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Table 1. Biological indicator performance data for stream sterilization processes. For use in monitoring the 250°F (121°C), gravity and 270°F (132°C) vacuum assisted steam sterilization process. Organism: Geobacillus stearothermophilus ATCC 7953
Population (mean/strip): 3.7x106 CFU Determined at time of manufacture. Population is reproducible only under the exact conditions under which it was determined.
Resistance Testing Data Test D-value (121°C): 1.6 minutes
Survival time (121°C): 7.3 minutes Kill time (121°C): 16.9 minutes
Survival/kill is verified and D-value is determined in a BIER vessel using a gravity cycle. D-values are determined by a fraction negative procedure after graded exposures to sterilization condi- tions. D-value is reproducible only under the exact conditions under which it is determined. User would not necessarily obtain the same results and would need to determine the biological indicators suitability for their particular use.
The D-value is defined as the decimal reduction value, which indicates the resis- tance of the BI. The larger the D value, the more resistant the microorganism is to destruction.1 D-value testing is determined in a biological indicator evaluation resis- tometer (BIER) test vessel that has a small chamber, no come-up-time or load. For BIs used for steam sterilization, this testing is done at 121°C (250°F). The D-value is the exposure time required to secure inactiva- tion of 90 percent of a population of test organisms under stated conditions. For example, if a BI used for steam steriliza- tion states that the D-value (121°C) is 1.6 minutes, it means 90 percent of the spore population is killed in the first 1.6 min- utes of a 121°C steam sterilization cycle. During the next 1.6 minutes, 90 percent of the remaining spore population is killed. By this data, you can tell that all spores do not die at the same time. There is a transi- tion period between all spores surviving and all spores being killed. During this transition period, when some negative and some positive BIs are obtained, the cycle is described as consisting of marginal steril- ization conditions.
Biological indicator performance is also defined by survival/kill values. This also relates to the resistance of the biological indicator. The survival time is the time at which all spores in the BI will still be alive. The kill time is the time at which all spores in the BI will be killed. The survival and
kill value can be determined by testing in a BIER test vessel or can be calculated based on the spore count and the D-value. BI performance data should be included in each package of product, usually in a Certificate of Analysis.
Evolution of biological indicators In the 1970s, self-contained BIs first became commercially available. Self-contained BIs had three major advantages over multi- component BIs. First, they eliminated the need to aseptically transfer the spore strip to a liquid growth media by combining the spore strip (or carrier) and a crush- able glass ampoule in the same container. This addressed the common contamina- tion problem of spore strips. Second, the addition of a pH dye, which turned yellow when microbial growth produced acidic by-products, was used to detect positives in place of observing for cloudy media indicating microbial growth. This greatly simplified interpretation of the results and put BI testing in the hands of the steriliza- tion departments rather than the micro- biology laboratory. The third advantage is faster read-out times. As refinements in recovery media were developed, they resulted in shorter required incubation times. These advantages have resulted in the elimination of spore strips that require aseptic transfer to media and incubation wherever possible. These advantages, plus
labor and time savings, have resulted in the widespread use of self-contained BIs. The need to verify the efficacy of the sterilization process in a shorter time period has been becoming more important because of the turnover demands on the sterilization department, the complexity of medical devices being introduced, and the need to save time and control costs. These needs drove the development of rapid readout BIs. Rapid readout BIs (indi- cators with enzyme-based early-readout capability) are identical to the original self-contained BIs with one major excep- tion: The glucose in the media has been removed and replaced by a glucoside (or equivalent glucosidase substrate) with a fluorescent indicator dye attached. Spores that have not been destroyed by a steriliza- tion process and are biologically active are demonstrated in a much shorter period of time because, as soon as the glucoside is broken down, the fluorescent dye becomes detectable in trace amounts. Spores do not need to multiply to release the dye from the glucose substrate. A proper ster- ilization process will sufficiently destroy cellular components so that microbes are no longer able to grow. Following a proper sterilization process, neither detectable enzymatic activity is present nor is the cell able to grow or multiply. An auto-reader detects the presence of the naturally occurring enzyme, which is an intrinsic component produced by the
Table 2. Biological indicator performance data for ethylene oxide sterilization processes. For use in monitoring the ethylene oxide sterilization process. Organism: Bacilus atrophaeus ATCC 9372
Population (mean/strip): 3.9x106 CFU Determined at time of manufacture. Population is reproducible only under the exact conditions under which it was determined.
Resistance Testing Data Test D-value (121°C): 3.4 minutes
Survival time (121°C): 15.99 minutes Kill time (121°C): 36.99 minutes
Survival/kill is verified and D-value is determined in a BIER vessel at 54°C, 60 percent RH, 600 mg ethylene oxide/liter. D-values are determined by a fraction negative procedure after graded exposures to sterilization conditions. D-value is reproducible only under the exact conditions under which it is determined. User would not necessarily obtain the same results and would need to determine the biological indicators suitability for their particular use.
hpnonline.com • HEALTHCARE PURCHASING NEWS • June 2023 41
Self-Study Test Answers:1. D, 2.C, 3. B, 4. D, 5.B, 6. B, 7. D, 8. A, 9. A, 10. B
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