Validating Autoclave Sterilizing Efficiency

Introduction

The autoclave is validated to ensure that it is functioning correctly and full sterilization is achieved after usage. This ensures that pharmaceutical products are free from microorganisms and they are fit for consumption. In addition, effectiveness of an autoclave determines the quality and purity of goods obtained, high standards are only achieved if the yields are free from microbial contaminants, particulates, and pyrogens.

Reasons for Re-evaluation

Failure of the equipment to work in regards to comparison with the positive control.

Responsibility

• Laboratory technologist and microbiologist will carry out the experiment.
• The executive manager checks the protocol and approves.
• The head of the microbiological department is responsible for accountability.

Reference documents

• Annexure -1- Validation protocol Initial Biological Indicator Count.
• Annexure – 11 Autoclave validation protocol.

Procedure

Organism: Bacillus stearothermophilus spores with a certified strength of 10⁶.

Strip Validation

The number of spores in one Bacillus stearothermophilus strip from a batch is counted for validation purposes before the start of the experiment. The strip is dipped aseptically into 100ml of normal saline. Thereafter, the solution is left to settle for 30 minutes before applying heat shock by immersing the tube in an 80⁰C water bath for about 15 minutes. Vortexing is then done to homogenize the spores’ suspension depending on the first count, one can serially dilute the solution further using normal saline to get several dilutions.

An aliquot of each solution is then taken from the different dilutions, this is done to ensure that more than 300 colonies forming unit/plate colony count is not obtained. Melted SCD agar medium, which has been cooled to 50⁰C is poured into several plates and swirled before letting the media to solidify. Thereafter, the petridishes are incubated at 55⁰C in an inverted position for 48-70 hours. The colonies in the plate are then counted and the average number per strip is calculated using the dilution factor. The strip is then utilized if the first count is between 75%-150% of the label claim. The results are recorded in the Annexure-1 form.

Load glasswares and other accessories into the autoclave chamber, then label required amount of strips and place them into it. The strips are then placed in the Petri dishes, but one should be left to act as a positive control. Running of the autoclave at 121⁰C and pressure of 15lb for 20 minutes then follows. All the strips are then removed after one cycle is over.

Sterile Soybean Casein Digest (SCD) medium is labeled by writing the date, location, and equipment identity number. Thereafter, the unexposed strip is transferred to an SCD medium and marked as positive control. One SCD medium tube is not inoculated to be negative control. All the tubes are then incubated at about 55⁰C for two weeks. One should observe the tubes with strips for growth or turbidity each day. A positive control should exhibit growth after 72 hrs.

Interpretation

The absence of growth in SCD medium tubes indicates that the autoclave is good for sterilization procedures. If growth is seen in any of the SCD medium tubes, gram staining is performed to ascertain whether the colony is for B. stearothermophillus. In addition, the colony morphological feature is also compared with that of the positive control for identification purposes. In case of failure, the noncompliance of the validation process is investigated and the equipment utilization is stopped. Revalidation takes place after the autoclave problem is rectified. The calibration card containing equipment name, identification number, a validation number, frequency, due date, and validation done by is affixed. In addition, the records and the results obtained are recorded in annexure 11.

Deviation

No deviation from the procedure occurred during the validation process.

Conclusion

The autoclave successfully sterilized the biological indicator spores since no growth was observed in the tubes after incubation. This implies that all microorganisms including B. stearothermophilus were inactivated. Thus, the validation process showed that the autoclave is performing well and it is fit for utilization.

Recommendations

Validation of the autoclave should be done at least twice a year to ascertain its efficacy.

Report

The filter papers were impregnated with spores of B. stearothermophilus standard culture after suspension. The strips had a mean of about 1000000 spores/strip before serial dilution. The tubes inoculated with the organism showed no growth even after a period of two weeks. On the other hand, positive control tubes had bacteria flourishing in it after 72 hours while in the negative control no growth was observed.

Annexure-1

Validation of Autoclave in quality control laboratory

Initial Biological Indicator Count

• Date of testing:
• Claimed initial count: 1.0 × 10⁶
• Name of the indicator organism: B. stearothermophilus
• Growth medium: Soybean Casein Digest Medium
• Incubation temp and period: 55⁰C

• Dilution Steps 100 ml →10ml → 10ml → 10ml
• Mean cfu /strip
• Actual initial count:
• Acceptance criteria: Initial count should not be more that 150% of the label claim and not less than 75 %.
• Analyst:
• Checked by:
• Date:
• Date:

Annexure-11

Microbiological Department

Autoclave Validation

• Organism: Bacillus steriothermophilus spores with a certified strength of 10⁶
• Label claim: 1.0 × 10⁶
• Medium: Soybean Casein Digest Medium
• Incubation temperature: 55⁰
• Sterilization: Temp. 121°C for 30 minutes
• Cycle parameters: I cycle for 20 minutes

Observations

• -ve = No growth
• + =growth
• Negative control: No growth
• Positive control: Growth
• Comment: All tubes had no growth except the positive control.
• Sign. Chief microbiologist
• Date of report
• Sign of quality control in charge