Understanding Biological Indicator Grow-Out Times - Pharmaceutical Technology

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Understanding Biological Indicator Grow-Out Times
This study used biological indicators containing   Geobacillus stearothermophilus spores and a new technology to continuously monitor incubated BIs and record nonsterile results.

Pharmaceutical Technology
Volume 34, Issue 1

MPN and poisson distribution analysis

Figure 6: Biological indicator (BI) incubation times with approximately 1, 2, 3, 4, and 5 spores per BI.
The results from the moist heat sterilization exposures where 30 to 80 nonsterile BIs were observed per 100 tested were further analyzed using MPN estimate of the average number of surviving CFU and the Poisson distribution prediction shown in Table II. Using the assumption discussed above, for the outcome of each sterilization exposure, a predicted number of surviving CFU was assigned to each observed grow-out time; the highest number of surviving CFU predicted in the Poisson distribution analysis was assigned to the shortest observed grow-out times. The next lowest number of surviving CFU was again assigned to the shortest remaining grow-out times; this process was repeated until all predicted values of surviving CFU > 1 were assigned. The remaining grow-out times were then all assigned a value of one surviving CFU. For example, the lot of BIs that had 80 nonsterile BIs after exposure would be predicted to have approximately two BIs with five CFU, six BIs with four CFU, 14 BIs with three CFU, 26 BIs with two CFU, 32 BIs with only one CFU, and the remaining 20 BIs with no viable CFU. Figure 6 illustrates the BI grow-out times for exposed BIs with five to one CFU predicted. A summary of these data are given in Figure 7 and Table IV.

Figure 7: Summary of biologicial indicator incubation for units from 105 to 1 spore.
The first nonsterile outcome recorded from BIs starting incubation with approximately 105 spores was 2:16. This was 37 minutes faster than the time for the first nonsterile BI from the group of BIs starting incubation with ~300 spores. This was three hours and three minutes faster than any BI predicted to have one spore. The time between the first positive and the last positive in each group of BI increases as the predicted population decreases.


Table IV: Grow-out times for biological indicators (BI) with varying numbers of CFU determined by enumeration (105) or prediction based upon Poisson distribution analysis.
First, it is clear that there is an inverse relationship between the number of surviving CFU on a BI and the overall grow-out time. This was predicted because the time required to attain a detectable cell density and/or cumulative metabolic activity requires less time when the starting level of viable CFU is higher than when it is lower. That is, a cell density of 106 cells/mL can be attained in half the time for a starting population of 103 cells/mL compared with a starting population of 1 cell/mL, germination and generation times being equal.

Second, the grow-out times appear to follow a normal distribution for BIs with several hundred surviving CFU; BIs with few surviving CFU have more highly variable grow-out times with a measurable percentage exhibiting relatively long times before being scored as nonsterile. Of the ~1000 nonsterile BIs in the group where 30 to 80 of 100 were nonsterile after exposure, 10 BIs, or ~1%, exhibited a prolonged grow-out time (> 11 hours).

Third, the data support the hypothesis that delayed outgrowth of BIs is observed and limited to situations where a significant number or the majority of exposed BIs have only one surviving CFU.

John R. Gillis, PhD,* is president of SGM Biotech, Inc., 10 Evergreen Dr., Bozeman, MT 59715,
. Gregg A. Mosley is president of Biotest Laboratories in Minneapolis. John B. Kowalski, PhD, is principal consultant of SteriPro Consulting, Sterigenics International, Inc., in Oak Brook, IL. Garrett Krushefski is scientific and technical services manager, Paul T. Nirgenau is a microbiological scientist, and Kurt J. McCauley is a R&D laboratory manager, all at SGM Biotech.

*To whom all correspondence should be addressed.

Submitted: Nov. 13, 2009. Accepted: Dec. 7, 2009.


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