Understanding Biological Indicator Grow-Out Times—Part II - Pharmaceutical Technology

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Understanding Biological Indicator Grow-Out Times—Part II
In Part II of a series, the authors report on the range and distribution of grow-out times for biological indicators exposed to sublethal sterilization processes.


Pharmaceutical Technology
Volume 37, Issue 6, pp. 52-59

Hydrogen peroxide vapor exposures. All H2O2 exposures were performed with self-contained BIs with 106 spores of G. stearothermophilus inoculated onto stainless steel discs (see Table I).


Table III: Time-to-nonsterile results for Geobacillus stearothermophilus biological indicators (BIs) exposed to hydrogen peroxide vapor.
Unexposed controls: Three lots of 100 BIs were incubated at 60 2 C to determine a baseline for the time required for BIs to exhibit nonsterile results (see Table III). The average time for the first nonsterile BI in the three lots was 2.98 hours. Ninety-five percent of all BIs tested exhibited nonsterile results by 5.3 hours. The incubation duration between the first nonsterile BI and 95% BIs nonsterile was 2.3 hours.

Calculated survival time exposures: Three lots of 100 BIs were exposed to the calculated survival time and incubated as previously described (see Table III). The average time for the first nonsterile BI in the three lots was 3.54 hours. Ninety-five percent of the BIs were nonsterile in 6.0 hours. This incubation duration from the first nonsterile BI to 95% nonsterile BIs was 2.46 hours. This result was very similar to that observed with the unexposed controls. Two of the three lots tested did not yield 100 nonsterile BIs; one lot had 99 nonsterile BIs and the second lot had 98 nonsterile BIs. It is believed that the calculated survival time exposure was inappropriately long due to inaccuracy of the D-value determination. If the D-value is overstated, the average number of surviving spores would be less than approximately 100 resulting in some of the BIs being sterilized in the survival time exposure.

One delayed nonsterile BI was observed in one of the lots tested. For this BI, the grow-out time was 4.11 hours longer than that of the adjacent nonsterile test result. This delayed nonsterile BI took 223% longer to exhibit nonsterility than 95% of the nonsterile BIs. This was the only delayed nonsterile BI observed for the calculated survival time exposures for any of the processes examined in this study.

FDA RIT protocol exposures: Three lots of 100 BIs were exposed to H2O2 vapor that resulted in 30 to 80 BIs nonsterile per lot (see Table III). The average time for the first nonsterile BI for the three lots was 4.7 hours. Ninety-five percent of the BIs were nonsterile by 9.9 hours of incubation. The duration of incubation from the first nonsterile BI to 95% nonsterile was 5.2 hours, which was nearly twice as long as that for the BIs exposed to the calculated survival time exposure.

There were also three delayed nonsterile BIs observed; one BI from one lot and two BIs from another lot. The delayed grow-out time was approximately 5.5 hours longer than that of the adjacent nonsterile test results. This incubation duration was 190% of the time to 95% of the nonsterile BIs.

It appeared that significant nonlethal spore damage occurred at this exposure condition, which was designed to yield a surviving population of 0 to 5 CFU (7–9). Using the probability table in Part I of this study, the authors projected that 66% of the 160 nonsterile BIs would contain only one surviving CFU (1). However, only 2.5% of the nonsterile BIs had a delayed response.

All grow-out time results for all H2O2 exposures are presented in vertical scatter plots in Figure 1, Panel B. They are also presented graphically in Figure 2, Panel B.


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