Validation Protocol to Determine the Shelf Life of Prepared Microbiological Media _ Pharmaceutical Guidelines

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 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 sterilization Representative volumes 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 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 respect to pH at the end of study 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 microbiological media on media  on storage with respect to change in pH and growth promotion test to ensure that at the time of usage the media has ha s capability 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 DOCUMENT

SOP for media media preparation preparation 5.0 RESPONSIBILITY

Microbiologist 6.0 PROCEDURE 6.1 PREPARATION OF MEDIA

6.1.1 6.1.2 6.1.3 6.1.4 6.1.5

Media shall be prepared as per the SOP for media preparation. preparation. Liquid media shall be distributed distributed in parts of 100 ml in 250 ml conical flasks / bottles. Solid media shall be poured in to sterilized petridishes petridishes after sterilization. All the media shall be labelled for name of the medium, date of preparation and signature. Media shall be stored in an incubator maintained at 20‐25 °C and 2‐8°C

6.2 RECORDING OF pH

6.2.1 6.2.2 6.2.3 6.2.4

After sterilization of media, pH shall be recorded recorded in the datasheet as ‘INITIAL pH’ On storage, at different time intervals mentioned in section 1.0, individual individual liquid media shall be checked for pH. pH. For each medium, pH observed at different different time intervals shall be recorded in the data sheet. Solid media should be checked for pH initially only.

6.3 GROWTH PROMOTION PROMOTION TEST

6.3.1 After preparation and sterilization 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 individual media shall be checked for growth promotion. promotion. 6.3.3 Record the results results of growth promotion test promotion test in datasheet for each medium. 6.4 CHECKING FOR PHYSICAL APPEARANCE AND CONTAMINATION CONTAMINATION

6.4.1 Check the solid agar media visually for dryness. dryness. Check the solid agar media as well as liquid media visually for any deformation deformation such as change in color sedimentation, precipitation, 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 : 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

Sr. No Name of the Medium 1 Bismuth Sulphite agar 2 Brilliant Green a ar

ANNEXURE – I Test Test org orga anis nism Observ serva atio tions Salmonella species Salmonella

Good growth, Black or green colonies. Good Good

rowth rowth Small Small Trans Trans arent arent and

species

colorless, or opaque, or white frequently surrounded by a pink or red zone colonies. Good growth, well‐developed, red colonies with or without black centers. Formation of acid and gas in the stab culture with or without concomitant blackening and the absence of acidity from the surface growth.

Xylose Lysine Deoxycholate agar Triple Sugar Iron agar

Salmonella species Salmonella species

5

Mannitol Salt agar

Staphylococcus aureus

Good growth, yellow surrounded by yellow zone.

colonies

6

Vogel ‐ Johnson Staphylococcus agar aureus

Good growth, Black surrounded by yellow zone.

colonies

7

Braid Parker agar

8

Cetrimide agar  

9

Pseudomonas agar Pseudomonas for Fluorescein aeruginosa

10

Pseudomonas agar Pseudomonas for Pyocyanin aeruginosa

11

Eosin Methylene Blue agar

Escherichia coli

12

MacConkey agar

Escherichia coli

13 14 15

MacConkey broth EE broth M‐endo

16

Giolitti Cantoni broth

17

Cetrimide broth

18

Peptone water Fluid Lactose medium

Escherichia coli Escherichia coli Escherichia coli Staphylococcus aureus ATCC 6538 Pseudomonas aeruginosa Bacillus subtilis Salmonella species OR Escherichia coli Bacillus subtilis OR Escherichia coli Bacillus subtilis

3 4

19

Staphylococcus aureus Pseudomonas aeruginosa

20

Soyabean Casein Digest medium

21

R2A agar

22

Fluid Thioglycolate medium

Bacillus subtilis

23

Soyabean Casein Digest agar

Bacillus subtilis

24

Sabouraud Dextrose agar

Candida albicans OR  Aspergillus niger 

25

Nutrient agar

Bacillus subtilis

26

Plate Count agar

Bacillus subtilis

Good growth, Black colonies surrounded by clear zone. Good growth , Generally greenish, shows greenish fluorescence when observed under Ultraviolet light Good growth, Generally colorless to yellowish, shows yellowish fluorescence when observed under Ultraviolet light Good growth, Generally greenish, shows blue fluorescence when observed under Ultraviolet light Good growth, Blue‐black colonies under transmitted light; with characteristic metallic sheen under reflected light. Good growth, Brick‐red colonies; may have surrounding zone of precipitated bile. Acid and gas production. Good growth with colur change pink to dark red with a green metallic surface sheen Good growth

Good growth , Generally greenish colouration Good growth in the form of turbidity Good growth in the form of turbidity

Good growth in the form of turbidity

The total viable count obtained should not be less than + 30 % of the actual count of the respective culture suspension Good growth in the form of turbidity

The total viable count obtained should not be less than + 30 % of the actual count of the respective culture suspension The total viable count obtained should not be less than + 30 % of the actual count of the respective culture suspension The total viable count obtained should not be less than + 30 % of the actual count of the respective culture suspension The total viable count obtained should not be less than + 30 % of the actual count of the respective culture

ANNEXUERE ‐ II

STORAGE CONDITION DATE: S.No

Name of  Test the organism Medium

1

RAA

2

SDA

3

SCA

INSTRUMENT ID: Inoculum Incubation used Temp cfu /ml

Incubation Period

Count obtained cfu/ml

STORAGE CONDITION DATE:

INSTRUMENT ID:

S.No

Name of  the Medium

1

SCM

2

FTG

3

CMM

4

FLM

5

TTB

6

MCB

7

CTB

8

SCB

9

GCB

10

EEB

11

RFM

12

BSP

Test organism

Inoculum used cfu /ml

Incubation Temp

Incubation Period

pH

Observation

STORAGE CONDITION DATE:

INSTRUMENT ID:

S.No

Name of  the 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

Test organism

Inoculum used

Done By:

Checked By:

Date:

Date:

Incubation Temp

Incubation Period

Observation

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