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Validation Protocol to Determine the Shelf Life of Prepared Microbiological Media

Learn how to validate the shelf life of prepared microbiological media used in microbiological analysis of samples in Pharmaceutical.
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 carryout 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 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 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 Test organism Observations
Medium
1 Bismuth Sulphite Salmonella Good growth, Black or green colonies.
agar species
2 Brilliant Green agar Salmonella Good growth, Small, Transparent and

may have surrounding zone of precipitated bile. 12 MacConkey agar Escherichia coli Good growth. or white ﴾frequently surrounded by a pink or red zone﴿ colonies. 7 Braid Parker agar Staphylococcus Good growth. or opaque. 3 Xylose Lysine Salmonella Good growth. 13 MacConkey broth Escherichia coli Acid and gas production. Generally greenish. well‐developed. red Deoxycholate agar species colonies with or without black centers. Black colonies agar aureus surrounded by yellow zone. Transparent and species colorless. 14 EE broth Escherichia coli Good growth with colur change 15 M‐endo Escherichia pink to dark red with a green metallic coli surface sheen 16 Giolitti Cantoni Staphylococcus Good growth broth aureus ATCC 6538 17 Cetrimide broth Pseudomonas Good growth . yellow colonies aureus surrounded by yellow zone. Small. Generally colorless to for Fluorescein aeruginosa yellowish. 8 Cetrimide agar Pseudomonas Good growth .2 Brilliant Green agar Salmonella Good growth. Blue‐black colonies Blue agar under transmitted light. Brick‐red colonies. shows yellowish fluorescence when observed under Ultraviolet light 10 Pseudomonas agar Pseudomonas Good growth. 5 Mannitol Salt agar Staphylococcus Good growth. for Pyocyanin aeruginosa shows blue fluorescence when observed under Ultraviolet light 11 Eosin Methylene Escherichia coli Good growth. Generally greenish. Generally greenish aeruginosa colouration 18 Peptone water Bacillus subtilis Good growth in the form of turbidity 19 Fluid Lactose Salmonella Good growth in the form of turbidity medium species OR Escherichia coli 20 Soyabean Casein Bacillus subtilis Good growth in the form of turbidity Digest medium OR Escherichia coli 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 Bacillus subtilis Good growth in the form of turbidity medium 23 Soyabean Casein Bacillus subtilis The total viable count obtained should Digest agar not be less than + 30 % of the actual ANNEXUERE ‐ II count of the respective culture suspension 24 Sabouraud Candida albicans The total viable count obtained should Dextrose agar OR not be less than + 30 % of the actual Aspergillus niger 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 . 4 Triple Sugar Iron Salmonella Formation of acid and gas in the stab agar species culture ﴾with or without concomitant blackening﴿ and the absence of acidity from the surface growth. with characteristic metallic sheen under reflected light. 6 Vogel ‐ Johnson Staphylococcus Good growth. Black colonies aureus surrounded by clear zone. aeruginosa shows greenish fluorescence when observed under Ultraviolet light 9 Pseudomonas agar Pseudomonas Good growth.

author and founder of Pharmaceutical Guidelines.No the Observation organism used Temp Period Medium 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: Ankur Choudhary is India's first professional pharmaceutical blogger. Sign‐up for the free email updates for your daily dose of pharmaceutical tips. suspension STORAGE CONDITION DATE: INSTRUMENT ID: Name of Inoculum Test Incubation Incubation Count obtained S. Need Help: Ask Question ◉ Offline .No the used pH Observation organism Temp Period Medium ﴾cfu /ml﴿ 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: Name of Test Inoculum Incubation Incubation S. a widely‐read pharmaceutical blog since 2008.No the used organism Temp Period ﴾cfu/ml﴿ Medium ﴾cfu /ml﴿ 1 RAA 2 SDA 3 SCA STORAGE CONDITION DATE: INSTRUMENT ID: Name of Inoculum Test Incubation Incubation S.

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