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July, 24 2024

Indicators of Success 

Insights into hydrogen peroxide and biological indicators

Validating successful decontamination with biological indicators can be challenging with any process, especially those using them with hydrogen peroxide. 

Choosing the Right Biological Indicator

Biological indicators (BIs) used in hydrogen peroxide processes cannot utilize any cellulose substrates for the inoculation of spores in the manufacturing process. Cellulose products, such as paper, can adversely react with H₂O₂ by forming a gelatinous material comparable to the consistency of a wet sponge. If such a substrate were used in the BI manufacturing process and during the H₂O₂ decontamination cycle, the cellulose would shield the spores from the H₂O₂, preventing them from being killed in the decontamination cycle. This is one reason why all biological indicator manufacturers opt for stainless-steel substrates for inoculation with spores. 

The second reason for choosing stainless steel is that it serves as an excellent surrogate material, similar to the hard surfaces found in hospital or pharmaceutical settings. Most guidelines recommend challenging decontamination cycles with biological indicators containing 10^6  spores from the Geobacillus stearothermophilus ATCC 12980 strain. Inoculating a high concentration of these spores within a small surface area is challenging. Although each manufacturer has proprietary methods for achieving a monolayer of spores on the surface, some stacking of spores is essential to the process. When this happens in certain H₂O₂ processes, there is an increased chance of unexpected positives when using a manufactured lot where spore stacking is more prevalent. Unexpected positives, or ‘rogue’ BIs, are terms given by end users who observe positives in validated cycles. 

Storage of Biological Indicators 

The storage of BIs used in H₂O₂ processes also varies by manufacturer. In most cases, there are both temperature and relative humidity requirements to maintain stability throughout the shelf-life, with relative humidity being the most critical. Some BI manufacturers have published studies on BIs stored under high RH conditions, with the results showing that storage under high RH (above 50%) impacts both population and resistance. The population declines, and the remaining spores increase in resistance. At times, end-users may encounter spores that are ‘hyper-resistant’ to the H₂O₂ process. 

How BIs are Certified 

Biological indicator manufacturers send customers Certificates of Analysis (COAs), certifying each manufactured lot of BIs for spore population and D-value. The decimal reduction time describes the amount of time required to kill 90% of a specific microorganism. This time period is referred to as the D-Value. The issue is that the D-value is determined in a very specific vessel or enclosure specific to each manufacturer. When it comes to the types of enclosures subsequently decontaminated by different hydrogen peroxide technologies, they vary from isolators, filling enclosures, and pass-throughs to other smaller vessels and larger laboratory spaces. For this reason, any resistance performance certified by the BI manufacturer becomes a reference value only for customers. Companies utilizing H₂O₂ for decontamination must then become familiar with how biological indicators perform in their specific enclosures and spaces. The D-value or resistance performance noted in those areas can be trended and compared to the Certificate of Analysis data to make future performance more predictable. 

Depending on the size of the enclosure or space as well as the number of BIs used, estimated spore log reduction may be used to target additional spore log reductions for future decontamination validations.  

BI Spore Population Log Reduction Probable Percent Positive BIs 
10^6 0 100% 
10^5 1 100% 
10^4 2 100% 
10^3 3 100% 
10^2 4 100% 
10^1 5 100% 
10^0 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 

Replicating BIs at each location can provide even greater accuracy of spore log reduction by using the Halvorson-Ziegler Most Probable Number equation. 

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Learn more about steramist validation

Biological indicators are the chosen tool for validating successful decontamination practices. The challenges of utilizing biological indicators can be overwhelming and knowing the next steps can be difficult. Trust the original decontamination experts at TOMI to assist you in navigating through these validation processes.