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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 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 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
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 colouration
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:
Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
Email: .moc.enilediugamrahp@ofni Need Help: Ask Question


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