1、Designation: E 1766 95 (Reapproved 2002)Standard Test Method forDetermination of Effectiveness of Sterilization Processes forReusable Medical Devices1This standard is issued under the fixed designation E 1766; the number immediately following the designation indicates the year oforiginal adoption or
2、, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method covers a reproducible procedure fortesting processes used to
3、sterilize reusable medical devices(instruments). This test method is not designed to validate asterilization process, but tests an established sterilization cycleor process. It is a practical test of the effectiveness of asterilization process applied to reusable medical devices. Bac-terial spores m
4、ore resistant to the test sterilant than the naturalbioburden of the instrument are used as the test organisms.Commercially available liquid suspensions of bacterial sporesare used to inoculate the instruments.1.2 This test method is intended for reusable medicaldevices cleaned in accordance with th
5、e device manufacturersinstructions and prepared for sterilization in accordance withthe instructions for the sterilization process being used.1.3 This test method assumes that cleaned, reusable medicaldevices will be free of visible soil but may have remainingadherent bioburden.Aworst-case bioburden
6、 can be representedby suspensions of bacterial endospores, which are commer-cially available for monitoring chemical or physical steriliza-tion processes. These endospores should have a verifiableresistance (D value) to the specific process and sterilant beingevaluated.21.4 It is impractical to test
7、 for the sterility of some devicesby immersion in growth medium because of their complexity,size, and availability (for long-term incubation) or adverseeffects on the devices from long-term immersion. Therefore,elution, rinsing, or swabbing techniques are used to recover testorganisms from inoculate
8、d devices.1.5 A recovery control will be included by inoculation of atest device and use of the elution methods without applying thesterilization process being tested. A minimal recovery of 106colony-forming unit (CFU)/mL per device is required for therecovery control.1.6 Results of the recovery con
9、trol and process test cycle arecompared to determine the effectiveness of the sterilizationprocess.1.7 Results of the recovery control and applied inoculum arecompared to determine the recovery efficiency, if desired.1.8 The procedure should reveal that tested devices are freeof recoverable microorg
10、anisms when five or more consecutivetests are conducted.1.9 A knowledge of microbiological techniques is requiredto conduct these procedures.1.10 The values stated in SI units are to be regarded as thestandard.1.11 This standard does not purport to address all of thesafety concerns, if any, associat
11、ed with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:3D 1193 Specification for Reagent WaterE 1054 Practices for Ev
12、aluating Inactivators of Antimicro-bial Agents Used in Disinfectant, Sanitizer, Antiseptic, orPreserved Products43. Terminology3.1 Definitions:3.1.1 bioburdenthe number and types of viable microor-ganisms that contaminate a device.3.1.2 CFUcolony-forming unit.3.1.3 inoculumthe number (usually specif
13、ied as CFUs)and type (genus and species) of viable microorganisms used tocontaminate a given sample or device.3.1.4 sporicidal agentany chemical or physical agent thatkills spores.3.1.5 sterilantany sterilizing agent.1This test method is under the jurisdiction of ASTM Committee E35 onPesticides and
14、Alternative Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved October 10, 2002. Published March 2003. Originallyapproved in 1995. Last previous edition approved in 1995 as E 1766 - 95.2Oxborrow, G. S., and Berube, R., “Sterility Te
15、stingValidation of SterilizationProcesses, and Sporicide Testing,” Disinfection, Sterilization, and Preservation,Block, S. S., 4th Edition, Lea and Febiger, Philadelphia, PA, 1991, pp. 10471058.3For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at s
16、erviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.4Withdrawn.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3.1.6 sterilea state of being free of liv
17、ing organisms.3.1.7 sterilization cycle or processa physical or chemicalprocess that has been demonstrated to meet applicable criteriafor sterilization as defined by AAMI.53.1.8 sterilizerany device using a chemical or physicalprocess that produces sterile materials.3.2 Definitions of Terms Specific
18、 to This Standard:3.2.1 applied inoculumthe estimated count of the suspen-sion of bacterial spores expressed as CFU/mL used to inoculatethe test devices. This value may be used if the efficiencies ofthe recovery methods are determined.3.2.2 process test cyclea complete sterilization cycle thatuses a
19、ll parameters of the sterilization process as dictated bythe manufacturer.3.2.3 recovery controlthe CFU recoverable from a devicefollowing inoculation and optional drying of the spore suspen-sion in or on the unprocessed device. The recovery of $106CFUs per device is required.3.2.4 recovery effcienc
20、ya measure of the recovery ofinoculated organisms from a device may be determined whennecessary. The recovery efficiency may be expressed as theratio of the CFU from the recovery control compared to theCFU of the applied inoculum. This value is multiplied by 100to express efficiency as a percent. It
21、 is recommended that aminimum of three tests be performed when estimating recoveryefficiency.3.2.5 reusable medical devicesany medical device that isclaimed to be usable after reprocessing.3.2.6 sporea bacterial endospore. (Strain identificationand the means used to identify whether the vegetative o
22、r sporestate is present should be indicated.)3.2.7 worst-casethe intentional exaggeration of one ormore parameters of a test compared to normal clinical condi-tions.4. Summary of Test Method4.1 Percent recovery of inoculum may be used to ensurereproducible inoculation and recovery techniques.4.2 The
23、 test method is performed by contaminating thecleaned reusable medical device with a bacterial endosporesuspension.4.3 After inoculation, and drying, if required, the device isprepared and processed according to the sterilant or sterilizermanufacturers instructions.4.4 Following the sterilization pr
24、ocess, the test devices aresampled using specified elution techniques to recover anysurviving spores.5. Significance and Use5.1 The test method is designed to demonstrate that allaccessible surfaces and internal recesses or lumina of previ-ously cleaned, reusable medical devices can be rendered free
25、 ofrecoverable microorganisms when processed in a specifiedsterilizer cycle.5.2 Surviving spores are recovered by swabbing, brushing,or irrigating with an elution fluid. Recovery methods may beenhanced by mechanical action, sonication, and repeatedflushing with elution fluid.NOTE 1The spore inoculat
26、ion technique described in this test methodis only one of the available procedures for testing the sterilization ofdevices. Spores on paper strips (biological indocators) are a traditionaltool used to develop and monitor sterilization cycles and are alsoappropriate for the evaluation of sterilizatio
27、n of medical devices.66. Apparatus6.1 Syringes, 10 to 50 mL, sterile.6.2 Sterile Cotton Swabs.6.3 Sterile Petri Dishes.6.4 Sterile Test Tubes, to hold 10 mL.6.5 Sterile Glass Bottles, to hold 50 mL.6.6 Steam Sterilizer.6.7 Water Bath,486 2C.6.8 Incubator(s),356 2C and 55 6 2C.6.9 Colony Counter.6.10
28、 Medical Device, precleaned in accordance with themanufacturers instructions.6.11 Disposable or Reusable Membrane Filter Apparatus,sterile, 0.45-m pore size.6.12 Micropipette, calibrated to dispense 5 to 20 L.6.13 Other devices or apparatus specified by the sterilant,medical device, or sterilizer ma
29、nufacturer.7. Reagents7.1 Purity of ReagentsReagent grade chemicals shall beused in all tests. Unless otherwise indicated, it is intended thatall reagents conform to the specifications of the Committee onAnalytical Reagents of the American Chemical Society wheresuch specifications are available.7Oth
30、er grades may be used,provided it is first ascertained that the reagent is of sufficientlyhigh purity to permit its use without lessening the accuracy ofthe determination.7.2 Purity of WaterUnless otherwise indicated, referencesto water shall be understood to mean reagent water as definedby Type III
31、 of Specification D 1193.7.3 Media:7.3.1 Type III or Better ASTM Water, for making broths andelution fluids.7.3.2 Sterile USP Fluid D8(elution fluid), containingpolysorbate 80 or stripping solution9containing 0.4 g KH2PO4,10.1 g Na2HPO4, and 1.0 g.5See “Guideline for Industrial Ethylene Oxide Steril
32、ization of Medical Devices”(ST27), AAMI, Arlington, VA, 1992, for typical criteria.6United States Pharmacopeia, XXIII, Rand McNally, Taunton, MA, 1995, pp.200206.7Reagent Chemicals, American Chemical Society Specifications, AmericanChemical Society, Washington, DC. For suggestions on the testing of
33、reagents notlisted by the American Chemical Society, see Analar Standards for LaboratoryChemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeiaand National Formulary, U.S. Pharmaceutical Convention, Inc. (USPC), Rockville,MD.8United States Pharmacopeia, XXII, “Sterility Tests, D
34、iluting and RinsingFluids,” Rand McNally, Taunton, MA, 1990, p. 1484.9Williamson, P., “Quantitative Estimation of Cutaneous Bacteria,” Skin Bacteriaand Their Role in Infection, Marbac, H. I., and Hildick-Smith, G., eds., McGraw-Hill, New York, NY, 1965.E 1766 95 (2002)27.3.3 Isooctylphenoxypolyethox
35、y Ethanol (Triton X-100),in 1-L distilled water adjusted to pH 7.8.7.3.4 Soybean-Casein Digest Broth, USP, single or doublestrength, with neutralizers for the test sterilant, as appropriate,and a volume acceptable for each test.7.3.5 Soybean-Casein Digest Agar, USP, single or doublestrength, with ne
36、utralizers for the test sterilant, if appropriate;10 to 50 mL in tubes or bottles, tempered to 48 6 2C.7.4 Test Organisms/Spore Suspension:7.4.1 For moist heat sterilization, standardized spore sus-pensions of Bacillus stearothermophilus containing nominally108CFU/mL and meeting USP resistance crite
37、ria for steamsterilization should be used.107.4.2 For chemical or dry heat sterilization, standardizedspore suspensions of Bacillus subtilis containing nominally 108CFU/mL and meeting USP resistance criteria for ethyleneoxide or dry heat sterilization should be used.97.4.3 For sterilants in which ei
38、ther of the above organismsmay be inappropriate, other indicator organisms may besubstituted, provided there is substantial evidence to indicatethat they are more appropriate.7.4.4 The origin of the spore strain, production, storage, andexpiration dates should be identified.7.5 Neutralizers (as appr
39、opriate)See Practices 1054 forrecommended neutralizers.8. Procedure8.1 Select the devices to be evaluated.8.2 Read the cleaning instructions for each medical deviceto be tested, and ensure that all required accessories areavailable. Clean and dry the device according to the devicemanufacturers instr
40、uctions.8.3 Inoculate the device and include the sites most difficultto sterilize. The number of sites may vary with the complexityof the medical device. The rationale used to identify and verifythe most difficult to sterilize sites must be documented.Inoculation procedures producing a recoverable c
41、ount of 106CFU spores per instrument should be used.8.4 Specific procedures are used for inoculation, elution,control testing, neutralization, growth promotion, and sterilitytesting.8.5 Inoculation of Devices to Determine the AppliedInoculumThese paragraphs describe the steps for enumerat-ing the CF
42、U of spores inoculated onto the medical device beingtested. The spores are not subjected to drying or othertreatments, as they might be when determining the recoverycontrol (see 8.6).8.5.1 Surface Site Inoculation:8.5.1.1 Micropipetter MethodInoculate the surface di-rectly by introducing a volume of
43、 spore suspension contain-ing$ 107spores to the site(s), and distribute over the inocu-lated site(s) with the tip of the pipet. Immerse the device inelution fluid or rinse immediately, or swab the inoculum into atest tube or other sterile container. Alternatively, recovery canbe determined by microp
44、ipetting aliquots of suspension di-rectly into tubes of elution fluid and enumerating the mixture.Mix thoroughly by vortexing. Make serial dilutions, and add1.0 mL of each sample (the original eluate as well as eachdilution) to individual tubes containing 20 mL of molten (46 to50C) agar, mix, and po
45、ur into sterile petri plate. Allow theagar to solidify and incubate at a temperature optimal for sporeoutgrowth. Examine plates for colonies at 48 h, and re-examinedaily for up to 7 days. Determine the mean number of CFUrecovered from each device by counting the appropriate plates,and calculate the
46、total CFU recovered from the inoculateddevice using the dilution factor.8.5.1.2 Swab MethodMoisten a sterile swab with sporesuspension, and swab the selected site. The number of sporesapplied onto the surface should be measured by immediatelyeluting and swabbing the inoculum from the device. Enumera
47、tethe eluate using standard dilution and plating techniques, asnoted in 8.5.1.1.8.5.2 Internal Site InoculationAs appropriate, connectany cleaning or irrigation attachment(s) recommended by themedical device manufacturer, and inoculate by irrigating theinternal lumina or recesses with inoculum. Usin
48、g a sterileirrigation device (syringe, pump, etc.), elute the suspensionfrom the device aseptically by irrigating the internal lumina orrecesses with several volumes of elution fluid. Collect allelution fluid, mix thoroughly, and determine the CFU usingstandard dilution and plating techniques, as no
49、ted in 8.5.1.1.8.6 Inoculation of Devices to Determine the RecoveryControlThese paragraphs describe the steps for enumeratingthe CFU of spores recoverable from the medical device after ithas been inoculated but just prior to being subjected to aprocess test cycle.8.6.1 Surface Site Inoculation:8.6.1.1 Micropipetter InoculationThe same inoculum andtechnique(s) used to determine the applied inoculum (see8.5.1.1) should be used to inoculate the medical device. Dry theinoculum if specified by the manufacturers instructions forpreparation of the ins
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