Hold time study protocol for sterilized media

HOLD TIME STUDY PROTOCOL FOR STERILIZED MEDIA

S. No Description
1.0 Protocol Approval
2.0 Objective
3.0 Purpose and Scope
4.0 Responsibility
5.0 Qualification Team
6.0 Training
7.0 Methodology
8.0 Acceptance Criteria
9.0 Qualification report
10.0 Approval of qualification report
11.0 Re-qualification
12.0 Discrepancy And Corrective Action Report
13.0 Abbreviation

1.0 PROTOCOL APPROVAL
This is a specific protocol for the validation of storage of sterilized media [Solid & Liquid] which shall be stored in the Microbiology laboratory.
Media are used for the product analysis testing and environment monitoring. The media are sterilized in the validated Steam Heat Sterilizer. After the sterilization process, they are unloaded in the aseptic area. The media are stored at room temperature in microbiology laboratory. The validation study consists of sterilization of media, holding them for a specified length of time after sterilization. The media testing at different time intervals for check the fertility & sterility level. The purpose of this study is to verify and establish the hold time for sterilized media before their actual usage.

2.0 OBJECTIVE
The Objective is to perform the validation of storage of sterilized media [Liquid & Solid] which shall be stored in the Microbiology laboratory.
3.0 PURPOSE AND SCOPE
The purpose of this activity is to evaluate the sterilized media storage [Solid & Liquid] in microbiology laboratory.
The scope is limited to evaluate the sterilized media storage [Solid & Liquid] in microbiology laboratory.
4.0 RESPONSIBILITIES
Department Responsibility
Microbiology Execution of Protocol & Report.
Recording of observation, collection of data and filling of report.
Microbiologist shall be responsible for performing the validation activity.
Engineering & Utility To ensure that all the required utilities are working as per respective SOPs.
To ensure that the related instruments and equipment’s are in calibrated and validated status.
To ensure that environmental conditions of all areas met the requirements.
Quality Control Review of the Validation report

Quality Assurance Verification of protocol completeness, accuracy and suitability for application.
Scheduling and conducting of validation runs.
Validation data compilation and review.
Review of the Validation report, documents and its compliance to meet the acceptance criteria of the protocol.
Identification of Executors: All the identified executors involved with this Protocol are to Record Name, Designation, Signature and Date.

5.0 QUALIFICATION TEAM

Officer /Executive – Microbiology

Head – Microbiology

Quality Assurance – Officer /Executive

6.0 TRAINING:
All concerned personnel shall be given training on validation of hold time study of media after sterilization. All necessary aspects related validation procedure shall be explained in this training programme. The training record shall be attached in Annexure
7.0 METHODOLOGY:
7.1 Sterilization of media:
7.1.1 Prepared the media as per SOP on “Preparation of media plates, slant and tube for microbiological Analysis”.
7.1.2 Sterilized the media as per SOP on “Operation Cleaning and Maintenance of Autoclave” for study of Hold Time for Media.
7.1.3 Unload in aseptic area media complete of sterilization cycle.
7.1.4 Preparation of Liquid (Broth) test medium
7.1.4.1 The SCDM, FTGM, MCB and RVSE medium shall be prepared as per the current SOP on “Preparation of media plates, slant and tube for microbiological Analysis” as per the required quantities for the validation purpose in 100 & 10 mL tube.
7.1.4.2 The necessary entries for the prepared media shall be documented in the respective media preparation records.
7.1.4.3 The medium shall be prepared and dispensed in 100 mL tubes as per the “Preparation of media plates, slants and tubes for microbiological analysis”.
7.1.4.4 The sterilized medium shall be unloaded and shall be stored in the aseptic area at 20-25oC for the validation study.
7.1.4.5 The sterilized medium shall be stored at 20-25oC for the maximum time period of 24 days and the time intervals for the validation study shall be of the following listed down time periods.

Day 0.
Day 03.
Day 06.
Day 09.
Day 12.
Day 15.
Day 18.
Day 21.
Day 24.

7.1.5 Preparation of Solid test media
7.1.5.1 The SCDA, SDA/SCA and Selective media shall be prepared as per the current SOP on “Preparation of media plates, slant and tube for microbiological Analysis” as per the required quantities for the validation purpose.
7.1.5.2 The necessary entries for the prepared media shall be documented in the respective media preparation records.
7.1.5.3 The medium shall be prepared and dispensed in 20-25 mL as per the “Preparation of media plates, slants and tubes for microbiological analysis”.
7.1.5.4 The media plate shall be stored in the aseptic area at 20-25oC for the validation study.
7.1.5.5 The sterilized medium shall be stored at 20-25oC for the maximum time period of 25 days and the time intervals for the validation study shall be of the following listed down time periods.

Day 0.
Day 03.
Day 06.
Day 09.
Day 12.

7.2 List of cultures for the challenge study.
4.1.1 The cultures as listed in Table-I shall be selected as the challenge organisms for the validation study.
All the eight organisms shall be selected for the validation study.
S. No. Name of Culture ATCC

S. No.	Name of Culture	ATCC
1	Staphylococcus aureus	ATCC 6538
2	Pseudomonas aeruginosa	ATCC 9027
3	Bacillus subtilis	ATCC 6633
4	Clostridium sporogenes	ATCC 11437
5	Escherichia colli	ATCC 8739
6	Salmonella	ATCC 6633
7	Candida albicans	ATCC 10231
8	Aspergillus brasiliensis	ATCC 16404

7.3 Preparation of challenge inoculum for performing the validation study [Inoculum of 10-100CFU/mL].
7.3.1 The cultures as listed in Table I shall be quantified for the inoculum for the countable range of 10-100CFU/mL as per the approved SOP on “Inoculum preparation” and shall be used within a period of 10± 1 day.
7.4 Test Procedure
7.4.1 The GPT shall be performed as per the current SOP on “Growth promotion, inhibition and indicative properties”.
7.4.2 The test organisms selected shall be of all organisms as per listed in Table-I.
7.4.3 GPT shall be performed for the sterilized & stored medium vials for the specified time intervals as per above mentioned.
7.4.4 The quantified culture suspension from the refrigerator shall be taken out and shall be allowed to acclimatize at room temperature.
7.4.5 The test performed as per explain culture with specific media in SOP of growth promotion, inhibition and indicative properties.
7.4.6 Take the media tube and performed the Growth promotion test as per growth promotion, inhibition and indicative properties.
7.4.7 The test performed under laminar air flow and shall be inoculated through sterilized micropipette tips using a calibrated micropipette of 10 – 100 CFU/ mL of each medium of the organism listed in Table –I.
7.4.8 The activity shall be performed in an aseptic condition in the LAF station of the microbial limit test room of the controlled area of the Microbiology laboratory.
7.4.9 The inoculated FTGM tube shall be incubated at 30 –35o C for a maximum period of 72 hrs. in case of bacteria.
7.4.10 The inoculated SCDM tube shall be incubated and at 20 –25o C for a maximum period of 72 hrs. in case of bacteria and 120 hrs. in case of Fungus & Yeast.
7.4.11 The inoculated MCB tube shall be incubate at 42-44oC for a maximum period of 24 hrs. inhibition for 48 hrs.
7.4.12 As a negative control shall be kept one tube of sterilized media incubate parallel of test.
7.4.13 The test repeats above mention time respectively.
7.4.14 Microbiologist shall observe the incubated tube for the growth in the form of turbidity.
7.4.15 The observed results shall be documented in the format and shall be attached as Annexure-II.
7.4.16 For solid media shall be performed growth promotion test as per respective sop of growth promotion, inhibition and indicative properties as per above mention time interval.
7.5 Procedure for pH & Physical condition
7.5.1 The pH and visual observation for colour shall be performed before the sterilization of the medium once.
7.5.2 The pH and physical condition for colour shall be performed after sterilization during the specified time intervals of the medium to notice any change in color and pH as record of annexure –III.
7.5.3 The pH testing & physical observation for colour shall be performed in the media preparation room of the Microbiology laboratory.
7.5.4 The observed results shall be documented in the format and shall be attached as Annexure-III.
8.0 ACCEPTANCE CRITERIA

The sterilized media sample at various time intervals shall show the growth after incubation at the specified temperature & time.
For solid media the growth obtained must not differ by a factor of 2 from the calculated value of a standardized inoculum.
The sterilized solid media at various time intervals shall show the growth obtained must not differ by a factor of 2 from the calculated value of a standardized inoculum during after incubation at the specified temperature & time.

9.0 QUALIFICATION REPORT

The validation report shall consist of a summary document, which shall describe the activities carried out during the validation exercise. The validation report shall also detail the results of the validation exercise and the inference drawn from it.
The investigation shall be carried out if any deviations from the established limits as per the protocol. The proper investigation shall be carried out to find out the root cause. Based on the root cause the corrective actions shall be recommended.
Any deviation observed during the Hold Time Study shall be reported and final decision shall be taken by Head-QA.
All the data shall be attached to the validation report as Annexure

10.0 APPROVAL OF QUALIFICATION REPORT
10.1 The report shall be evaluated and proper references / conclusions / recommendations shall be recorded by quality assurance.
10.2 The media monitoring report shall be evaluated and finally approved by quality assurance.
11.0 RE-QUALIFICATION:
11.1 Re-qualification of all individuals shall take place once every two years.
12.0 DISCREPANCY AND CORRECTIVE ACTION REPORT
12.1 Document any discrepancies observed during the personnel qualification record. Include the corrective actions of the same. When all the discrepancies are satisfactorily resolved or an approved action plan is developed which ensures that the discrepancy will be resolved.
13.0 ABBREVATION
Abbreviation Definitions
HTS Hold Time Study
SOP Standard Operating Procedure
QA Quality Assurance
QC Quality Control
NA Not Applicable
No. Number
% Percentage
°C Degree Centigrade
Cfu Colony Forming Unit
SCDA Soyabean Casein Digest Agar
MLT Microbial Limit Test
cm Centimetre
LAF Laminar Air Flow

Name of Media						Date of Preparation
Media Sterilization Lot No.						Date of Sterilization
Hold time started on						Interval time
Incubator ID No.						Tested by
Date of testing						Date of Observation
Name of Cultures		Standardized Inoculum		Observation					Percentage %	Remark
				Tube/Plate-I			Tube/Plate-II







Inhibitory Properties
Name of Culture			Standardized Inoculum	Observation					Percentage %	Remark
				Plate/Tube-I			Plate/Tube-II

Detail of Culture Suspension
Name of Culture	Date of Preparation				Valid upto			Report No.		Remark







Ø The growth obtained must not differ by a factor of 2 from the calculated value of a standardized inoculum. Ø In case of broth medium there should be visible growth appears within the specified incubation time and temperature. Remarks:The above hold time mediacomplies / does not comply as per the In house Specification. Observed By Checked By Approved By Date: Date: Date: