Steam Sterilizer – Qualification & Validation Procedure

Standard Operating Procedure (SOP) for procedure for Validation / Qualification of Steam Sterilizer used in microbiology laboratories.

Validation of Steam Sterilizer

1.0   Purpose:

    • To lay down the procedure for Validation of Steam Sterilizers.

2.0   Scope:

    • This Standard Operating Procedure is applicable at microbiology section of Quality Control department.

3.0   References & Annexures:
steam sterilizer

    • References:
    • Health Technical Memorandum (HTM) 2010
    • Annexures:
    • Validation Report of steam sterilizer by Biological Indicator (Annexure- 1).
    • Result of Bowie-Dick Type test Kit (Annexure- 2)
    • Validation Status Label (Annexure- 3)

4.0   Responsibilities:

    • Microbiologist / Executive – Quality control.
    • Responsible for following the Procedure for Validation of Steam Sterilizers as per this SOP.
    • Quality Control (QC) Head.
    • Ensure proper control and compliance of the SOP.
    • Quality Assurance (QA) Department:
    • To review the SOP.
    • To ensure the implementation of SOP.
    • Regulatory Affairs, Quality Head and Plant Head :
    • To review and approve new or revised SOPs.

5.0   Procedure for Validation of Steam Sterilizer:

Note: Executive/Officer-Microbiology shall inform the external agency selected for validation as per the schedule for performing the validation as per the plan mentioned below.

  • Empty Chamber Heat Distribution Studies – Validation of Steam Sterilizer :

    • Purpose
    • To establish the uniformity of temperature distribution in the Steam Sterilizer chamber and identify the location of the “coolest point”.
    • Test Instrument
    • Calibrated temperature data logger with minimum 10 calibrated RTD probes (Resolution ± 0.1 °C)
    • Procedure
    • Conduct the test three times and ensure that it has a cold start each time.
    • Keep the chamber empty for all cycles.
    • Fix minimum10 RTDs in the chamber, through the access port to cover the throughout sterilizer chamber, ensure that one probe should be fix near to the drain point.
    • The reference number allocated to each probe shall be mentioned in reports.
    • Use Teflon tape to secure probes in position.
    • Ensure that the tips do not touch any metallic surface.
    • Attach the probes to the multiple probe temperature data logger.
    • Close the door(s) of Steam Sterilizer/ Autoclave.
    • Set the cycle parameters as established in Cycle development studies.
    • Adjust the Sterilization Time to 20/30 minutes or as per requirement.
    • Set the data logger to display the temperature of all the probes and record the temperature for every one minute interval.
    • Operate the Steam Sterilizer as per SOP titled, Operating Procedure for Steam Sterilizer. Start the sterilization cycle and data logger at the same time.
    • Analyze the results and locate the coolest point, as the position in the proximity of the probe, showing the minimum temperature, during the complete cycle.
  • Loaded Chamber Heat Distribution Studies – Validation of Steam Sterilizer :

    • Purpose
    • To determine the heat distribution at maximum load in sterilizer.
    • To establish that the heat distribution at the “slowest to heat point” of the specific load, at the established sterilization phase cycle time.
    • Test Instrument
    • Calibrated temperature data logger with minimum 10 No. calibrated RTD probes (Resolution 0.1 °C)
    • Procedure
    • Conduct the test three times and ensure that it has a cold start each time.
    • Keep the chamber loaded with full load for all cycles.
    • Fix minimum10 RTDs in the chamber, through the access port to cover the throughout sterilizer chamber, ensure that one probe should be fix near to the drain point.
    • The reference number allocated to each probe shall be mentioned in reports.
    • Use Teflon tape to secure probes in position.
    • Ensure that the tips do not touch any metallic surface.

    • Attach the probes to the multiple probe temperature data logger.
    • Close the door (s) of Steam Sterilizer/ Autoclave.
    • Set the cycle parameters as established in Cycle development studies.
    • Adjust the Sterilization time to 20/30 minutes or as per requirement.
    • Set the data logger to display the temperature of all the probes, and record the temperature after every one minute interval.
    • Operate the Steam Sterilizer/ Autoclave as per respective standard operating system. Start the sterilization cycle and data logger at the same time.
    • Analyze the results and locate the coolest point, as the position in the proximity of the probe, showing the minimum temperature, during the complete cycle.
  • Loaded Chamber Heat Penetration Study – Validation of Steam Sterilizer :

    • Purpose
    • To determine the heat distribution at maximum load in sterilizer to establish that the steam penetration at the “slowest to heat point” of the specific load, at the established sterilization phase cycle time, meets the specified Fo Value (NLT 12 min).
    • Test Instrument
    • Calibrated temperature data logger with minimum 10 No. calibrated RTD probes (Resolution ± 0.1 °C)
    • Procedure

    • Carry out the test using the maximum load intended to be sterilized using the Steam Sterilizer.
    • Each experiment shall have a cold start each time.
    • Set the operation values of parameters as established during cycle development studies and the Sterilization Time as 20/30 minutes or as per requirement.
    • Load the Steam Sterilizer Chamber and record the Load pattern diagram in report.
    • Dip the RTDs in Medium or place such that the sensor is in contact with the material to be sterilized.
    • Use Teflon tape to secure RTD probes in position.
    • The mapping of probes should be done to ensure proper representation of the full load.
    • Allocate a reference number to each probe and state in the diagram.
    • Attach the RTD probes to the multiple probe data logger.
    • Close the doors of Steam Sterilizer/Autoclave.

    • Set the data logger to display the temperature of all the probes, after every 1 minute and record the same manually.
    • Operate the Steam Sterilizer/ Autoclave as per SOP titled Operating Procedure for Steam Sterilizer.
    • Start the sterilization cycle and data logger at the same time.
    • Record the temperature of all the probes.
    • Record and analyze the results to locate the slowest to heat point as the position in the proximity of the RTD, showing the minimum temperature, during the cycle.
    • Calculate the Fo value at the “slowest to heat” point, using the following formula:    

Fo =                 t X (To – Tr)

             z

      • Where  t    =  time interval of temperature measurement (1 minutes here)
      • To  =    exposure temperature at any given instant
      • Tr  =     process reference temperature (121.5/121 ° C)
      •  z    =    A resistance value of 10 ° C assumed for the Steam Sterilizer
    • Repeat the experiment three times to ensure reproducibility of location of slowest to heat point, and Fo Value at coolest point, in the specific load.
    • On the basis of the results of the calculated Fo Value increase or decrease the Sterilization Cycle Time to achieve a desired Fo Value.
    • Repeat the experiment three times to assure the reproducibility of Fo value at coolest point in the steam sterilizer chamber.
  • Suitability by Heat labile physical indicator Validation of Steam Sterilizer :

    • Purpose
    • To establish an assurance level for completion of sterilization using chemical indicators.
    • Indicator used
    • OK (Proper sterilization strip) or Steam Clox Indicator or as per availability.
    • Procedure
    • Place strip of OK (Proper sterilization) / Steam Clox Indicator in the chamber of autoclave.
    • Run Steam Sterilizer/Autoclave at 121.5/121ºC and 15 lbs pressure as per SOP.
    • After completion of sterilization cycle remove the physical indicators from the autoclave.
    • Observe the colour change in the indicator the colour should change from pink to green in Steam Clox and from cream to black in Proper Sterilization OK indicator or as per manufacturer recommendation.
    • Attach the indicator with the record after check and write the date along with checked by sign.
  • Biological Challenge Test – Validation of Steam Sterilizer :

    • Purpose
    • To study the destruction of a resistant microbial challenge by a Moist Heat Cycle, at the Sterilization Cycle Time established by Fo studies.
    • Test Spores
    • Prospore vials of Geobacillus stearothermophilus
    • Procedure

    • Carry out the test using the loads for which Fo (thermal) has been established.
    • Place Minimum 10 vials of self contained Geobacillus stearothermophilus at minimum 10 different locations by covering the coolest locations like drain point and put stickers on each indicating date and time of validation, equipment ID and location of ampoule inside the Autoclave (Every Cycle of Heat Penetration)
    • Run a complete Steam Sterilizer cycle at the cycle parameters established during Fo studies for the particular load.
    • After completion of the sterilization cycle remove biological indicator from the Autoclave.
    • Retain one vial without sterilization as positive controls.
    • Cool the ampoules to about 60°C and incubate all the vials at 55 to 60°C for 48 hours or as per manufacturer recommendation within 4 Hours.
    • Sterilization cycle is considered to be perfect complete and validated if the colour change in the vials are as follows up to the completion of incubation period-
    • Sterilized vials – Clear and Purple in Colour or as per manufacturer instruction.
    • Unsterilized Vials – Turbid and yellow in Colour or as per manufacturer instruction.
    • Enter all the observations in the format maintained for calibration of equipment as Annexure-1.
  • Steam Penetration Study by Bowie-Dick Test – Validation of Steam Sterilizer :

    • Purpose
    • To study of steam penetration evenly in steam sterilizer chamber.
    • Test Kit Used
    • Bowie-Dick type test pack
    • Procedure
    • Keep the Bowie-Dick type test pack in the lower shelf of steam sterilizer near drain & set the Bowie- Dick cycle for as per vendor recommendation.
    • Run the cycle as per the SOP for operation of steam Sterilizer or autoclave for as per vendor requirement.
    • After completion of cycle remove the test pack from chamber, remove the cover and paper sheets around test paper.
    • Observe the Bowie- Dick test paper for even colour change of the indicator which is distributed on test paper.
    • Record the result in Annexure-2 and attach this test paper with Annexure-2.
    • The test should be considered satisfactory if a uniform colour change throughout the indicator.
    • If there is a failure check the previous load and the previous results.
    • Details of Cycle:

S.N. Type of Cycle No. of cycle
1. Empty 3
2. Heat Distribution Minimum Load 3
3. Heat Distribution Maximum Load 3
4. Heat Penetration Minimum Load 3
5. Heat Penetration Maximum Load 3
6. Bowie-Dick 3
    • Frequency of calibration:

Source Parameter Standard Acceptance
Criteria
Frequency
External Heat distribution Calibrated Thermocouple ± 3.0º C Yearly
External Heat Penetration Calibrated Thermocouple ± 3.0º C Yearly
In House Physical Indicator OK/ Steam Clox or as per availability Colour change Every Cycle
In House Biological Challenge Test Geobacillus stearothermophilus Prospore vials No turbidity

After 48 hrs. incubation

Half Yearly
In House Bowie-Dick Test  3M/ Any Standard Company Colour Change Half Yearly
  • Documentation:

    • Outside agency shall submit a detailed report for validation of autoclave containing all data of heat distribution and heat penetration studies.
    • Microbiologist shall fill the details of validation status on the validation tag pasted on the equipment as per Annexure-3
    • Report any discrepancy observed during calibration to Head – Quality Control and notify the defect to the maintenance department or service engineer of the equipment and Affix an Under Maintenance label on the equipment.

6.0   Abbreviations and Definition of Terms Validation of Steam Sterilizer :

    • Abbreviations :
    • RTD :   Resistance Temperature Detectors
    • SOP  :   Standard Operating Procedure
    • Definition of Terms :
    • D Value : The D Value is the time (in minutes) required to reduce the microbial population by 90% or 1 log cycle (i.e. to a surviving fraction of 1/10).
    • Z Value : The z value is the temperature required for one log reduction in the D value.
    • Fo Value : The Fo Value is the equivalent time that a monitored article is exposed to the desired temperature, e.g. 121 ° C for steam sterilization.

Annexure- 1 : Validation Report of steam sterilizer by Biological Indicator

Validation Report No.   :
Equipment Name          :

Equipment ID              :

Location                       :

Sterilization Parameter:

Date of Incubation       :

Date of Observation     :

Incubation Temperature:

Load Pattern                 :

Indicator Used   :

Lot No.             :

Exp Date           :

Z-value             :

D-value             :

Survives Time    :

Killed Time        :

Prospore Count   :

Mfg By              :

S. No. Location Details Observation
Date
+ve Control

Note :

  1. Test Ampoule shows Turbidity/Change of colour to Yellow – Test Invalid
  2. Positive control Ampoule shows Turbidity/Change of colour to Yellow –Test Valid
  3. Test Ampoule retains its Purple colour/No Turbidity –Test Valid

Annexure- 2 : Result of Bowie-Dick Type test Kit.

Date of Sterilization : Ster.-Hold Time :
Sterilization Cycle No. : Autoclave ID No. :
BOWIE-DICK TYPE TEST KIT
Manufacturer : Mfg date :
Lot Number : Exp. date :
Observation :
Conclusion: Steam penetration is SATISFACTORY / NOT SATISFACTORY
Acceptance Criteria: The colour of Bowie-Dick test paper should be changed from……..
 

Annexure- 3 : Validation Status Label.

VALIDATION STATUS LABEL

Name of Equipment
Equipment ID
Date of Validation
Next Due Date
Calibrated By  

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Janki Singh is experienced in Pharmaceuticals, author and founder of Pharma Beginners, an ultimate pharmaceutical blogging platform. Email: [email protected]

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