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SOP for Validation of Autoclave

Standard operating procedure to validate the steam sterilizer using biological indicators having spores of Bacillus stearothermophilus ATCC 7953.


       To lay down the procedure for validation of autoclave in order to verify that the laboratory autoclave is capable of sterilizing the load at the specified working temperature and pressure.

2.0  SCOPE

       This SOP shall be applicable to Quality Control Dept.




       Senior Manager Quality Assurance


5.1  Validation of autoclave is done once in three months using the ‘MERCK Sterikon Plus Bioindicator System’.
5.2  The bioindicator system consists of ampules containing spores of Bacillus stearothermophilus ATCC 7953.
5.3  These ampoules are stored under refrigeration when not in use.
5.4  On the day of validation remove three ampules from the refrigerator and allow them to come to room temperature.
5.5  Take two glass beakers and place an ampule each, in each beaker, to avoid contamination due to accidental breakage during sterilization.
5.6  Place the two beakers, containing an ampule each, into the autoclave, along with the material to be autoclaved.
5.7  The position of the two ampules.
5.8  Switch on the autoclave and carry out the autoclaving cycle as per the procedure described in SOP
5.9  After autoclaving, remove the ampules and allow them to cool to room temperature..
5.10  Mark the two ampules and incubate at 60 ± 2° C for 48 hrs.
5.11  The third non-sterilized ampule is incubated along with the sterilized ampules, to serve as a control.
5.12  If sterilization is effective, the contents of the sterilized ampules remains clear, red violet color.
5.13  If sterilization is ineffective, the contents of the sterilized ampules turn turbid, yellow in color.
5.14  The contents of the control ampule should also turn yellow.
5.15  Record and store the results obtained.
5.16  Label the sterilized ampules and the control ampule, mentioning the date of validation, and store in the refrigerator, for reference.
5.17   NOTE:
5.17.1  Each ampule of the ‘MERCK Sterikon plus Bioindicator System’ consists of nutrient broth, sugar, a pH indicator and spores of  Bacillus stearothermophilus ATCC 7953.
5.17.2  The thermal resistance of the spores is such that they are totally killed after 15 minutes, when heated in compressed steam at a temperature of 121°C ± 0.5°C.
5.17.3  If sterilization is adequate, the Bacillus stearothermophilus spores get killed and the contents of the ampule remain a clear red violet color.
5.17.4  If sterilization is inadequate, the spores survive. Within 24 hours of incubation, the contents of the ampule turn turbid due to microbial growth and the color turns yellow due to production of acid, as a result of sugar fermentation.

Related: Performance Evaluation of Biological Indicators

5.18  Precautions:

5.18.1  Ensure that the ampules are stored in the refrigerator, when not in use.
5.18.2  Ensure that the ampules are not used beyond the date of expiry printed in the pack.
5.18.3  Ensure that the ampules are at room temperature before loading them into the autoclave.


6.1  SOP : Standard Operating Procedure
6.2  QC : Quality Control
6.3  Dept. : Department

Also see: Low Temperature Sterilization Process (115°C)

Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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