h55i BIOLOGICAL INDICATORS— RESISTANCE …

1 H55iBIOLOGICAL INDICATORS RESISTANCE PERFORMANCETESTSC hangetoread:TOTAL VIABLE SPORE COUNT&For paper carrier BIOLOGICAL indicators, remove three specimensof the relevant BIOLOGICAL indicators&1S (USP31)from their originalindividual containers. Disperse the paper into component fibers byplacing the test specimens in a sterile 250-mL cup of a suitableblender containing 100 mL of chilled, sterilizedPurified Waterandblending for&a time known to be adequate&1S (USP31)to achieve ahomogeneous is not unusual for blending times of 15minutes or more to be required for optimal (USP31)Transfer a 10-mL aliquot of the suspension to a sterile, screw-capped 16-6125-mm tube. ForBiological Indicator for SteamSterilization, Paper Carrier,heat the tube containing the suspensionin a water bath at 958to 1008for 15 minutes (heat shock), startingthe timing when the temperature reaches 958. ForBiologicalIndicator for Dry-Heat Sterilization, Paper Carrier,and forBiological Indicator for Ethylene Oxide Sterilization, Paper Carrier,heat the tube containing the suspension in a water bath at 808to 858for 10 minutes, starting the timing when the temperature&of thespore suspension&1S (USP31)reaches 808.

2 Cool rapidly in an ice-waterbath at 08to 48. Transfer two 1-mL aliquots to suitable tubes, andmake appropriate serial dilutions in sterilizedPurified Water,thedilutions being selected as calculated to yield preferably 30 to 300colonies, but not less than 6, on each of a pair of plates when treatedas described below. Where the BIOLOGICAL indicator has a low sporeconcentration, it may be necessary to modify the dilution series andto use more plates at each dilution. Prepare a separate series of platesfor each aliquot. Place mL of each selected dilution in each oftwo 15-6100-mm Petri dishes. Within 20 minutes, add to eachplate 20 mL ofSoybean Casein Digest Agar Medium&&1S (USP31)that has been melted and cooled to 458to 508. Swirl to attain ahomogeneous suspension, and allow&it&1S (USP31)to the plates in an inverted position at 558to 608forBiological Indicator for Steam Sterilization, Paper Carrier,and at308to 358forBiological Indicator for Ethylene Oxide Sterilization,Paper Carrierand forBiological Indicator for Dry-Heat Steriliza-tion, Paper Carrieror at the optimal recovery temperature specifiedby the manufacturer.

3 Examine the plates after 24 and 48 hours,recording for each plate the number of colonies; and&use&1S (USP31)the number of colonies&observed&1S (USP31)after 48 hours tocalculate the results. Calculate the average number of spores perspecimen from the results, using the appropriate dilution factor. Thetest is valid if the log number of spores per carrier at 48 hours isequal to or greater than the log number after 24 hours in each Indicator for Steam Sterilization, Self-Contained,aseptically remove the&three carriers&1S (USP31)from the container,and proceed as directed forBiological Indicator for SteamSterilization, Paper Indicators for Moist Heat, Dry Heat, andGaseous Modes of Sterilization, Nonpaper Carriers,asepticallyremove the three carriers from their original packaging or each carrier in a suitable sterile container containing 100 mL ofchilledPurified Water,and sonicate or shake on a reciprocal shakerfor an appropriate time.

4 Fifteen minutes or more may be required foroptimal recovery. A previous study should be conducted that ensuresthat the recovery method results in at least 50% to 300% recovery ofthe labeled spore viable count. Transfer a 10-mL aliquot of thesuspension to a sterile, screw-capped 16-6125-mm tube. Heat thetubes containing suspensions ofBacillus atrophaeus, Bacillussubtilis,andBacillus coagulansat 808to 858for 10 minutes. Heatthe tubes containing a suspension ofGeobacillus stearothermophilusat 958to 1008for 15 minutes. Start the timing when the lowesttemperature of the stated temperature ranges is reached. Cool rapidlyin an ice-water bath at 08to 48. Transfer two 1-mL aliquots tosuitable tubes, and make appropriate serial dilutions selected dilutions should be those that will preferablyyield 30 to 300 colonies but not fewer than 6 on each pair of plateswhen treated as described below. When the BIOLOGICAL indicator has alow spore concentration, it may be necessary to modify the dilutionseries and to use more plates at each dilution.

5 Prepare a separateseries of plates for each aliquot. Place mL of each selecteddilution in each of two 15-6100-mm Petri dishes. Within 20minutes add the aliquot to each plate containing 20 mL of agar thathas been melted and cooled to between 458and 508. Swirl to attain ahomogeneous stearothermophilus, B. atrophaeus, B. subtilis, ,useSoybean Casein Digest Agar Mediumand incubatethe plates in an inverted position aerobically at the followingrespective temperatures for each microorganism: 558to 608, 308to358, and 488to 528, or at the optimum temperature specified by thebiological indicator manufacturer. Examine the plates after 24 and 48hours. Record the number of colonies observed on each the average number of spores per carrier from the results,using the appropriate dilution factor. The test is valid if the lognumber of spores per carrier at 48 hours is equal to or greater thanthe log number after 24 hours in each Indicators for Moist Heat, Dry Heat, and GaseousModes of Sterilization, Liquid Spore Suspensions, , B.

6 Atrophaeus, B. subtilis,andB. coagulansasbiological indicators, prepare an appropriate serial dilution of theoriginal spore suspension in chilledPurified Watercontained in asterile, screw-capped 16-6125-mm tube, and proceed with theviable spore count procedures specified underBiological Indicatorsfor Moist Heat, Dry Heat, and Gaseous Modes of Sterilization,Nonpaper (USP31)Changetoread:D-VALUE DETERMINATION&Conduct all the tests described in this section under asepticconditions, using sterilized equipment for nonthermophilic microor-ganisms. D-value determination forG. be performed in a controlled but (USP31)Apparatus&The test equipment for the determination of microbial resistanceis described in substantial detail in ISO 18472,Sterilization ofHealth Care Products BIOLOGICAL and Chemical Indicators details of individual BIOLOGICAL Indicator Evalua-tion Resistometers (BIERs) vary with the specifics of their designand the particular sterilization process in conjunction with whichthey are used.

7 Provided that the performance of the BIER vesselmeets the requirements of the ISO standard for exposure of thebiological indicator, design differences are (USP31)ProcedureCarry out the tests for D value at each of the applicable sets ofsterilization conditions for which the packaged BIOLOGICAL indicatorunder test is labeled for use. Take a sufficient number of groups ofspecimens of BIOLOGICAL indicators in their original individualcontainers, each group consisting of&not less than 5&1S (USP31)specimens. The number of groups provides a range of observationsfrom not less than one labeled D value below the labeled survival1 ANSI/ AAMI/ ISO 18472 : 2006, Sterilization of Health Care Products BIOLOGICAL and Chemical Indicators Test Equipment. Association for theAdvancement of Medical Instrumentation (AAMI), 1110 Road, Suite220, Arlington, VA 22201-4795 First Supplement, USP NFMicrobiological Tests/h55iBiological Indicators1time through not less than one labeled D value above the labeled killtime.

8 Place each group on a separate suitable specimen holder thatpermits each specimen to be exposed to the prescribed sterilizingcondition at a specific location in the sterilizing chamber&of (USP31)Check the&BIER&1S (USP31)apparatus for operatingparameters using specimen holders without specimens. Select aseries of sterilizing times in increments from the shortest time for thespecimens to be tested. The differences in sterilizing times over theseries are as constant as feasible, and the difference between adjacenttimes is no greater than 75% of the labeled D procedures for the use of BIER vessels for the evaluation ofmicrobial RESISTANCE are defined in a series of ISO standards underthe 11138 5 The appropriate standard should be followed forthe BIOLOGICAL indicator. The test methods and carriers used with theBIER may be adapted to the specifics of the BIOLOGICAL indicator. Themethod and apparatus used for paper carriers may differ from thosefor other carriers and will be substantially different from those usedfor suspensions of BIOLOGICAL D-value exposure conditions for alternative material carriersare the same as the conditions used to determine the D value forpaper carriers.

9 If the manufacturer s label permits usage of thebiological indicator carrier with multiple sterilization methods, thendata on D value, survival time, and kill time will need to be providedby the manufacturer for each sterilization method. It is possible thatbiological indicators inoculated onto carriers other than paper will beused for gaseous or vapor sterilization/decontamination methodssuch as vapor phase hydrogen peroxide and chlorine physical conditions for the evaluation of biologicalindicators for use with vapor phase hydrogen peroxide or chlorinedioxide have not been defined. In the case of chlorine dioxide,concentration of the gas, relative humidity, and temperature arecritical process control conditions that can be accurately manufacturer of BIOLOGICAL indicators marketed for use withchlorine dioxide should state the conditions under which the D-valuedetermination was conducted so that the user can at least discern theresistance of a lot of BIOLOGICAL indicators as compared to their ownanticipated use conditions.

10 The situation with vapor phase hydrogenperoxide is a more complex one. Various equipment manufacturershave proposed different decontamination or sterilization , there is no standard process for the conduct of vapor phasehydrogen decontamination or surface sterilization. It follows, then,that there are no industry standard BIOLOGICAL indicator evaluationmethods for vapor hydrogen peroxide, and it has been reported thatthere may not be a direct correlation between vapor concentrationand rate or even effectiveness of BIOLOGICAL indicator , it is difficult to accurately assess relative humidity,which is often defined as a critical process parameter, in the presenceof vapor hydrogen peroxide. For these reasons it is more reasonableto consider RESISTANCE of BIOLOGICAL indicators to be a relative orcomparative measure from the manufacturer rather than a true Dvalue. It follows that, depending upon equipment and processesemployed, it may be impossible for an end user to duplicate thebiological indicator RESISTANCE tests performed by the (USP31)&ForBiological Indicators for Moist Heat, Dry Heat, andGaseous Modes of Sterilization, Liquid Spore Suspensions,conductD-value determinations for each of the microorganisms that areprovided as a liquid spore crop suspension.