ValidationProtocoltoDeterminetheShelfLifeofPreparedMicrobiologicalMedia

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 2025C and 28C. 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 2025 C and 28C
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
2 Brilliant Green agar Salmonella Good growth, Small, Transparent and
species colorless, or opaque, or white
frequently surrounded by a pink or
red zone colonies.
3 Xylose Lysine Salmonella Good growth, welldeveloped, red
Deoxycholate agar species colonies with or without black centers.
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.

5 Mannitol Salt agar Staphylococcus Good growth, yellow colonies

aureus surrounded by yellow zone.

6 Vogel Johnson Staphylococcus Good growth, Black colonies

agar aureus surrounded by yellow zone.

7 Braid Parker agar Staphylococcus Good growth, Black colonies

aureus surrounded by clear zone.
8 Cetrimide agar Pseudomonas Good growth , Generally greenish,
aeruginosa shows greenish fluorescence when
observed under Ultraviolet light
9 Pseudomonas agar Pseudomonas Good growth, Generally colorless to
for Fluorescein aeruginosa yellowish, shows yellowish
fluorescence when observed under
Ultraviolet light
10 Pseudomonas agar Pseudomonas Good growth, Generally greenish,
for Pyocyanin aeruginosa shows blue fluorescence when
observed under Ultraviolet light
11 Eosin Methylene Escherichia coli Good growth, Blueblack colonies
Blue agar under transmitted light; with
characteristic metallic sheen under
reflected light.
12 MacConkey agar Escherichia coli Good growth, Brickred colonies; may
have surrounding zone of precipitated
bile.
13 MacConkey broth Escherichia coli Acid and gas production.
14 EE broth Escherichia coli Good growth with colur change
15 Mendo 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 , 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
 suspension
STORAGE CONDITION
DATE: INSTRUMENT ID:
Name of Inoculum
Test Incubation Incubation Count obtained
S.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.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.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:

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