediately carry out growth promotion test on all of them as per the SOP for growth promotion. Record the results in datasheet.

6.3.2  On storage, at different time intervals as mentioned in section 1.0, individual media shall be checked for growth promotion.

6.3.3  Record the results of growth promotion test in datasheet for each medium.

6.4 CHECKING FOR PHYSICAL APPEARANCE AND CONTAMINATION

6.4.1  Check the solid agar media visually for dryness.

Check the solid agar media as well as liquid media visually for any deformation such as a change in color sedimentation, precipitation, for microbial contamination etc.

7.0 ACCEPTANCE CRITERIA

The maximum storage period over which a medium comply the following criteria shall be taken into consideration for deciding the shelf life of the particular medium.

A)  pH may not vary from the given range of pH in Annexure - II

B)  Growth promotion test shall comply when done initially as well as during the particular storage period.

i)  Liquid media: Should show growth in the form of turbidity. If liquid medium is opaque, then after incubation in this medium further carryout streaking on selective agar media, where the characteristic growth shall be observed as Annexure – I.

ii)  General /Enrichment agar media: The total viable count obtained should not be less than + 30 % of the actual count of the respective culture suspension.

iii)  Selective agar media : Should show characteristic growth comparable to as described in

Annexure –I.

C)  The Solid agar media should not get dry. The liquid, as well as solid agar media, should not show any deformation and contamination. 

Related: Maintenance of Microbial Cultures

ANNEXURE – I
Sr. No	Name of the Medium	Test organism	Observations
1	Bismuth Sulphite agar	Salmonella species	Good growth, Black or green colonies.
2	Brilliant Green agar	Salmonella species	Good growth, Small, Transparent and colorless, or opaque, or white (frequently surrounded by a pink or red zone) colonies.
3	Xylose Lysine Deoxycholate agar	Salmonella species	Good growth, well-developed, red colonies with or without black centers.
4	Triple Sugar Iron agar	Salmonella species	Formation of acid and gas in the stab culture (with or without concomitant blackening) and the absence of acidity from the surface growth.
5	Mannitol Salt agar	Staphylococcus aureus	Good growth, yellow colonies surrounded by yellow zone.
6	Vogel - Johnson agar	Staphylococcus aureus	Good growth, Black colonies surrounded by yellow zone.
7	Braid Parker agar	Staphylococcus aureus	Good growth, Black colonies surrounded by clear zone.
8	Cetrimide agar	Pseudomonas aeruginosa	Good growth, Generally greenish, shows greenish fluorescence when observed under Ultraviolet light
9	Pseudomonas agar for  Fluorescein	Pseudomonas aeruginosa	Good growth, Generally colorless to yellowish, shows yellowish fluorescence when observed under Ultraviolet light
10	Pseudomonas agar for  Pyocyanin	Pseudomonas aeruginosa	Good growth, Generally greenish, shows blue fluorescence when observed under Ultraviolet light
11	Eosin Methylene Blue agar	Escherichia coli	Good growth, Blue-black colonies under transmitted light; with characteristic metallic sheen under reflected light.
12	MacConkey agar	Escherichia coli	Good growth, Brick-red colonies; may have a surrounding zone of precipitated bile.
13	MacConkey broth	Escherichia coli	Acid and gas production.
14	EE broth	Escherichia coli	Good growth with colour change
15	M-endo	Escherichia coli	pink to dark red with a green metallic surface sheen
16	Giolitti Cantoni broth	Staphylococcus aureus ATCC 6538	Good growth
17	Cetrimide broth	Pseudomonas aeruginosa	Good growth, Generally greenish coloration
18	Peptone water	Bacillus subtilis	Good growth in the form of turbidity
19	Fluid Lactose medium	Salmonella  species OR Escherichia coli	Good growth in the form of turbidity
20	Soyabean Casein Digest medium	Bacillus subtilis       OR Escherichia coli	Good growth in the form of turbidity
21	R2A agar	Bacillus subtilis	The total viable count obtained should not be less than + 30 % of the actual count of the respective culture suspension
22	Fluid Thioglycolate medium	Bacillus subtilis	Good growth in the form of turbidity
23	Soyabean Casein Digest agar	Bacillus subtilis	The total viable count obtained should not be less than + 30 % of the actual count of the respective culture suspension
24	Sabouraud Dextrose agar	Candida albicans OR Aspergillus niger	The total viable count obtained should not be less than + 30 % of the actual count of the respective culture suspension
25	Nutrient agar	Bacillus subtilis	The total viable count obtained should not be less than + 30 % of the actual count of the respective culture suspension
26	Plate Count agar	Bacillus subtilis	The total viable count obtained should not be less than + 30 % of the actual count of the respective culture suspension

                                                                    ANNEXUERE - II

STORAGE CONDITION
Date:                                                    Instrument ID:
S.No	Name of the Medium	Test organism	Inoculum used (cfu /ml)	Incubation Temp	Incubation Period	Count obtained (cfu/ml)
1	RAA
2	SDA
3	SCA
STORAGE CONDITION
Date:                                                    Instrument ID:
S.No	Name of the Medium	Test organism	Inoculum used (cfu /ml)	Incubation Temp	Incubation Period	pH	Observation
1	SCM
2	FTG
3	CMM
4	FLM
5	TTB
6	MCB
7	CTB
8	SCB
9	GCB
10	EEB
11	RFM
12	BSP

STORAGE CONDITION
Date:                                                    Instrument ID:
S.No	Name of the Medium	Test organism	Inoculum used	Incubation Temp	Incubation Period	Observation
1	MEA
2	TSI
3	VRB
4	CBA
5	BPA
6	EMB
7	MSA
8	VJA
9	PAP
10	PAF
11	CTA
12	MCA
13	BGA
14	XLD
15	BSA

Done By:                                                                                                Checked By:

Date:                                                                                                       Date:

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