Validating successful decontamination using biological indicators can be difficult using any method, but it can be particularly difficult when hydrogen peroxide is used.
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Choosing the Right Biological Indicator
Cellulose substrates cannot be employed by biological indicators (BIs) in hydrogen peroxide operations to inoculate spores during manufacturing. Paper and other cellulose products can react negatively with H2O2 by generating a gelatinous substance that is similar in consistency to a wet sponge.
The cellulose in such a substrate would protect the spores from the H2O2 during the cleaning cycle and the BI production process, preventing their destruction.
This is one of the reasons that all producers of biological indicators use stainless-steel surfaces for spore inoculation.
The second reason is that stainless steel is an excellent surrogate material, much like the hard surfaces used in pharmaceutical or medical settings.
Most guidelines suggest using biological indicators containing 106 spores from the strain of Geobacillus stearothermophilus ATCC 12980 to challenge decontamination cycles. It is difficult to inoculate many of these spores in a tiny surface area.
Some spore stacking is necessary for the procedure, although each manufacturer has unique techniques for creating a monolayer of spores on the surface. Using a manufactured lot, where spore stacking is more common, increases the likelihood of unexpected positives when this occurs in specific H₂O₂ processes.
End users who notice positives in verified cycles refer to these as "rogue" or unexpected positives.
Storage of Biological Indicators
Manufacturers also differ in how BIs used in H2O₂ operations are stored. Relative humidity is the most critical factor, but temperature is also necessary to preserve stability during shelf life.
Some BI producers have reported studies on BIs stored at high relative humidity (over 50%). The findings indicate that storage at high relative humidity affects both population and resistance.
As the population decreases, the remaining spores become more resistant. End users can occasionally encounter spores that are "hyper-resistant" to the H₂O₂ process.
How BIs are Certified
Biological indicator producers send clients Certificates of Analysis (COAs), which attest to the spore population and D-value of each manufactured batch of biological indicators. The time needed to eradicate 90% of a certain bacterium is expressed as decimal reduction time.
The D-Value is the name given to this time frame. The problem is that each manufacturer's D-value is established in a particular vessel or enclosure.
The kinds of enclosures that are subsequently cleaned by various hydrogen peroxide methods range from pass-throughs, filling enclosures, and isolators to other smaller vessels and bigger lab areas.
Because of this, any resistance performance verified by the BI maker serves as a benchmark for clients. Companies using H₂O₂ for decontamination need to learn how biological indicators function in specific spaces and enclosures.
To increase the predictability of future performance, the resistance performance or D-value recorded in those locations can be trended and compared to the Certificate of Analysis data.
Estimated spore log reduction may target additional spore log reductions for subsequent decontamination validations, depending on the enclosure or space size and the number of BIs used.
Source: SteraMist Disinfection and Decontamination Technology
| BI Spore Population |
Log Reduction |
Probable Percent Positive BIs |
| 106 |
0 |
100% |
| 105 |
1 |
100% |
| 104 |
2 |
100% |
| 103 |
3 |
100% |
| 102 |
4 |
100% |
| 101 |
5 |
100% |
| 100 |
6 |
63% |
| 10-1 |
7 |
10% or 1:10 |
| 10-2 |
8 |
1% or 1:100 |
| 10-3 |
9 |
0.1% or 1:1000 |
| 10-4 |
10 |
0.01% or 1:10,000 |
| 10-5 |
11 |
0.001% or 1:100,000 |
| 10-6 |
12 |
0.0001% or 1:1,000,000 |
The Halvorson-Ziegler Most Probable Number equation can further increase the accuracy of spore log reduction by replicating BIs at each place.
Biological indicators are the chosen tool for verifying effective decontamination procedures. Using biological indicators can provide daunting obstacles, and deciding what to do next can be challenging. You can rely on TOMI's original decontamination specialists to help you navigate these validation procedures.
This information has been sourced, reviewed and adapted from materials provided by SteraMist Disinfection and Decontamination Technology.
For more information on this source, please visit SteraMist Disinfection and Decontamination Technology.