1、Designation: E2755 10Standard Test Method forDetermining the Bacteria-Eliminating Effectiveness of HandSanitizer Formulations Using Hands of Adults1This standard is issued under the fixed designation E2755; the number immediately following the designation indicates the year oforiginal adoption or, i
2、n the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method is designed to determine the activity ofhand sanitizers (also kno
3、wn as hand rubs, hygienic hand rubs,or hand antiseptics) against transient bacterial flora on thehands.1.2 Performance of this procedure requires the knowledgeof regulations pertaining to the protection of human subjects(see 21 CFR Parts 50 and 56).1.3 This test method should be performed by persons
4、 withtraining in microbiology, in facilities designed and equippedfor work with potentially infectious agents at biosafety level2.21.4 UnitsThe values stated in SI units are to be regardedas standard. No other units of measurement are included in thisstandard.1.5 This standard does not purport to ad
5、dress all of thesafety concerns, if any, associated 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. For more specificprecautionary statements, see 8.2.2.
6、Referenced Documents2.1 ASTM Standards:3E1054 Test Methods for Evaluation of Inactivators of An-timicrobial AgentsE1174 Test Method for Evaluation of the Effectiveness ofHealth Care Personnel Handwash FormulationsE2276 Test Method for Determining the Bacteria-Eliminating Effectiveness of Hygienic Ha
7、ndwash and Han-drub Agents Using the Fingerpads of Adults2.2 Other Standards:AATCC Test Method 147 2004 Antibacterial Activity As-sessment of Textile Materials: Parallel Streak Method421 CFR Parts 50 and 56 Protection of Human Subjects;Institutional Review Boards53. Terminology3.1 Definitions:3.1.1
8、antiseptic, na material for use on living tissue thateither destroys microorganisms or suppresses their growth.3.1.2 hand sanitizer, nan antimicrobial gel, foam, liquid,spray, or wipe, used on hands that are not visibly soiled toreduce the number of transient microorganisms, which isapplied by rubbi
9、ng, and does not require a post-treatment waterrinse. Such agents may also be referred to as hand rubs,hygienic hand rubs, or hand antiseptics.3.1.3 healthcare personnel handwash, na cleanser orwaterless agent intended to reduce transient bacteria on thehands.3.1.4 neutralization, nthe process for i
10、nactivating orquenching the activity of a microbicide, often achieved throughphysical (for example, filtration or dilution) or chemical means,or both.3.1.5 resident microorganisms, nmicroorganisms that sur-vive and multiply on the skin, forming a stable population.3.1.6 test bacteria, nan applied in
11、oculum of bacteria thathas characteristics which allow it to be readily identified. Testbacteria are used to simulate a transient topical microbialcontaminant. This may also be referred to as a test organism,marker organism, simulant, or contaminant.3.1.7 test material, na product or formulation whi
12、chincorporates an antimicrobial ingredient(s).3.1.8 transient microorganisms, nmicroorganisms thatcontaminate the skin but do not form a stable population.4. Summary of Test Method4.1 This test method uses adult subjects who have provideda written informed consent and whose hands have been1This test
13、 method is under the jurisdiction of ASTM Committee E35 onPesticides and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved June 1, 2010. Published July 2010. DOI: 10.1520/E275510.2CDC-NIH, Biosafety in Microbiological a
14、nd Biomedical Laboratories, 5thed., U.S. Department of Health and Human Services, U.S. Government PrintingOffice, Washington, DC, 2007.3For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume inf
15、ormation, refer to the standards Document Summary page onthe ASTM website.4Available from American Association of Textile Chemists and Colorists(AATCC), P.O. Box 12215, Research Triangle Park, NC 27709, http:/www.aatcc.org.5Available from U.S. Government Printing Office Superintendent of Documents,7
16、32 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http:/www.access.gpo.gov.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.determined to be free from any apparent damage at the time ofparticipation in the study. Subjects a
17、re to refrain from use ofany antimicrobials for at least one week prior to the initiationof the test procedure (see Section 11).4.2 Subjects hands are artificially contaminated with 0.2mL of a high-titer suspension of the test bacteria which isdistributed over all surfaces of the hands and fingers t
18、o producea minimum baseline recovery level of 108cfu/hand. BecauseSerratia marcescens is relatively sensitive to drying, the hightiter suspension is prepared by growing in broth with vigorousaeration, followed by a 10-fold concentration with centrifuga-tion. Staphylococcus aureus is more resistant t
19、o drying and istherefore not concentrated after growth with vigorous aerationin broth.4.3 Test material effectiveness is measured by comparingthe number of test bacteria recovered from contaminated handsafter use of the test material to the number recovered fromcontaminated hands not exposed to the
20、test material. Activityof the test material is measured following a single applicationand may also be measured after multiple consecutivecontamination/application cycles in a single day.5. Significance and Use5.1 Hand hygiene is considered one of the most importantmeasures for preventing the spread
21、of infectious microorgan-isms. Hand sanitizers reduce the microbial load on the handswithout the use of soap and water, and are thus an importanttool in the practice of good hand hygiene. Hand sanitizers arerecommended for use on hands that are not visibly soiled. Theyare formulated to be applied fu
22、ll strength to dry hands, “rubbedin” until dry, and are not rinsed off.5.2 This test method is specifically designed to evaluate thebacteria-eliminating activity of hand sanitizers fromexperimentally-contaminated hands. It is intended to be analternative to Test Method E1174, which was designed prim
23、a-rily to evaluate antimicrobial handwashing agents that arelathered with the aid of water and then rinsed off. When usingTest Method E1174 to evaluate hand sanitizers, inadequatedrying of the hands after contamination dilutes the test productand can compromise activity, leading to an underestimatio
24、n ofeffectiveness. By applying a higher titer test bacteria suspen-sion in a smaller volume, soil load on the hands is minimizedand hands are completely dry prior to application of the testmaterial. These modifications result in a better approximationof the in-use conditions for hand sanitizers and
25、thus provide amore reliable indication of their performance in the field.5.3 This test method can be used to test any form of handsanitizer, including gels, rubs, sprays, foams, and wipes ac-cording to label directions at typical “in-use” doses.5.4 Susceptibility to biocides can vary among different
26、species of bacteria and major differences have been notedbetween gram-negative and gram-positive organisms. This testmethod provides the option to use either a gram-negativebacterium (Serratia marcescens) or a gram-positive bacterium(Staphylococcus aureus) as the test organism. S. marcescens isused
27、as a test organism in both Test Method E1174 and TestMethod E2276. S. aureus is a highly relevant pathogen inhealthcare, institutional, and community settings. Moreover,hands are an important vehicle in the transfer of S. aureusbetween people and the environment, and in the transferbetween individua
28、ls.5.5 This test method may be used as an alternative to TestMethod E2276, which limits the test bacteria to the fingerpadsand does not incorporate actual use conditions such as frictionduring hand decontamination.5.6 The investigator should be aware of potential healthrisks associated with the use
29、of these organisms and precau-tions similar to those referenced in Section 8 should be taken.6. Apparatus6.1 CentrifugeFor the sedimentation of S. marcescens forconcentration.6.2 Centrifuge TubesSterile, for sedimentation of S.marcescens for concentration.6.3 Colony CounterAny of several types may b
30、e used; forexample, Quebec colony counters and similar devices. Auto-mated, computerized plater/counter systems may also be used.6.4 GlovesSterile, loose-fitting, unlined, powder-freegloves possessing no antimicrobial properties. Perform a zoneof inhibition test, such asAATCC Test Method 147, to eva
31、luatethe antibacterial activity.6.5 Handwashing SinkSufficient in size to permit hand-washing without the touching of hands to sink surface or othersubjects.6.5.1 Water Faucet(s)Located above the sink at a heightto permit hands to be held higher than the elbow during thewashing procedure.6.5.2 Tap W
32、ater Temperature Regulator and TemperatureMonitorTo set and maintain the tap water temperature at40 6 2C.6.6 IncubatorCapable of maintaining temperatures of35 6 2C and 25 6 2C. The latter temperature ensures ad-equate pigment production for S. marcescens on solid media.6.7 Miscellaneous LabwareConti
33、nuously adjustable pi-petters (1-mL and 0.2-mL capacity) and sterile pipette tips,sterile serological pipettes (5.0-mL capacity), sterile culturetubes, sterile disposable Petri dishes, sterile syringes, Erlenm-eyer flasks, and beakers.6.8 Sampling ContainersSterile or sterilizable containershaving t
34、ight closures and sufficient capacity to hold 75 mLsampling solution (see 7.7).6.9 Shaking IncubatorRotary platform shaking incubatorcapable of maintaining 35 6 2C and capable of shaking at 250r/min. Alternatively, use an incubator capable of maintaining35 6 2C and able to accommodate a portable rot
35、ary shaker,capable of shaking at 250 r/min.6.10 SterilizerAny steam sterilizer capable of processingculture media and reagents.6.11 Timer (Stop-Clock)Type that can be read for minutesand seconds.6.12 TourniquetsChildrens size or any style capable ofsecuring gloves to the wrist.6.13 Vortex MixerAny v
36、ortex that will ensure propermixing of culture tubes.E2755 1027. Reagents and Materials7.1 Antibiotic OintmentA topical, triple-antibiotic oint-ment for application to the hands after the final decontamina-tion.7.2 Cleansing WashA mild, proven non-antimicrobialliquid soap. May be purchased commercia
37、lly or preparedaccording to the instructions provided in Test Method E1174.7.3 Chlorhexidine Skin CleanserAntiseptic skin cleansercontaining 4 % chlorhexidine gluconate (w/v) for hand decon-tamination.7.4 Culture Media:7.4.1 BrothSoybean-casein digest broth (tryptic soybroth) is recommended.7.4.2 Ag
38、ar Plating Media:7.4.2.1 S. aureus Plating MediumHardyCHROM (trade-mark), Staph aureus, available from Hardy Diagnostics, isrecommended. Other indicator media for S. aureus or MRSAmay be appropriate but should be validated prior to use.NOTE 1S. aureus forms smooth, deep pink to fuchsia-colored colo-
39、nies. The growth of most other organisms, including Staphylococcusepidermidis are partially to completely inhibited.7.4.2.2 S. marcescens Plating MediumSoybean-casein di-gest agar (tryptic soy agar) is recommended.7.5 Dilution FluidSterile Butterfields buffered phosphatediluent6(or other suitable di
40、luent) adjusted to pH 7.2 6 0.1 andcontaining an effective inactivator for the test material, ifnecessary.NOTE 2Inactivator is only required if neutralization of the testmaterial cannot be achieved upon dilution into the sampling solution (see7.7).7.6 Ethanol Solution70 % ethanol in water (v/v) for
41、handdecontamination.7.7 Sampling SolutionDissolve 0.4 g KH2PO4, 10.1 gNa2HPO4, 1.0 g isooctylphenoxypolyethoxyethanol (for ex-ample, Triton X-100), and appropriately validated neutralizers,if necessary, in distilled water. Adjust pH to 7.8 6 0.1 with 0.1N HCl or 0.1 N NaOH and bring volume to 1 L wi
42、th distilledwater. Sterilize in an autoclave and aseptically dispense 75-mLportions into sterile sampling containers (see 6.8).7NOTE 3A neutralizer validation should be conducted according toTest Methods E1054 prior to the study. Test Methods E1054 provides a listof neutralizers appropriate for comm
43、only used antimicrobial agents. Insome cases (for example, some alcohol-based hand sanitizers) neutraliza-tion is achieved by dilution alone.7.8 Test MaterialUse directions provided with the testmaterial. If directions are not provided, use the directions givenin this method.8. Test Bacteria8.1 Serr
44、atia marcescens (ATCC 14756). This strain forms astable red pigmentation at 25C.8.2 Staphylococcus aureus (ATCC 6538 (methicillin-sensitive) or ATCC 33591 (methicillin-resistant) is an alterna-tive test bacteria. S. aureus is differentiated from residentmicroorganisms (including Staphylococcus epide
45、rmidis) withchromogenic indicator medium (see 7.4.2.1). (WarningApplication of microorganisms to the skin may involve ahealth risk. Determine the antibiotic sensitivity profile of thetest bacteria prior to applying to the skin. After the test hasbeen completed, decontaminate the subjects hands and f
46、ollowproper procedures to reduce infection risk (12.1-12.4). If aninfection occurs, provide the antibiotic sensitivity profile to theattending clinician.)9. Preparation of Test Bacteria Suspension9.1 Method 1 (for S. marcescens):9.1.1 A homogeneous bacterial suspension is used to inocu-late the subj
47、ects hands. Prepare a stock culture of S. marce-scens (ATCC 14756) by inoculating approximately 5 mL ofsoybean-casein digest broth (see 7.4.1) from a cryogenic stockor lyophilized vial or pellet and incubate for 25 6 1hat3562C. Inoculate the appropriate volume of soybean-casein digestbroth with 1 mL
48、 of the stock culture of S. marcescens/125 mLof broth to yield the volume necessary to complete the study(that is, 2 mL per hand contamination (see 11.3) per testsubject). The volume of the broth culture should not exceedabout one fourth of the capacity of the Erlenmeyer flask toensure adequate aera
49、tion. Incubate for 25 6 1hat356 2Cwith shaking at 250 r/min to yield a titer of approximately 1.03 1010cfu/mL.NOTE 4The frozen or lyophilized stock should be at least two but nomore than four 24-h soybean-casein digest broth (see 7.4.1) transfers fromthe original ATCC culture.9.1.2 Transfer the culture to appropriate sized sterile centri-fuge tubes or bottles and centrifuge at conditions appropriate tosediment the culture completely (recommended conditions are7000 G for 10 min). Decant the supernatant and resuspend thepellet to o
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