Inoculum hold time study in pharmaceutical Microbiology Laboratory

1.0 Introduction:

In routine practice inoculums are prepared once in a week. In this case the inoculums used later on by keeping at 2-8°C. But whether the inoculums viability is affected by storage at the 2-8°C in the refrigerator needs to be monitored. So it is necessary to evaluate the quality of the inoculums during hold time, with respect to the Total Viable count. In routine practices we are not getting the count which is specified in the pharmacopeia i.e. NMT 100 CFU. So to achieve the same with aliquots of the original inoculums it needs to be checked for the proportionate count.

2.0 Objective:

2.1 The objective of this exercise is to assure that the quality of the inoculums is not differ by factor 2 even after the storage at the 2-8°C for 15 days hold time from the date of the preparation of Inoculum.2.2 To establish the data for usage by storage of the inoculums even after the preparation of Inoculum.2.3 To establish the effectiveness of inoculums during storage used for the test.2.4 To establish the data for aliquots of the dilution of the culture, gives the proportionate count.

3.0 Scope:

This protocol is applicable for the study of the storage hold time of inoculums at 2-8°C.

4.0 Responsibility:
4.1 Quality Control:-To carry out the study.
-To prepare, review and to monitor execution as per validation protocol.
-To Prepare report.

4.2 Quality Assurance:-To review and approve validation protocol as per cGMP concept.
-To monitor execution activity as per Protocol.

5.0 Pre-requisites for Hold time study validation:

5.1 Qualified analyst / Trained analyst5.2 Availability of working cultures & reference cultures.5.3 Qualified instruments, Media, Calibrated glasswares.5.4 Standard Operating Procedures:5.4.1 SOP for Growth Promotion Test of Microbiological media5.4.2 SOP for receiving, storage and preparation of microbial culture media5.4.3 SOP for Culture Dilution Preparation5.5 List of cultures requirements:

Sr. No.	Name of Culture	Strain No.
1	Escherichia coli	ATCC 8739
2	Salmonella abony	NCTC 6017
3	Pseudomonas aeruginosa	ATCC 9027
4	Staphylococcus aureus	ATCC 6538
5	Candida albicans	ATCC10231
6	Bacillus subtilis	ATCC 6633
7	Aspergillus niger	ATCC16404

6.0 Qualified instruments:1. Incubators2. Refrigerator3. Laminar air flow system4. Autoclave5. Weighing Balance6. pH meter7. Colony counter8. Water Bath

7.0 Media:

1. Soyabean Casein Digest agar2. Sabouraud Dextrose Agar3. Buffered Sodium Chloride Peptone water4. 0.9 % saline water

8.0 Methodology:Counting validity: Prepare and the Standardize inoculums as per the SOP for Culture Dilution Preparation.  After incubation record the observations and checked for viable count. Select the dilution which shows countable count (30-300 CFU/ml). Keep the inoculums at 2-8°C and standardize the inoculums after 5, 9, 15, 18 days and Record the observation.

Validity with respect to the proportionate count: Take selected dilution of the 15 day old culture which shows countable count (30-300 CFU/ml). Standardized that dilution by using the aliquots of 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml and 1.2ml and record the observation.
Find out % recovery by using following Formula:
% recovery = Obtain Count    X 100                              Initial Count

Acceptance criteria:

Total viable count: Count should not differ by factor 2 (50-200%) with comparison to initial count. Proportionate count: The aliquots of the dilution of the culture should give the proportionate count which should not differ by factor 2 (50-200%) of the proportionate initial count.

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