There is no universally recognized method to establish an acceptable minimum incubation time (MIT) of less than seven days for biological indicators (BIs) used to monitor the effectiveness of sterilization processes. A method to qualify a reduced incubation time (RIT) of less than seven days was published by FDA in the Center for Device and Radiological Health (CDRH) Guide for the Validation of Biological Indicator Incubation Time in 1986 (1). The international community, however, has been reluctant to accept this method due to the lack of published results to support the chosen test requirements and acceptance criteria. In 2007, the ISO/TC198 Working Group 4 (WG4), Biological Indicators, initiated a New Work Item Proposal (NWIP) to develop an ISO Technical Specification (TS) that would contain a method for validation of an MIT for BIs. The position taken by ISO WG4 was that any proposed method should be supported by results published in a peer-reviewed technical journal. At the request of the ISO WG4, the Association for the Advancement of Medical Instrumentation (AAMI) BI Working Group 4 formed an ad hoc committee in 2008 to develop a plan for such research and the publication of the results. To date, two articles have been published that contain results that support a rationale for a method to determine an MIT for BIs (2, 3).
This article is a result of the ad hoc committee’s efforts and proposes a method to establish an MIT for BIs. The method is based on an analysis of the grow-out times for more than 10,000 BIs exposed to a variety of sterilization processes (2, 3). The BIs used in these studies were manufactured in conformance to ISO 11138-1 and specific subsections, as appropriate, and released for commercial use (4). The manufacturer’s incubation instructions were followed as appropriate.
Study design and key findings
The studies included several BI configurations, including both self-contained BIs and conventional spore strips. The spore carriers included both paper and stainless steel. The two most common species of bacterial endospores used in the manufacture of BIs, Geobacillus stearothermophilus and Bacillus atrophaeus, were tested. The growth media for the studies were controlled and included media ampuls for self-contained BIs and tubed media for spore strips as previously described (2, 3).
Separate groups of BIs were evaluated under the following conditions:
- Exposed to the various sterilization processes in a manner that resulted in 30 to 80% of the BIs testing nonsterile (positive for growth). This quantal zone result, in which a fraction of the BIs are sterile and a fraction are nonsterile, meets the requirement of the FDA CDRH protocol
- Exposed using the times calculated from the Calculated Survival Time (CST) formula published in ISO 11138-1, ISO 14161, and the
United States Pharmacopoeia (4-6).
All exposed BIs were incubated until a nonsterile result was observed or for a maximum of seven days. A nonsterile result was based on evidence of microbial growth such as turbidity and/or a change in color of the pH indicator in the growth medium.
Key findings from these grow-out time studies that are relevant to identification of a results-based MIT method are listed as follows:
- The ranges of grow-out times observed approximate a normal distribution irrespective of the type of sterilization process and associated lethal mechanism(s) or the spore species on the BI.
- An inverse relationship exists between the number of surviving spores on an exposed BI and the observed grow-out time. BIs that have a greater number of surviving spores have a shorter grow-out time than those that have fewer surviving spores.
- Prolonged grow-out times were found for a small percentage (~1.1%) of the BIs tested. Such prolonged grow-out times were observed with all of the sterilization processes (moist heat, EO, H2O2 vapor, and ClO2 gas) and only occurred when the sterilization exposure gave a quantal-zone result.
- BIs demonstrating prolonged grow-out times were not observed when exposed to a sterilization exposure at the CST. BIs exposed in this manner had faster and more consistent grow-out times when compared to BIs exposed under conditions that gave quantal-zone results.
Rationale for proposed MIT method
One of the issues negatively affecting the widespread acceptance of the FDA CDRH protocol is the lack of test results to support the method itself and its acceptance criteria. The FDA CDRH protocol requires the performance of three sterilization exposures, each of which use 100 BIs and must result in 30 to 80% of the BIs testing nonsterile. Critics view the 30 to 80% values and the additional requirement for greater than 97% correlation of the RIT results to the seven-day incubation time results as arbitrary. The international community has consistently raised the issue of why 30 to 80% survival is appropriate and why a 100% correlation to the seven-day incubation time results is not required. No rationale or supporting test results have been presented to justify the use of the 30 to 80% nonsterile and the greater than 97% correlation values.
The exposure of microorganisms in general, and spores in particular, to a sterilization process results in a range of effects characteristic of the sterilization process itself and the extent of treatment. As the extent of treatment progresses, a BI has fewer and fewer surviving spores until, at some treatment level, there are no surviving spores. In the quantal zone, some of the BIs have only one surviving spore and others have a low number of survivors.
For exposed BIs that have only one surviving spore, one of two conditions can exist: the spore has not been damaged in a manner that significantly affects either germination time or post-germination growth rate, or damage has occurred that significantly affects one or both of these characteristics. When a sterilization exposure gives a quantal-zone result of 50% of the BIs testing nonsterile, the Poisson distribution predicts approximately 35 BIs will have only one surviving spore and the remaining 15 will have more than one surviving spore (2). From the results of the previously reported grow-out time studies, it is clear that most of the BIs with only one surviving spore, and, most likely, all the BIs that have more than one surviving spore, exhibit grow-out times within a relatively narrow range. Also, a very low number of BIs would be expected to exhibit a prolonged grow-out time; prolonged times were observed for 2/458 (0.44%) and 3/309 (0.97%) BIs for moist heat and EO sterilization processes, respectively.
BIs that have prolonged grow-out times are more likely to occur when the exposure conditions result in a lower percentage being nonsterile, for example 35% versus 75%. For a 35% nonsterile result, the average number of viable spores per exposed BI is 0.43 versus 1.39 when 75% of the BIs test nonsterile. As the percent of nonsterile BIs decreases, proportionally more of the nonsterile BIs will have only a single viable spore. For a 35% nonsterile result, approximately seven of the 35 nonsterile BIs have two or more surviving spores; for a 75% nonsterile result, approximately 40 of the 75 nonsterile BIs have two or more surviving spores (2).
To develop a viable MIT method, the use of a sterilization exposure that results in a consistent low number of surviving spores is an important prerequisite. Treatment of BIs in a resistometer at an exposure time specified by the CST calculation will result in a more consistent and reproducible method to determine an MIT as opposed to producing 30 to 80% nonsterile BIs.
The CST, as indicated in Equation 1, is an exposure time that takes into account both the spore population level and the D10 value and is intended to result in 100% of the BIs testing nonsterile (4-6). A BI subjected to a CST exposure would be expected to have approximately 100 surviving spores; the results of the previous grow-out time studies showed that when BIs were exposed to the CST, no prolonged grow-out times were observed in 1297 CST exposures.
CST = (D10value) x (log10 population level - 2) Eq. 1.
where the CST and D10 value are in minutes. The extent of treatment for a CST exposure is marginally less than that used to obtain the 30 to 80% nonsterile result required by the FDA CDRH protocol. BIs treated in a CST sterilization exposure will challenge the incubation medium and conditions with ~100 spores that have received a significant lethal insult (~99.99 % of the spores inactivated due to the lethal action(s) of the sterilizing agent). This level of microorganisms is similar to that used in the microbiological-medium growth promotion testing and bacteriostasis/fungistasis testing where inoculations are performed with low levels (≤ 100) of test microorganisms (7).
Proposed MIT method
Based on the results of the testing summarized in the two previously published studies and the subsequent analysis of the data, the following method for determination of an MIT for BIs is proposed:
- Obtain a minimum of 100 BIs from each of three lots, which are produced from different spore crops.
- Determine the D10 value and population for each lot.
- Calculate the survival time for each BI lot using Equation 1.
- Expose each lot of BIs at the CST in separate resistometer runs.
- Use recovery methods specified by the BI manufacturer (or specified growth medium) and defined environmental conditions.
- Incubate until all BIs test nonsterile or until seven days incubation time has been completed. (Note: If all BIs do not test nonsterile after seven days of incubation, reconfirm D10 value and population.)
- Record the time for all BIs to test nonsterile.
- The MIT is the longest time found for a nonsterile result for the three BI lots.
For determination of the CST, it is recommended that the D10 value be calculated using the Holcomb-Spearman-Karber method (8). This method uses all of the fraction negative results from the series of sterilization exposures to calculate the “mean time to sterility” and associated D10 value. A D10 value calculated in this manner is more likely to be the most accurate and provide the best D10 value estimate from a statistical perspective.
In developing a new method for MIT determination, it is important to consider the evolution of the practices for monitoring and control of sterilization processes during the past 25 or more years. For example, early moist-heat sterilizers had only a chamber pressure gauge and a thermometer in the drain line to monitor the process. Active electromechanical control of the process did not exist, and, consequently, the results of BI testing were critical to determining process efficacy.
Today, we operate under the umbrella of process validation. Validation requires comprehensive documentation of the physical/chemical aspects of each sterilization cycle. The documentation must be sufficient to show that the process was executed as expected and that the process specification was met in its entirety. Modern sterilizers, in contrast to early vessels, have sophisticated monitoring and control systems that provide detailed run records for each sterilization process.
In current practice, results of BI testing are subordinate to the recorded physical/chemical results; a sterilization run is nonconforming if the physical/chemical requirements are not met even though all BIs are inactivated. The use of BIs in the current context of sterilization process-monitoring control must be matched to our current understanding of BI outgrowth.
Extensive grow-out time testing for BIs exposed to four different sterilization processes under controlled conditions has given a very clear picture of the kinetics of spore germination and outgrowth for both unexposed BIs and BIs exposed to differing extents of sterilant treatment. Unexposed BIs grow out the most rapidly and exhibit the least variation in grow-out time. As would be expected, BIs exposed to a sterilization process exhibit longer grow-out times on average and also show more variability in grow-out times. The longer average grow-out time is clearly related to the presence of a lower number of viable spores after sterilant exposure and also likely due to some damage to the spore metabolic and/or genetic apparatus.
BIs exposed to a sterilant that results in a quantal-zone outcome demonstrate more variability than unexposed BIs but also show a low frequency of BIs with a prolonged grow-out time. The MIT method proposed in this paper using the CST sterilization exposure takes into account this increased variability but properly avoids the occurrence and inappropriate influence of “outlier” results. In contrast, an unforeseen consequence of the 30-80% survival window of the FDA CDRH protocol is that the RIT is driven by the outlier results of large percentages of BIs having only a single spore. This effect can be seen in the fact that a shorter incubation time can be achieved if the average nonsterile results are closer to 80% versus 30%, which is a result attributed to the difference in the fraction of nonsterile BIs having only a single surviving spore. This consequence is clearly undesirable for a test method and demonstrates a lack of robustness and reproducibility.
The adoption of the proposed MIT method would not be predicted to reduce the effectiveness of BIs for monitoring and control of sterilization processes but rather provide a means for reproducible determination of BI grow-out time.
- FDA, Guide for the Validation of Biological Indicator Incubation Time (Rockville, MD, June, 1986).
- J.R. Gillis et al., Pharm. Tech. online, “Understanding Biological Indicator Grow-Out Times,” www.pharmtech.com/BIgrowout, Jan. 2, 2010, accessed May 6, 2013.
- J.R. Gillis, et al., Pharm. Tech. 37 (6) 52-59 (2013).
- ISO 11138-1:2006 Sterilization of Health Care Products--Biological Indicators--Part 1: General Requirements (ISO, Geneva, 2006).
- ISO 14161:2009 Sterilization of Health Care Products--Biological Indicators--Guidance for the Selection, Use and Interpretation of Results (ISO, Geneva, 2009).
- USP 36-NF 31 (US Pharmacopeial Convention, Rockville, MD 2013), p. 2659-2662.
- USP General Chapter <61>, “Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests” (US Pharmacopeial Convention, Rockville, MD 2013).
- I.J. Pflug, R.G. Holcomb, and M.M. Gomez, “Thermal Destruction of Microorganisms,” in Disinfection, Sterilization, and Preservation, S. Block, Ed. (Lippincott, Williams, & Wilkins, Philadelphia, PA, 2001), pp. 79-129.