For paper carrier biological indicators, remove three specimens of the relevant biological indicators from their original individual containers. Disperse the paper into component fibers by placing the test specimens in a sterile 250-mL cup of a suitable blender containing 100 mL of chilled, sterilized Purified Water and blending for a time known to be adequate to achieve a homogeneous suspension. It is not unusual for blending times of 15 minutes or more to be required for optimal recovery. Transfer a 10-mL aliquot of the suspension to a sterile, screw-capped 16- × 125-mm tube. For Biological Indicator for Steam Sterilization, Paper Carrier, heat the tube containing the suspension in a water bath at 95 to 100 for 15 minutes (heat shock), starting the timing when the temperature reaches 95. For Biological Indicator for Dry-Heat Sterilization, Paper Carrier, and for Biological Indicator for Ethylene Oxide Sterilization, Paper Carrier, heat the tube containing the suspension in a water bath at 80 to 85 for 10 minutes, starting the timing when the temperature of the spore suspension reaches 80. Cool rapidly in an ice-water bath at 0 to 4. Transfer two 1-mL aliquots to suitable tubes, and make appropriate serial dilutions in sterilized Purified Water, the dilutions being selected as calculated to yield preferably 30 to 300 colonies, but not less than 6, on each of a pair of plates when treated as described below. Where the biological indicator has a low spore concentration, it may be necessary to modify the dilution series and to use more plates at each dilution. Prepare a separate series of plates for each aliquot. Place 1.0 mL of each selected dilution in each of two 15- × 100-mm Petri dishes. Within 20 minutes, add to each plate 20 mL of Soybean–Casein Digest Agar Medium that has been melted and cooled to 45 to 50. Swirl to attain a homogeneous suspension, and allow it to solidify. Incubate the plates in an inverted position at 55 to 60 for Biological Indicator for Steam Sterilization, Paper Carrier, and at 30 to 35 for Biological Indicator for Ethylene Oxide Sterilization, Paper Carrier and for Biological Indicator for Dry-Heat Sterilization, Paper Carrier or at the optimal recovery temperature specified by the manufacturer. Examine the plates after 24 and 48 hours, recording for each plate the number of colonies; and use the number of colonies observed after 48 hours to calculate the results. Calculate the average number of spores per specimen from the results, using the appropriate dilution factor. The test is valid if the log number of spores per carrier at 48 hours is equal to or greater than the log number after 24 hours in each case. For Biological Indicator for Steam Sterilization, Self-Contained, aseptically remove the three carriers from the container, and proceed as directed for Biological Indicator for Steam Sterilization, Paper Carrier.

For Biological Indicators for Moist Heat, Dry Heat, and Gaseous Modes of Sterilization, Nonpaper Carriers, aseptically remove the three carriers from their original packaging or container. Place each carrier in a suitable sterile container containing 100 mL of chilled Purified Water, and sonicate or shake on a reciprocal shaker for an appropriate time. Fifteen minutes or more may be required for optimal recovery. A previous study should be conducted that ensures that the recovery method results in at least 50% to 300% recovery of the labeled spore viable count. Transfer a 10-mL aliquot of the suspension to a sterile, screw-capped 16- × 125-mm tube. Heat the tubes containing suspensions of Bacillus atrophaeus, Bacillus subtilis, and Bacillus coagulans at 80 to 85 for 10 minutes. Heat the tubes containing a suspension of Geobacillus stearothermophilus at 95 to 100 for 15 minutes. Start the timing when the lowest temperature of the stated temperature ranges is reached. Cool rapidly in an ice-water bath at 0 to 4. Transfer two 1-mL aliquots to suitable tubes, and make appropriate serial dilutions in Purified Water. The selected dilutions should be those that will preferably yield 30 to 300 colonies but not fewer than 6 on each pair of plates when treated as described below. When the biological indicator has a low spore concentration, it may be necessary to modify the dilution series and to use more plates at each dilution. Prepare a separate series of plates for each aliquot. Place 1.0 mL of each selected dilution in each of two 15- × 100-mm Petri dishes. Within 20 minutes add the aliquot to each plate containing 20 mL of agar that has been melted and cooled to between 45 and 50. Swirl to attain a homogeneous suspension.

For G. stearothermophilus, B. atrophaeus, B. subtilis, and B. coagulans, use Soybean–Casein Digest Agar Medium and incubate the plates in an inverted position aerobically at the following respective temperatures for each microorganism: 55 to 60, 30 to 35, and 48 to 52, or at the optimum temperature specified by the biological indicator manufacturer. Examine the plates after 24 and 48 hours. Record the number of colonies observed on each plate. Calculate the average number of spores per carrier from the results, using the appropriate dilution factor. The test is valid if the log number of spores per carrier at 48 hours is equal to or greater than the log number after 24 hours in each case.

For Biological Indicators for Moist Heat, Dry Heat, and Gaseous Modes of Sterilization, Liquid Spore Suspensions, using G. stearothermophilus, B. atrophaeus, B. subtilis, and B. coagulans as biological indicators, prepare an appropriate serial dilution of the original spore suspension in chilled Purified Water contained in a sterile, screw-capped 16- × 125-mm tube, and proceed with the viable spore count procedures specified under Biological Indicators for Moist Heat, Dry Heat, and Gaseous Modes of Sterilization, Nonpaper Carriers.

Conduct all the tests described in this section under aseptic conditions, using sterilized equipment for nonthermophilic microorganisms. D-value determination for G. stearothermophilus and B. coagulans can be performed in a controlled but unclassified environment.