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

Results and discussion

Moist-heat exposures. All moist-heat exposures were performed with self-contained BIs with 105 spores of G. stearothermophilus inoculated onto paper carriers (see Table I).


Table II: Time-to-nonsterile results for Geobacillus stearothermophilus biological indicators (BIs) exposed to moist heat.
Unexposed controls: Nine lots of 100 and one lot of 80 BIs were incubated at 60 2 C to determine a baseline for time to nonsterile results (see Table II). The first nonsterile BI was detected at 2.27 hours. Ninety-five percent of all BIs exhibited nonsterile results by 3.9 hours. The incubation duration between the first nonsterile BI and 95% BIs nonsterile was 1.63 hours. There was a slight tail in the curve due to a small number of BIs exhibiting somewhat longer grow-out times. It was concluded that this distribution of grow-out times was due to natural variation often seen in biological systems.

Calculated survival time exposures at 121 C: Five lots of 100 BIs were exposed to the calculated survival time process and incubated as previously described (see Table II). The average time for the first nonsterile BI for the five lots tested was 2.98 hours. Ninety-five percent of all BIs exposed to these conditions were nonsterile in 4.9 hours. The 4.9 hours of incubation required to observe 95% of the BIs as nonsterile was 20% longer than that found with the unexposed controls. No delayed nonsterile BI's were observed in this series of exposures.

FDA RIT protocol exposures: Three lots of 100 of BIs were exposed to a moist-heat sterilization process that resulted in 30 to 80 nonsterile BIs for each of the lots (see Table II).

The average time for the three lots exposed at 132 C for the first nonsterile BI was 3.42 hours. Ninety-five percent of the BIs exposed to 132 C were nonsterile at 7.0 hours. The incubation duration from first nonsterile BI to 95% nonsterile was 3.58 hours, which was nearly twice as long as that observed for the calculated survival time exposure.

At 134 C, the average time for the three lots tested for the first nonsterile BI was 3.27 hours, which was slightly less than that observed at 132 C. Ninety-five percent of the BIs exposed to 134 C were nonsterile in 8.0 hours, which was one hour longer than at 132 C. The incubation duration from the first nonsterile BI to 95% nonsterile was 4.73 hours, which was about one hour longer than that observed for the exposures at 132 C.

At 135 C, the average time in the three lots for the first nonsterile BI was 3.38 hours. Ninety-five percent of the BIs exposed to 135 C were nonsterile at 7.9 hours. The incubation duration from the first nonsterile BI to 95% nonsterile was 4.73 hours, which was also approximately one hour longer than that observed for the exposures at 132 C.

There were two delayed nonsterile BIs observed, one exposed at 132 C and one exposed at 134 C. The BI exposed to 132 C grew out between 8.27 hours and 120 hours of incubation. The BI exposed to 134 C grew out between 9.38 hours and 168 hours of incubation. The actual grow-out times for these two BIs is believed to be less than 120 and 168 hours, but these BIs were not monitored after 10 hours so the exact grow-out time was not determined.

Using the probability table shown in Part I of this study, the authors projected that 61% of the 335 nonsterile BIs exposed at 132 C and 134 C were likely to contain only one surviving spore (1). The two delayed grow-out results accounted for only 0.6% of the nonsterile BIs.

The grow-out time results for the moist-heat exposures at 121 C, 132 C, 134 C, and 135 C are presented in vertical scatter plots (see Figure 1, Panel A). The vertical scatter plots illustrate the variability in grow-out time for the different exposure temperatures and different lots of BIs. The difference in grow-out time results between the survival time and RIT exposures is clearly illustrated.

The 121 C calculated survival time exposure results were much more consistent from first to last nonsterile BI grow out. There were a few BIs that were slower to grow out, but the delay was not significant. Lots 1, 2, 3, and 4 had one to three BIs that were noticeably slower in grow out; the delay average was approximately 0.5 hours.

All of the RIT exposure results were much more variable than the results for the calculated survival exposures. The grow-out time results for the moist-heat exposures are graphically illustrated in Figure 2, Panel A.


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