Autoclave Validation Methods – Objective, Procedure, Result

Why we need to Validate an Autoclave?

Validating an autoclave is crucial for ensuring the sterilization process is effective and reliable. Here’s why validation is necessary:

1. Ensures Sterility: The primary purpose of an autoclave is to sterilize medical instruments, laboratory equipment, and other items. Validation confirms that the autoclave is consistently achieving the conditions (temperature, pressure, time) required to kill all forms of microbial life, including spores.
2. Compliance with Regulations: Many industries, particularly healthcare, pharmaceuticals, and food production, are governed by strict regulatory standards. Validating an autoclave is often a legal requirement to ensure compliance with these standards, such as those set by organizations like the FDA, CDC, or ISO.
3. Prevents Contamination: Improper sterilization can lead to contamination, which can compromise patient safety in healthcare settings or lead to product recalls in industries like pharmaceuticals. Validation helps prevent these risks by ensuring the autoclave is functioning correctly.
4. Quality Assurance: Validation is part of a broader quality assurance process. It provides documented evidence that the autoclave performs reliably over time, which is essential for maintaining high standards of quality in manufacturing and clinical practices.
5. Detects Equipment Issues: Regular validation can help detect issues with the autoclave, such as calibration errors, malfunctions, or wear and tear, before they result in failed sterilization cycles. This proactive approach helps maintain equipment efficiency and safety.
6. Consistency and Reproducibility: Validation ensures that every sterilization cycle is consistent and reproducible, providing confidence that every batch processed in the autoclave meets the required sterility standards.
7. Supports Risk Management: In any setting where sterility is critical, the risk of using non-validated equipment can be significant. Validation minimizes these risks by providing evidence that the autoclave is performing as expected, reducing the likelihood of sterilization failures.

Methods for Autoclave Validation

Autoclave validation is a critical process in ensuring that sterilization procedures are effective and reliable. This is particularly important in the pharmaceutical industry, where sterility is paramount for safety and compliance. The validation process involves a series of tests designed to verify that the autoclave operates correctly under various conditions and consistently achieves the desired sterility. Below are the primary methods used for autoclave validation:

1. Bowie-Dick Test for Steam Penetration
   • Purpose: This test assesses the autoclave’s ability to remove air from the chamber and allow steam to penetrate the load effectively.
   • Procedure:
     1. Perform the Bowie-Dick test over three trials.
     2. A specially designed test pack or indicator sheet is placed in the autoclave.
     3. The autoclave is run through a standard cycle, and the results are observed.
   • Expected Outcome: Successful completion of this test indicates that the steam is effectively penetrating the load, ensuring even sterilization.
2. Empty Chamber Heat Distribution Studies
   • Purpose: This test measures the uniformity of temperature distribution within the autoclave when it is empty.
   • Procedure:
     1. Conduct three trials with temperature mapping probes placed at various locations within the sterilizer chamber.
     2. The autoclave is run without any load to assess the evenness of heat distribution.
   • Expected Outcome: The temperature throughout the chamber should remain consistent, ensuring that all areas reach the required sterilization temperature.
3. Loaded Chamber Heat Distribution and Penetration Studies
   • Purpose: This test verifies that the autoclave can achieve uniform temperature distribution and effective steam penetration when fully loaded.
   • Procedure:
     1. Perform three trials for each specific sterilization load, following a fixed loading pattern.
     2. Temperature mapping probes are placed at various locations within the load.
     3. Include Biological Indicators (Geobacillus stearothermophilus spore vials with a spore count of 10^6 or more) in the innermost layers of the load.
   • Expected Outcome: The temperature should be uniformly distributed across the load, and the Biological Indicators should confirm successful sterilization.
   Specific Load Types Tested:
   • Sterile Area Garments:
     • Composed of garments such as boilersuits, headgear, booties, and gloves.
     • The load should achieve uniform sterilization throughout.
   • Glassware:
     • Includes items such as stainless steel manifold holders, sampling units, and various sampling bottles.
     • The test ensures that even complex items like glassware receive thorough sterilization.
   • Media:
     • Consists of different culture media in various containers.
     • Ensures that media, often sensitive to sterilization conditions, are correctly sterilized without compromising their integrity.
4. Estimation of the F0 Value
   • Purpose: The F0 value represents the equivalent time in minutes at 121°C (250°F) required to kill a specific population of microorganisms. It is a key parameter in assessing sterilization effectiveness.
   • Procedure:
     1. During the sterilization hold period, measure the F0 value at each temperature mapping probe.
     2. Compare the measured F0 values with the expected standards to ensure they meet the necessary criteria.
   • Expected Outcome: The F0 value should be sufficient to confirm that all microorganisms, including resistant spores, are effectively killed.

Method	Acceptance Criteria
Bowie-Dick Test for Steam Penetration	The Bowie-Dick Test indicator should show a uniform color change. Any non-uniform change or air entrapment (bubble) spot indicates inadequate air removal from the sterilization chamber.
Empty Chamber Heat Distribution Studies	The temperature at each temperature mapping probe should be within the range of 121°C to 124°C during the sterilization hold period, demonstrating uniform heat distribution.
Loaded Chamber Heat Distribution & Penetration Studies	The temperature at each temperature mapping probe within the load should be within the range of 121°C to 124°C during the sterilization hold period, indicating effective steam penetration and heat distribution.
Bio-challenge Studies	No bacterial growth should be observed during the incubation period of 48 hours at 55°C to 60°C. This ensures that the sterilization process has achieved the required sterility assurance level.
Estimation of F0 Value	The calculated minimum F0 value should exceed the biological F0 value for the biological indicator vial. The biological F0 value is calculated based on the D value, spore concentration, and desired level of non-sterility.

Compilation and Evaluation of Data

• Purpose: After completing all validation tests, the data generated must be compiled and analyzed.
• Procedure:
  1. Gather results from the Bowie-Dick tests, empty chamber studies, loaded chamber studies, and F0 value estimations.
  2. Evaluate the data to ensure the autoclave meets all specified acceptance criteria.
• Expected Outcome: Successful compilation and evaluation of data confirm that the autoclave can reliably sterilize various components under the tested conditions.

Method 1 – Bowie-Dick test Method for Steam Penetration

The Bowie-Dick test is a critical procedure designed to evaluate the efficiency of air removal and steam penetration in a steam sterilizer, particularly in pre-vacuum autoclaves. This test ensures that the sterilization process is capable of achieving uniform conditions necessary for effective sterilization across the entire load. Below is a detailed explanation of the Bowie-Dick test, its procedure, and the criteria for evaluating its results.

Objective of Bowie-Dick test for Steam Penetration

The primary objective of the Bowie-Dick test is to verify that the autoclave’s vacuum pulses are sufficient to remove trapped air from the sterilization chamber and load. Effective air removal is crucial as any residual air pockets can prevent steam from penetrating all parts of the load, leading to uneven temperature distribution and incomplete sterilization. The test specifically assesses whether the sterilizer can maintain the required conditions, typically 121°C for 17 minutes, necessary for effective sterilization.

Significance of the Test

• Air Entrapment Detection: The presence of air in the chamber can form bubbles within the Bowie-Dick test pack. These bubbles create regions with lower temperature and moisture levels, which are not conducive to sterilization. The test indicator within the pack will reflect this by displaying a different color in areas where air is trapped, indicating potential issues with air removal.
• Cycle Optimization: If the test reveals issues with air removal, it suggests that the sterilization cycle parameters need to be reviewed and possibly adjusted to ensure effective sterilization in subsequent cycles.

Procedure of Bowie-Dick test for Steam Penetration

The Bowie-Dick test is conducted in a sequence of steps designed to ensure accurate assessment:

1. Parameter Recording:
   • Record the set parameters for the Bowie-Dick test cycle, including temperature and time settings, to ensure consistency and traceability.
2. Placement of Test Pack:
   • Place one Bowie-Dick test pack near the drain of the sterilization chamber. The drain is typically the area most prone to inadequate air removal, making it a critical point for testing.
3. Cycle Selection:
   • On the autoclave’s control panel, select the Bowie-Dick cycle. This cycle is specifically designed to test air removal efficiency.
4. Operation of Steam Sterilizer:
   • Operate the steam sterilizer and allow the cycle to run its course. During the cycle, the autoclave performs a series of vacuum pulses to evacuate air and replace it with steam.
5. Preservation of Test Results:
   • Preserve the printout taken during the Bowie-Dick test cycle along with the Bowie-Dick test indicator. These records are essential for compiling observations and evaluating the performance of the sterilizer.

Acceptance Criteria for Bowie-Dick test for Steam Penetration

• Uniform Color Change: The Bowie-Dick test indicator should exhibit a uniform color change across the entire test sheet. A consistent color change indicates that air was successfully removed from the chamber and steam penetrated evenly throughout the load.
• Non-Uniform Change or Air Entrapment: If the indicator shows a non-uniform color change or specific areas with air entrapment (bubbles), this signals inadequate air removal. Such results necessitate a review of the sterilization cycle parameters and possibly further adjustments to ensure proper sterilization.

Observation and Results

The observations made during the Bowie-Dick test are meticulously recorded, including the color changes on the indicator sheet. The results are analyzed to evaluate the efficiency of the sterilization process. A successful test confirms that the autoclave can reliably remove air and ensure even steam distribution, which is essential for achieving complete sterilization.

Method 2 – Empty Chamber Heat Distribution Method

Empty chamber heat distribution studies are a critical part of autoclave validation, ensuring that the sterilizer can uniformly distribute heat across the entire chamber during the sterilization cycle. This validation step is essential for confirming that the autoclave can achieve and maintain the required sterilization temperature, typically between 121°C and 124°C, under specified steam pressure conditions. Below is a comprehensive explanation of the procedure, objectives, and criteria for evaluating these studies.

Objective of Empty Chamber Heat Distribution Method

The primary objective of empty chamber heat distribution studies is to verify that the autoclave is capable of reaching and sustaining the sterilization temperature of 121°C throughout the chamber during the sterilization hold period. The study aims to demonstrate that heat is evenly distributed within the chamber, and no cold spots exist where the temperature might fall below the required threshold. Uniform heat distribution is crucial for ensuring that all parts of the load, when introduced, receive the same level of sterilization.

Procedure of Empty Chamber Heat Distribution Method

The procedure for conducting empty chamber heat distribution studies is methodical and involves several key steps:

1. Parameter Recording:
   • Before initiating the test, record the set parameters for the sterilization cycle to be used during the heat distribution study. This includes the target temperature, pressure settings, and cycle duration.
2. Temperature Probe Placement:
   • Insert a minimum of 16 temperature mapping probes into the chamber through the sterilizer’s port. The port should be sealed with silicone sealant to prevent steam leakage. The probes must be suspended in various positions within the chamber, ensuring that they do not touch any metallic surfaces, which could skew the readings. The exact position of each probe should be documented in a schematic diagram for accuracy.
3. Connection to Data Logger:
   • Connect the temperature probes to a suitable autoclave data logger. This device will scan and print the actual temperatures observed at different locations within the chamber over time, providing a detailed temperature profile of the sterilization cycle.
4. Operation of Steam Sterilizer:
   • Start the steam sterilizer and simultaneously activate the data logger to begin recording the temperature data throughout the sterilization cycle. The data logger will track the temperature at each probe location during the entire cycle.
5. Data Collection:
   • Upon completion of the sterilization cycle, collect the printout from the sterilizer and the data logger. This data, including the recorded temperatures at each probe location, should be preserved as part of the study documentation. The data is then downloaded, analyzed, and printed for further evaluation.
6. Reproducibility Test:
   • If the results from the initial test are acceptable, perform three consecutive runs to demonstrate the reproducibility of the cycle and the sterilizer. Consistent results across multiple runs confirm the reliability of the sterilizer’s heat distribution.
7. Data Compilation:
   • Compile all data generated during the qualification tests for a comprehensive evaluation of the system. This step ensures that the sterilizer meets the necessary criteria for uniform heat distribution.

Acceptance Criteria

• Uniform Temperature Distribution: During the sterilization hold period, the temperature at each temperature mapping probe should be within the range of 121°C to 124°C. This range ensures that the sterilizer is maintaining the required conditions for effective sterilization.
• Cold Spot Identification: Any location where the temperature falls below 121°C is considered a cold spot. The presence of cold spots indicates a failure in uniform heat distribution, necessitating adjustments to the sterilization process or equipment.

Observations and Results

The observations and results of the heat distribution studies are meticulously recorded, analyzed, and documented. The data is used to confirm that the autoclave can consistently achieve uniform heat distribution, which is essential for ensuring that the sterilization process is reliable and effective.

Method 3 – Loaded Chamber Heat Distribution & Penetration Method

Loaded chamber heat distribution and penetration studies are essential in validating the effectiveness of an autoclave in sterilizing materials when fully loaded. These studies ensure that steam can penetrate all parts of the load, reaching the required sterilization temperature consistently throughout the process. Below is a detailed explanation of the objectives, procedures, and criteria for these studies.

Objective

The primary objective of loaded chamber heat distribution and penetration studies is to confirm that steam effectively penetrates the innermost portions of the load during sterilization. The goal is to ensure that the entire load reaches and maintains the required temperature of 121°C throughout the sterilization hold period, under a steam pressure of 1.1 to 1.2 kg/cm². Any failure to reach this temperature at any point within the load indicates a cold spot, which may require reevaluation of the load configuration or size. Therefore, the study is crucial for confirming that the uniform heat distribution observed in empty chamber studies is not compromised when the autoclave is loaded.

Procedure

The procedure for conducting loaded chamber heat distribution and penetration studies involves several steps designed to thoroughly assess the sterilization process:

1. Parameter Recording:
   • Begin by recording the set parameters for the sterilization cycle, including target temperature, pressure, and cycle duration. This information is documented in an annexure to ensure accurate monitoring throughout the study.
2. Temperature Probe Placement:
   • Insert a minimum of 16 temperature mapping probes into the chamber through the sterilizer’s designated port. The port must be sealed with silicone sealant to prevent steam leakage. The probes should be strategically placed inside the load components at points expected to be the most challenging for steam penetration. Alongside the probes, place biological indicators at 12 different locations to further assess the sterilization efficacy. The positions of both probes and biological indicators should be recorded in a schematic diagram for reference.
3. Data Logger Connection:
   • Connect the temperature probes to a suitable data logger, capable of scanning and printing the actual temperature readings over time. This device will provide a detailed temperature profile for the entire sterilization cycle, allowing for a thorough analysis of the heat distribution and penetration within the loaded chamber.
4. Operation of Steam Sterilizer:
   • Operate the steam sterilizer, initiating the sterilization cycle, and simultaneously start the data logger. The data logger will record the temperature at each probe location throughout the cycle, providing real-time insights into the temperature dynamics within the load.
5. Data Collection:
   • Once the sterilization cycle is complete, collect the printout from both the sterilizer and the data logger. The recorded temperatures at each probe location are preserved in the annexure for detailed analysis. Additionally, aseptically collect the exposed biological indicators and send them to the microbiology lab for incubation and observation of results. This step is critical for confirming that the biological load has been effectively sterilized.
6. Reproducibility Test:
   • If the results from the initial test meet the acceptance criteria, conduct three consecutive runs to demonstrate the reproducibility of the sterilization cycle and the reliability of the autoclave. Consistency across these runs confirms the stability and effectiveness of the sterilization process when the chamber is fully loaded.
7. Data Compilation:
   • Compile all the data generated during the qualification tests for a comprehensive evaluation of the system. This step ensures that the autoclave meets the necessary criteria for uniform heat distribution and penetration within a loaded chamber.

Acceptance Criteria

• Uniform Temperature Distribution & Penetration: The temperature at each temperature mapping probe should be within the range of 121°C to 124°C during the entire sterilization hold period. This range confirms that the sterilizer is maintaining the required conditions for effective sterilization across the entire load.
• Cold Spot Identification: Any location where the temperature falls below 121°C is considered a cold spot, indicating inadequate steam penetration. Such findings may necessitate adjustments to the load configuration or size, followed by a repeat of the cycle to ensure complete sterilization.

Observations and Results

The observations and results of the loaded chamber heat distribution and penetration studies are meticulously recorded, analyzed, and documented. These results are crucial for validating that the autoclave can consistently achieve uniform heat distribution and steam penetration within a fully loaded chamber, thereby ensuring the effectiveness and reliability of the sterilization process.

Method 4 – Bio-challenge Method

Bio-challenge studies are a critical component of validating steam sterilization processes, particularly in the pharmaceutical industry. These studies are designed to ensure that the sterilization process effectively eliminates microbial contaminants, thereby providing a high level of sterility assurance. Below is a detailed explanation of the objectives, procedures, and acceptance criteria for bio-challenge studies.

Objective

The primary objective of bio-challenge studies is to validate the effectiveness of the steam sterilization process by challenging it with a known biological indicator, typically Geobacillus stearothermophilus spore vials. These spore vials, containing a spore population of not less than 10^6 spores per vial, are used to determine the Sterility Assurance Level (SAL). The SAL is a measure of the probability of a single viable microorganism surviving the sterilization process. For a sterilization process to be considered effective, it must achieve an SAL of 10^6, meaning the likelihood of a surviving spore is extremely low. If the biological indicator shows any growth after incubation, it indicates a failure in the sterilization cycle, necessitating a review of the cycle parameters.

Procedure

The bio-challenge study procedure is a systematic process that involves several steps to ensure accurate assessment of the sterilization process:

1. Initial Spore Count Determination:
   • Begin by determining the initial counts of the biological indicator spores. This step ensures that the spore population meets the required threshold for the study, typically not less than 10^6 spores per vial.
2. Collection and Incubation of Exposed Indicators:
   • After completing the loaded chamber heat distribution and heat penetration studies, collect the exposed biological indicators using sterile forceps and scissors. Place the collected indicators into a 100 ml beaker and send them to the microbiology laboratory for incubation. The vials should be incubated at a temperature of 55 to 60°C for 48 hours. This incubation period allows for any surviving spores to grow, thereby indicating whether the sterilization process was effective.
3. Control Vials:
   • Alongside the exposed indicators, maintain one vial as a negative control (provided by the manufacturer of the biological indicator) and one vial as a positive control (an unexposed biological indicator). The negative control confirms that the medium is sterile, while the positive control verifies that the spores are viable and capable of growth under the incubation conditions.
4. Daily Observation and Recording:
   • Observe the vials daily for any signs of growth. Typically, a color change from purple (indicating sterility) to yellow (indicating non-sterility) is observed. Record these observations on a daily basis in the annexure. This step is crucial for tracking the results of the bio-challenge study and identifying any potential failures in the sterilization process.
5. Data Compilation:
   • Compile all data generated during the bio-challenge study for a comprehensive evaluation of the sterilization system. This compilation includes the initial spore counts, daily observations, and final results of the biological indicator incubation.

Acceptance Criteria

• No Bacterial Growth: The key acceptance criterion for bio-challenge studies is the absence of bacterial growth in the biological indicator vials after the 48-hour incubation period at 55 to 60°C. The absence of growth (indicated by the vials remaining purple) confirms that the sterilization process effectively eliminated the Geobacillus stearothermophilus spores, thereby achieving the required SAL.

Observations and Results

The observations and results of the bio-challenge studies are meticulously recorded and analyzed to ensure the reliability of the sterilization process. Any signs of bacterial growth necessitate a thorough review of the sterilization cycle parameters and may indicate the need for adjustments to achieve the desired sterility assurance level.

Method 5 – Estimation of F0 Value Method

The estimation of the F0 value is a critical aspect of validating the efficacy of a sterilization process, particularly in steam sterilization. The F0 value is a measure of the cumulative sterilizing effect of the process, reflecting the degree to which microbial spores are inactivated. Here, we will outline the objective, procedure, calculation method, and acceptance criteria for determining the F0 value.

Objective

The objective of estimating the F0 value is to ensure that the calculated F0 value during the sterilization cycle meets or exceeds the biological F0 value at all temperature mapping locations. This ensures that the sterilization process is effective across the entire load and at all relevant locations within the sterilizer chamber.

Procedure

1. Temperature Recording:
   • During the sterilization hold period, record the temperature at each temperature mapping probe. This data is crucial for accurate F0 value calculation. The recorded temperatures are entered into an annexure for further analysis.
2. Calculation of F0 Value:
   • Using the recorded temperatures, calculate the F0 value at each temperature mapping probe. This calculation involves applying a specific formula to determine the effectiveness of the sterilization process at different temperatures.
3. Documentation:
   • Enter the calculated F0 values into the annexure. This documentation is necessary for evaluating the effectiveness of the sterilization process and for subsequent data analysis.
4. Data Compilation:
   • Compile all data generated during the qualification test for a comprehensive evaluation of the sterilization system. This includes both the calculated F0 values and any other relevant observations.

Calculation Method

To estimate the F0 value, use the following formula:

F0=dt×10(T-121/z)

Where:

• D121: Time interval between two consecutive temperature measurements (in minutes).
• T: The observed temperature at that particular time.
• z: The temperature change required to change the heat resistance of Geobacillus stearothermophilus spores (typically 10°C).

Acceptance Criteria

• Minimum F0 Value: The calculated minimum F0 value must be greater than the biological F0 value obtained from bio-challenge studies. This ensures that the sterilization process is capable of achieving the necessary sterility assurance level.The biological F0 value can be calculated using the following equation: F0=D121×(logA−logB)
• Where:
  • D121: The D value of the biological indicator at 121°C, which represents the time required to achieve a one-log reduction in spore population.
  • A: The concentration or spore population of the biological indicator.
  • B: The desired level of non-sterility (usually 10^6 spores).

Observations and Results

• Record and analyze the results to verify that the calculated F0 values meet or exceed the biological F0 value. This ensures that the sterilization process is sufficiently robust to achieve the desired level of sterility.

References

• Garibaldi BT, Reimers M, Ernst N, Bova G, Nowakowski E, Bukowski J, Ellis BC, Smith C, Sauer L, Dionne K, Carroll KC, Maragakis LL, Parrish NM. Validation of Autoclave Protocols for Successful Decontamination of Category A Medical Waste Generated from Care of Patients with Serious Communicable Diseases. J Clin Microbiol. 2017 Feb;55(2):545-551. doi: 10.1128/JCM.02161-16. Epub 2016 Dec 7. PMID: 27927920; PMCID: PMC5277525.
• https://www.pharmaguideline.com/2011/01/autoclave-validation.html
• https://paulyeatman.net.au/wp-content/uploads/2016/08/practical-guide-to-autoclave-validation.pdf
• https://riskmanagement.sites.olt.ubc.ca/files/2020/04/LAB-SOP-001-AUTOCLAVE-VALIDATION-PROTOCOL.pdf
• https://consteril.com/wp-content/uploads/2017/06/Consteril_Autoclave_Validation.pdf
• https://safety.umbc.edu/autoclave-validation/