Sterilization Validation | Protocol and Process

Sterilization aims to destroy all viable forms of microbes from finished products, workplaces, or surgical instruments.

This process is done on a routine basis in the industry for the safety of the end user or patient.

There are many types of sterilization, and their effectiveness is determined by sterilization validation.

What is sterilization validation?

It is a method used to verify the efficacy of sterilization of finished products after the sterilization process is complete.

• Different methods like heat, radiation, filtration, and chemical gases are employed for sterilization.

• However, one cannot be sure if the method was effective and if all the forms of microbes present in the samples have been killed.

To ensure this efficiency, sterilization validation is designed.

• The method primarily relies on specific biological indicators or bacterial probes to determine whether they are dead due to sterilization.

• Critically, a highly resistant microbial culture is used for the validation.

• Different sets of microbes are prescribed for different sterilization methods.

For Steam sterilization

• A strip containing spores of Geobacillus thermophilus bacteria is used for autoclave sterilization validation.

• The probes containing the bacterial spores are placed along with the product for moist heat sterilization in different corners of the autoclave before the start of sterilization.

• Then sterilization is carried out for a specified time, and once the process is done, these probes are taken out and cultured in media.

• If there is no bacterial growth from the strip in the culture medium within 24 to 48 hours, it indicates that the steam sterilization process was effective.

• Here, the bacteria are chosen as they are highly resistant to heat compared to other bacteria.

Dry heat sterilization

• Validation of dry-heat sterilization is performed using spores of Bacillus subtilis.

• As above, the probe containing spores of this bacterium is placed in the hot-air oven along with other items to be sterilized.

• After sterilization, the inserted probe is removed and placed in a nutrient medium to observe bacterial growth.

• The absence of bacterial growth within 24 to 48 hours indicates effective sterilization.

For Gaseous sterilization by ethylene oxide

• Gaseous sterilization uses ethylene oxide, a toxic gas, to kill the microbes.

• This method is preferred to sterilize heat-sensitive material.

• The probe containing Bacillus subtilis var. niger is used to validate this method.

• As before, the probe is inserted into the material, and post-sterilization, the probe is cultured to check for growth.

Radiation sterilization

• This a physical sterilization technique where either non-ionizing radiation is used for sterilization.

• The validation is done by using spores of the bacterium Bacillus pumilus.

• Since radiation is lethal, bacteria resistant to this radiation are used.

• Post-sterilization, the probes containing spores of Bacillus pumilus are checked for nutritional growth.

For filtration sterilization

• Serratia marcescens bacteria are used to check the efficiency of filtration sterilization. It has a diameter of 0.5-0.8 μm.  

• The filtration technique relies on the filter’s ability to prevent microbes from passing into the filtrate.

• A liquid containing a Serratia marcescens culture is passed through the filter, and the filtrate is added to the culture to check for growth.