Materials and methods
BIs. A list of the BIs used in the studies is shown in Table I, which lists the spore species, spore crops, sterilization process, BI configuration, spore carrier, spore population, and
BI lots used. Seventeen different spore crops were used that were produced over a period of five years; samples were tested
from 29 commercial batches of BIs. The BIs tested had spores inoculated onto paper or stainless-steel carriers. Spores inoculated
onto glass fiber discs were not tested. Specific types and lots of microbiological media were used for grow-out time testing
to minimize variability associated with the recovery media aspect of the testing. Each BI lot was produced with a separate
media lot; therefore, 29 different media lots were included in the testing.
Table I: Biological indicator (BI) test sample matrix.
Incubation of BIs.
Unexposed controls and exposed BIs were incubated for seven days and the time to exhibit a nonsterile result was recorded.
G. stearothermophilus BIs were incubated at 60 ± 2 °C in a Smart-Well incubator system (Mesa Laboratories). Grow-out of a BI was detected by measuring
color change in the growth medium and was recorded in 0.01-hour increments. The test BIs were removed from the Smart-Well
incubator after 24 hours and placed in a conventional incubator at 60 ± 2 °C for the remainder of the seven days. Bacillus atrophaeus BIs were incubated in a conventional incubator at 37 ± 2 °C and were monitored for nonsterility by visual inspection at intervals
between 30 and 60 minutes when nonsterile BIs were frequently occurring. Intervals were extended to several hours when there
were less frequent changes in numbers of nonsterile BIs. When it appeared that the majority of the nonsterile BIs were detected,
observation intervals were significantly decreased for the remainder of the seven days. The delayed nonsterile BI would be
detected, but the precise time delay would not be known.
Delayed nonsterile definition. A BI was classified as a delayed nonsterile unit if the timing of the observed growth was 150% or greater than the incubation
time needed for 95% of the other BIs in the test set to indicate the nonsterile response.
Equipment. Moist-heat, H2O2 vapor, and EO gas sterilization processes were performed in resistometers that met the requirements of ISO 18472: 2006 (6).
All CIO2 exposures were performed in a custom resistometer manufactured by ClorDiSys. There are no published guidelines describing
a resistometer for chlorine dioxide gas exposures.
Unexposed controls: Unexposed BIs were used as a control. Sets of 100 BIs for each for the various BI configurations were incubated to provide
a baseline response for each spore type, carrier, media, and incubation condition.
Calculated survival time exposure: The calculated survival time was based on the resistance of the specific spores/carrier combination to a particular sterilization
mode (3–5). This exposure was designed to reduce the number of viable spores by three to four orders of magnitude, which resulted
in each BI having approximately 100 surviving spores. The formula for calculation of the survival time exposure is:
Survival time exposure = D-value x (log10 of the population – 2)
Because the calculated survival time exposure was intended to yield a spore concentration of approximately 100 spores per
BI, all BIs were expected to be nonsterile. If all BIs were not nonsterile, then one had to review the two input values required
for this calculation—the D-value and the population count. The D-value is a more complicated value to measure accurately and
if the D-value is overstated, the calculated survival time exposure would be longer in duration and thus might result in some/all
of the BIs testing as sterile. For a surviving population of approximately 100 spores/BI, all exposed BIs should be nonsterile
100% of the time.
FDA RIT protocol exposure: The FDA RIT protocol exposure requires a sublethal process that, on groups of 100 BIs, yields at least 30 and no more than
80 positive BIs. The most probable number of spores per BI when 30 out of 100 BIs tested are nonsterile is 0.357. The most
probable number of spores per BI when 80 out of 100 BIs tested are nonsterile is 1.609. This results in a practical range
of 0–5 surviving CFU/BI. Additionally, some BIs in every set of 100 would not have any surviving spores (6).