Validation Protocol for Hold Time Study of Prepared Inoculum Suspension

1.0 Objective

Objective of this protocol is to provide documented evidence through the scientific data to establish and verify the stability of prepared microbial suspension upon holding for a period of time used in microbial testing.

2.0 Scope

The scope of this protocol is to evaluate the hold time of prepared inoculum suspension (CFU/ml) upon holding up to 15 days. This protocol shall also define the storage conditions effectiveness of inoculum stored at 2-8° C.

3.0 Reference Document

Following documents are referred during preparation of the protocol.

3.1  SOP for Preparation of Microbiological Culture Media

3.2  Maintenance of Microbial Cultures

3.3  SOP for Preparation of Culture Inoculum

4.0 Responsibility

Responsibilities of Quality Assurance and Quality control Microbiology department involved in activities related to the validation protocol are defined below

4.1 Quality Assurance

Review and approval of protocol

4.2 Quality Control

Preparation, review and execution of protocol

5.0 Procedure

5.1 Pre-Requisite

•  Sterilized glassware

•  Prepared inoculum suspension of Bacillus subtitles, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella enterica, Aspergillus niger, Candida albicans, Clostridium sporogenes and Enterobacter aerogenes.

5.2 Procedure For Hold Time Study

5.2.1  Subculture from preserved culture of specified organism into 10 ml soybean casein digest medium.

5.2.2  Subculture the anaerobic organism in 10 ml of cooked meat medium and incubate at the following temperature.

5.2.2.1  30-35ºC for 24 hours for aerobic bacterial culture. (Bacillus subtitles, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella enterica, Enterobacter aerogenes, Environment isolates (Bacillus pumilus).

5.2.2.2  30-35ºC for 48-72 hours for aerobic bacterial culture. (Mycobacterium smegmatis)

5.2.2.3  30-35ºC for 24-48 hours for anaerobic bacterial culture. (Clostridium sporogenes)

5.2.2.4  20-25ºC for 24-48 hours for culture of yeast. (Candida albicans)

5.2.2.5  20-25° C for 48-72 hours for culture of mold. (Aspergillus niger)

5.2.3  Assume that the broth culture has 10⁹ organisms /ml in-case of aerobic bacteria and 10⁸ organism/ml in case of anaerobic bacteria/yeast and mold.

5.2.4  Prepare serial dilution of each culture in 0.1% peptone saline to arrive at the dilution that may contain approximately 10-100 CFU/ml. Ensure that each dilution is thoroughly mixed during diluting process. 

5.2.5  Prepare the working dilution according to use. Label the dilution and store at 2-8° C.

5.2.6  Pipette 1.0 of each dilution into sterile petridishes in duplicate.

5.2.7  Perform the testing by pour plate method. Use Soybean casein digest agar for bacterial cultures and Soybean casein digest agar / Sabouraud dextrose agar for yeast and mold.

5.2.8  Incubate at the following temperature.

5.2.8.1  30-35ºC for 24 hours for aerobic bacterial culture. (Bacillus subtitles, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella enterica, Enterobacter aerogenes, Environment isolates (Bacillus pumilus).

5.2.8.2  30-35ºC for 48-72 hours for aerobic bacterial culture. (Mycobacterium smegmatis)

5.2.8.3  30-35ºC for 24-48 hours for anaerobic bacterial culture. (Clostridium sporogenes)

5.2.8.4  20-25ºC for 24-48 hours for culture of yeast. (Candida albicans)

5.2.8.5  20-25° C for 48-72 hours for culture of mold. (Aspergillus niger)

5.2.9  Calculate the viable bacterial / Yeast and mold count and calculate the volume of working dilution, which contains between 10-100 CFU.

5.2.10  Label the dilution indicating count/ml, date and store at 2-8° C. 

5.2.11  Perform the testing for working culture at initial and thereafter every 3^rd day up to 15 days storage. During the study culture shall be stored at 2-8° C up to hold time study. When holiday falls on testing day, perform the test on next working day.

6.0 Acceptance Criteria

The total viable count obtained should be ± 30 % of the actual count of the respective culture suspension.

7.0 Conclusion

After complete evaluation of the hold time study for prepared inoculum suspension a final hold time study summary report shall be prepared which should essentially contain discussion and conclusion which clearly determine the hold time period for inoculum.

8.0 Abbreviations

SOP - Standard operating procedure

° C - Degree Celsius

CFU - Colony forming units

% - Percentage

ml - Millilitre

Annexure I

Count Observation

S. No	Organisms	Count Observed (CFU/ml)
		Initial	3rd day	7th day	10th day	12th day	15th day
1	Bacillus subtitles
2	Staphylococcus aureus
3	Pseudomonas aeruginosa
4	Escherichia coli
5	Salmonella enterica
6	Enterobacter aerogenes
7	Clostridium sporogenes
8	Candida albicans
9	Aspergillus niger

Annexure-II

Percentage Recovery

S. No	Organisms	Percentage Recovery
		Initial	3rd day	7th day	10th day	12th day	15th day
1	Bacillus subtitles
2	Staphylococcus aureus
3	Pseudomonas aeruginosa
4	Escherichia coli
5	Salmonella enterica
6	Enterobacter aerogenes
7	Clostridium sporogenes
8	Candida albicans
9	Aspergillus niger

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