Hydrogen peroxide sterilization biological indicators

The biological indicators are made up of standardized bacterial spores inoculated onto different types of supports which, in addition to allowing their appropriate use, emulate the process that leads to the reduction of the microbial load.

The biological indicators for sterilization with vaporized hydrogen peroxide (vH₂O₂) look like:

Steel discs, rods etc. inoculate with spores.
Self-contained devices containing the disk with the spores and the ampoule with the culture broth
Vials of spores in aqueous suspension.

Spores used:

Geobacillus stearothermophilus 7953
Geobacillus stearothermophilus 12980

Biological indicators for sterilization with hydrogen peroxide are regulated by:

ISO 11138-1:2017 Sterilization of health care products — Biological indicators Part 1: General requirements

Focus: validity of genetic tests on Geobacillus stearothermophilus

In recent years, some customers have started to perform genetic tests on the spores contained in biological indicators, and this has highlighted the difficulty of obtaining reliable and unambiguous results with regard to Geobacillus stearothermophilus, which shows extremely high genetic similarity to other Geobacillus species, including G. thermoleovorans and G. kaustophilus, as well as others. Many species share highly conserved genetic regions; in Geobacillus, genetic markers often do not have the variability necessary to distinguish species with 100% certainty.

To obtain exact differentiation, a multifaceted approach is often required that combines genetic, biochemical, and phenotypic analyses and thus allows precise differentiation of Geobacillus species; however, this is a complex and costly approach.

Comunicazione Verifica Genetica Geobacillus stearothermophilus

Biological Indicators APEX-Mesa

Biological Indicators Hydrogen peroxide Crosstex

Biological Indicators Hydrogen peroxide True Indicating

Steel discs, bars

They are steel supports inoculated with Geobacillus stearothermophilus spores and used to optimally verify sterilization with hydrogen peroxide.

The disc is usually enclosed in a paper sachet, which must be placed inside the load and subjected to the sterilization cycle, during which the hydrogen peroxide comes into contact with the spores.

Once finished, the disc should be removed from the sachet and incubated for 7 days in Soybean Casein Digest Broth (TSB). Any cloudiness of the medium indicates bacterial growth and therefore sterilization failure. Broths with pH indicators are available which, by changing color with bacterial growth, make it possible to validate a significant shortening of the incubation time.

“Self-contained” biological indicators (SCBI)

A valid alternative to discs is given by "self-contained" biological indicators, in which a steel disc with spores is placed inside a small plastic test tube with a perforated cap, together with a glass ampoule containing a broth. of culture with color change indicator.

The advantages of SCBI are two: the post-sterilization incubation time is significantly shortened and there is no manipulation of the spores since the strip remains inside the plastic tube, where it is incubated. The indicator must be placed inside the load and subjected to the sterilization cycle, during which the hydrogen peroxide comes into contact with the spores. At the end, the test tube is crushed, breaking the internal flask, which then releases the broth, moistening the disk. We proceed with the incubation and verification of any color change.

Spore suspensions

Spores suspended in aqueous solution for particular needs, for example verifying the sterilization of thin or complex-structured medical devices. Proceed by inoculating an aliquot of the suspension onto the sample and allowing it to dry. The object must then be packaged as usual and subjected to sterilization.

At the end, the object must be incubated in culture broth to check for any bacterial growth. It should be considered that the D-value indicated on the certificate refers to the tests performed on the suspension inoculated on paper strips and that the resistance could change significantly depending on the type of support on which the suspension is inoculated. In fact, spores can adhere to surfaces, forming densities in areas that appear porous or wrinkled at a microscopic level. This aspect is even more evident if you intend to inoculate the suspension in a liquid, whose effects on the resistance of the spore can be completely unexpected.