SOP Title: Validation of Laboratory Autoclave located in Microbiology Laboratory
- Objective:
To lay down the procedure for Validation of Laboratory Autoclave in Microbiology section of Quality Control Department.
- Scope
This procedure is applicable for Validation of Laboratory Autoclave section of Quality Control Department.
- Responsibility:
3.1 Quality Control: To prepare and review the SOP. To follow the procedures for Validation of Laboratory Autoclave in Microbiology section as per this SOP.
- Quality Assurance Department: To review and approve the SOP and Annexure.
- Accountability:
Head Quality Control Department, Head Quality Assurance Department.
- Procedure:
| Sr. No. | Procedure | |
| 5.1 | Safety Precautions: | |
| 5.1.1 | Ensure that the appropriate protective equipments are used during handling of Autoclave. | |
| 5.1.2 | Check the water level before each cycle in the Autoclave. | |
| 5.1.3 | Ensure that the instrument is cleaned and free from dust and is disconnected from the mains after use. | |
| 5.2 | Procedure to carry out validation of Autoclave : | |
| 5.2.1 | Protocol for Autoclave validation should be prepared. | |
| 5.2.2 | Validation of Autoclave is carried out for
1) Heat distribution/penetration study with temperature Mapping. 2) Sterilization study of Biological Indicators. |
|
| 5.3 | Heat distribution study with temperature Mapping: | |
| Acceptance criteria: The load penetration study carried out in Autoclave should comply with the acceptance criteria of respective heat distribution study for Empty load, half load and full load. | ||
| Frequency: Initial once after installation and then once in a Year. | ||
| 5.3.1 | To ensure that the autoclave is capable to attain the temperature of 121°C during the sterilization hold at a specified hold period with the steam pressure on the pressure gauge is 15 psi. | |
| 5.3.2 | Ensure that the autoclave temperature and pressure gauges are calibrated. Check the calibration status of Data logger and sensors used for calibration. | |
| 5.3.3 | Protocol for validation and revalidation of autoclave to be prepared as per SOP. | |
| A. | Heat Distribution study at 121°C / 15 psi in Empty chamber | |
| 5.3.3 | Empty cycle load heat distribution study shall be performed. | |
| 5.3.4 | Fill purified water in autoclave upto the mark before starting. | |
| 5.3.5 | Set the parameters for the sterilisation cycle set to be operated. | |
| 5.3.6 | Insert minimum 8 to 12 nos. of sensors or suitable numbers depending upon the volume of autoclave into the chamber through the port provided for Autoclave. | |
| 5.3.7 | Place one sensor near the autoclave controller probe, one sensor near the drain and one sensor in the middle position, distribute the remaining probes uniformly in the chamber. Place a chemical indicator (Autoclave sterilization indicator) tape/strip and a biological indicator near each of the first three sensors mentioned above. Define Annexure for sensor locations. | |
| 5.3.8 | Seal the logger port with silicon sealant so that steam leakage does not take place. | |
| 5.3.9 | Connect the sensors to a suitable calibrated data logger which can scan and print the actual temperature with time. | |
| 5.3.10 | Set the Autoclave to attain the desired temperature of 121°C at 15 psi during the complete sterilisation hold period. | |
| 5.3.11 | Start the autoclave cycle after ensuring all the necessary precautions are taken. In Empty load cycle the chamber must be totally empty except for data logger sensors and chemical and Biological indicators. | |
| 5.3.12 | Start the data logger recording once the cycle is started; record the actual temperature at an interval of 1 minute during sterilization cycle of 15 minutes or 30 minutes by using a data logger. After completion of sterilization, record the data in datalogger until temperature drops down to 100 °C. | |
| 5.3.13 | The temperature spread should be within the range of 121°C to 124°C during sterilisation for set temperature of 121°C at 15 psi pressure for 15 minutes and 30 minutes. | |
| 5.3.14
|
Review the temperature data for all sensors printed on datalogger print, check and compare the temperature observed at different location. | |
| 5.3.15
|
For Sterilization study of Biological Indicators refer point No. 5.4. | |
| 5.3.16 | For Heat Distribution study details record in Annexure. | |
| 5.3.17 | Define Annexure for the list of cycles and for sterilization parameters. | |
| B. | Heat Distribution/Penetration study at 121°C / 15 psi for Media(Half Load/Full Load) | |
| 5.3.18 | Media Sterilization cycle is validated for 121°C at 15 psi pressure for 15 minutes. | |
| 5.3.19 | Place one sensor near the autoclave controller sensor, one sensor near the drain and one sensor in the middle position, distribute the remaining sensors uniformly in the chamber. Place a chemical indicator (Autoclave sterilization indicator) tape/strip and a biological indicator near each of the first three sensors mentioned above. Define sensor locations. | |
| 5.3.20 | Use same numbers of sensors as used in the Empty load distribution cycle. | |
| 5.3.21 | Carry out the autoclave cycles as per Empty load method. Refer Point No.5.3.4 to 5.3.17. | |
| 5.3.22 | Prepare Solid and Liquid media for carrying out the media cycles as per SOP. Prepare preferably SCDA and R2A agar medium as solid media. SCDM and MacConkeys broth shall be prepared as liquid media. If these media are not available, any other media can also be used. Enter Media prepared in Annexure. | |
| 5.3.23 | Half load refers to the the media load which occupies half or minimum space in the Autoclave during sterilization cycle. Record in Annexure. | |
| 5.3.24 | Full load refers to the Media load which occupies the maximum loadable space in autoclave during sterilization cycle. Record contents of cycle in Annexure. | |
| 5.3.25 | Place sensors in between the media glasswares and in the media at different locations to map the temperature distribution and penetration of steam. | |
| 5.3.26 | For Steam sterilization study of Biological Indicators refer point No. 5.4 | |
| 5.3.27 | For Heat Distribution study record in Annexure. | |
| C. | Heat Distribution/Penetration study at 121°C / 15 psi for Garments/ Glasswares/Accessories.(Half Load/Full Load) | |
| 5.3.28 | Garments/ Glassware/Accessories Sterilization cycle is validated for 121°C at 15 psi for 30 minutes as to sterilize the garments and other items for maximum duration. | |
| 5.3.29 | Place one probe near the autoclave controller probe, one probe near the drain and one probe in the middle position, distribute the remaining sensors uniformly in the chamber. Place a chemical indicator (Autoclave sterilization indicator) tape/strip and a biological indicator near each of the first three sensors mentioned above. | |
| 5.3.28 | Use same numbers of sensors as used in the Empty load distribution cycle. | |
| 5.3.30 | Carry out the autoclave cycles as per Empty load method. Refer point No. 5.3.4 to 5.3.17. | |
| 5.3.31 | Half load refers to the the Garment/Glassware/ Accessories load which occupies half or minimum space in the Autoclave. | |
| 5.3.32 | Full load refers to the Glasswares/ Garments/ Accessories which occupies the maximum loadable space in autoclave. Record in Annexure for loading pattern and contents of cycle. | |
| 5.3.33 | Place sensors in between the Garments and glasswares at different locations to map the temperature distribution and penetration of steam. | |
| 5.3.34 | For Analysis of Biological Indicators refer point No. 5.4 | |
| 5.3.35 | For Heat Distribution study record in Annexure. | |
| D. | Heat Distribution/Penetration study at 115°C / 10 psi for Media.(Full Load) | |
| 5.3.36 | Media Sterilization cycle for some media like Rappaport is to be validated for 115°C / 10 psi pressure for 15 minutes. | |
| 5.3.37 | Follow the same procedure as media cycle point No. 5.3.19 to 5.3.25 | |
| E. | Heat Distribution study at 121°C at 15 psi for Media Discard autoclave.(Full Load) | |
| 5.3.38 | Media Decontamination/Sterilization cycle is validated for 121°C at 15 psi for 30 minutes. | |
| 5.3.39 | Prepare Solid and Liquid media (Refer point No. 5.3.22) for carrying out the media decontamination cycles. Glassware should be included for this type of cycle. Record media prepared in Annexure. | |
| 5.3.40 | Perform the validation as per Empty cycle method point No. No.5.3.4 to 5.3.17. | |
| 5.4 | B) Steam Sterilization study of Biological Indicators | |
| Frequency: Once a year during validation, every six months as to check sterilization efficiency or as per validation requirements. | ||
| 5.4.1 | Place 3 spore strips/ampoules of Biological indicator Geobacillus stearothermophilus (Bacillus Stearothermophilus) ATCC 7953 in the most difficult to sterilize positions in the chamber. | |
| 5.4.2 | Sterilize in cycles as per procedure for individual cycle. | |
| 5.4.3 | After sterilisation, incubate each spore strip in a individual 10 ml Soyabean Casein Digest Medium test tube for 7 days at 55°C to 60°C. For media preparations refer SOP QC 034. Keep positive and Negative control with the test strips. Positive control (Unexposed) should be incubated in SCDM along with test strips for 7 days at 55°C to 60°C. Negative control (Unexposed) Indicators to be incubated at 30°C to 35oC for 7 days for checking the spore growth if any in normal conditions. Check the Biological Indicator tubes daily for 7 consecutive days and record in Annexure. | |
| 5.4.3 | The unexposed spore strip (positive control) shows growth (turbidity), indicates that spores are viable. | |
| 5.4.4 | Negative control shows no growth (turbidity) means spores are not activated. | |
| 5.4.5 | If the positive control shows turbidity and all three test samples are not showing turbidity after 7 days of incubation period, then the Autoclave is working satisfactory as per the requirements. | |
| 5.4.6 | In case of ampoules, incubate the ampoules after sterilization at 55°C to 60°C for 48 hours. Keep positive and Negative control with the test ampoules. Incubate positive control (Unexposed) along with test ampoules. Incubate the negative control (Unexposed) at 30°C -35°C for 48 hours. | |
| 5.4.7 | Exposed ampoules should not show any colour changes. | |
| 5.4.8 | The unexposed ampoule (positive control) shows colour change, indicates that spores are viable. | |
| 5.4.9 | Negative control should show no colour change. | |
| 5.4.10 | Record the data on Annexure | |
| 5.4.11 | If validation fails then do not use the Autoclave. Inform the Head of Quality control and the Head of Quality assurance, raise an incident and investigate the reasons & take immediate action to rectify the same and perform Revalidation. | |
| 5.4.12 | Decontaminate the spore strips in autoclave before disposing off. Refer SOP for Decontamination of culture media and Microbial waste. | |
- Definitions / Abbreviations:
- Definitions
- Abbreviations :
| Abbreviation | Expansion |
| psi | Pressure square inch |
*Note – Ready to use SOP available in “DOWNLOAD” Section.