Sterilization Validation | Protocol and Process

Sterilization aims to destroy all the 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 sterilizations, and the specific mode is selected based on the requirement of the product to be sterilized.

However, besides the actual method of sterilization, it is essential to ensure that the process is effective.

What is sterilization validation?

It is a method employed to check the efficacy of sterilization of finished products after the process of sterilization 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, sterilization validation is designed.

The method primarily relies on specific biological indicators or probes containing bacteria to see if they are dead due to sterilization.

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

And there are different sets of microbes prescribed for different methods of sterilization.

For Steam sterilization

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

Sterilization of medical equipment in an autoclave

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 on 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 cultured media in 24 to 48 hours, it indicates that the steam sterilization process was effective.

Here, the bacteria is chosen as it is highly resistant to heat compared to other bacteria.

Dry heat sterilization

Validation for dry heat sterilization is done using the spores of the Bacillus subtilis species.

Similar to the above, the probe containing spores of this bacteria is placed inside the hot air oven along with other items meant to be sterilized.

After the sterilization process, the probe inserted is removed and placed in nutrition media to observe for the growth of bacteria.

Lack of bacterial growth in 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 in 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 bacteria 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 is used to check the efficiency of filtration sterilization. It has a size of 0.5 to 0.8 μm in diameter.  

Filtration through a syringe fitted with a filter

The filtration technique relies on the ability of the filter to prevent the passage of microbes into the filtrate.

A liquid suspended with a culture of Serratia marcescens is allowed to pass through the filter, and the filtrate is taken into the culture to check for growth.

How to validate sterile medical device

Medical devices made of metal are subjected to either the incineration method, where the metal is heated to red hot condition, or to autoclave method.

If sterilized by incineration method, one can validate by placing the device in sterilized nutrition to observe for the growth of bacteria.

If by autoclave, it can be ensured by using the spores of Geobacillus thermophilus during the sterilization process.

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