ASTM E2276-2010 Standard Test Method for Determining the Bacteria-Eliminating Effectiveness of Hygienic Handwash and Handrub Agents Using the Fingerpads of Adults《使用成人用指垫测定卫生洗手和擦手剂.pdf

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1、Designation: E2276 10Standard Test Method forDetermining the Bacteria-Eliminating Effectiveness ofHygienic Handwash and Handrub Agents Using theFingerpads of Adults1This standard is issued under the fixed designation E2276; the number immediately following the designation indicates the year oforigin

2、al adoption or, in 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.INTRODUCTIONHands can spread many types of pathogens directly (1)2or by trans

3、fer of such organisms to othersurfaces and objects during casual contact (2,3). Therefore, regular and proper decontamination ofhands by caregivers and food-handlers in particular is crucial for infection control. Hygienic handantisepsis is meant to reduce the load of transient microflora on hands,

4、thereby reducing the risk ofdisease transmission. Such reduction in the bacterial load may be due to a combination of bacterialinactivation and removal of viable bacteria from the skin. In this method the test bacterial suspensionis placed on the thumb- and fingerpads of adults to simulate the conta

5、mination of hands with transientmicroflora, the inoculum on the fingerpads is allowed to dry and is then treated with test and controlsolutions. Since in each test all ten digits on any given subject can be used, the protocol permits theinclusion of the required controls and several replicates of th

6、e test formulation in the same trial.1. Scope1.1 This test method is designed to determine the activity ofhygienic handwash and handrub (4) agents against transientbacterial flora on hands and is not meant for use with surgicalhand scrubs or preoperative skin preps.1.2 Performance of this procedure

7、requires the knowledgeof regulations pertaining to the protection of human subjects.31.3 The test method should be performed by persons withtraining in microbiology in facilities designed and equipped forwork with infectious agents at biosafety level 2 (5).1.4 The values stated in SI units are to be

8、 regarded asstandard. No other units of measurement are included in thisstandard.1.5 This standard does not purport to address 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 de

9、termine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:4D1129 Terminology Relating to WaterE1115 Test Method for Evaluation of Surgical Hand ScrubFormulationsE1173 Test Method for Evaluation of Preoperative, Precath-eterization, or Preinjection Sk

10、in PreparationsE1174 Test Method for Evaluation of the Effectiveness ofHealth Care Personnel Handwash FormulationsE1838 Test Method for Determining the Virus-EliminatingEffectiveness of Liquid Hygienic Handwash and HandrubAgents Using the Fingerpads of AdultsE2613 Test Method for Determining Fungus-

11、EliminatingEffectiveness of Hygienic Handwash and Handrub AgentsUsing Fingerpads of Adults3. Terminology3.1 DefinitionsFor definitions of general terms used inthis test method, refer to Terminology D1129.3.2 Definitions of Terms Specific to This Standard:1This test method is under the jurisdiction o

12、f ASTM Committee E35 onPesticides and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved April 1, 2010. Published May 2010. Originallyapproved in 2003. Last previous edition approved in 2003 as E2276 031. DOI:10.1520/E22

13、76-10.2The boldface numbers in parentheses refer to the list of references at the end ofthis standard.3Federal Register, Vol 46, No. 17, Jan. 27, 1991.4For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStan

14、dards volume information, refer to the standards Document Summary page onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3.2.1 active ingredient, na substance added to a formula-tion specifically for the inhibition o

15、r inactivation of microor-ganisms.3.2.2 dry control, na control to determine the number ofcolony forming units of the test bacterium remaining viableafter the initial drying of the inoculum on the skin.3.2.3 handrub, na liquid or gel which is applied byrubbing to decontaminate lightly soiled hands b

16、etween hand-washings and generally do not require a post-treatment waterrinse; such agents usually contain alcohol alone or with otheractive ingredients.3.2.4 hard water, nwater with a standard hardness ofcalcium carbonate.3.2.5 hygienic handwash agent, nan agent generally usedfor handwashing by per

17、sonnel in hospitals, other health-carefacilities, day-care centers, nursing homes, and food-handlingestablishments to eliminate transient microorganisms fromintact skin.3.2.6 input control, na control to determine the number ofcolony forming units of the test bacterium placed on each digit.3.2.7 neu

18、tralization, na process which results in quench-ing the antimicrobial activity of a test substance. This may beachieved through dilution of the test substance to reduce theantimicrobial activity, or through the use of chemical agents,called neutralizers, to eliminate antimicrobial activity.3.2.8 non

19、medicated soap, na soap or detergent that ismild to the skin and does not contain any antimicrobialchemicals.3.2.9 soil load, na solution of one or more organic and/orinorganic substances added to the suspension of the testorganism to simulate the presence of body secretions, excre-tions or other ex

20、traneous substances.3.2.10 test substance or test formulation, na formulationwhich incorporates antimicrobial ingredients.3.2.11 test organism, nan applied inoculum of an organ-ism that has characteristics which allow it to be readilyidentified. The test organism is used to simulate a transienttopic

21、al microbial contaminant. It may also be referred to as amarker organism, bacterial simulant/surrogate or bacterialcontaminant.3.2.12 test vehicle, nthe test formulation without anactive ingredient.3.2.13 transient microbiota, nmicroorganisms from theenvironment that contaminate but do not normally

22、colonize theskin.4. Summary of Test Method4.1 This test method is conducted on a group of adults whohave provided informed consent and the skin of whose handshas been determined to be free from any apparent damage.Subjects are to refrain from using any products containingantimicrobial agents for one

23、 week prior to the test. A knownvolume of the test bacterial suspension is placed on a demar-cated area on each fingerpad and the inoculum allowed to dry.The contaminated area then is exposed to the control (standardhard water) or test substance for the desired contact time andorganisms remaining on

24、 the fingerpad are eluted and theeluates assayed for viable bacteria. Percent or log10reductionsin the numbers of viable bacteria after treatment with thecontrol and test substance are then determined. The fingerpadmethod gives results that are comparable to those obtainedusing a whole-hand procedur

25、e (6). If two different formulationsare being compared in the same test, one of them may bedesignated as a reference and used in place of the hard watercontrol. If desired, one also may use tap water in parallel withthe hard water control to determine the influence of waterhardness on the test produ

26、cts bacteria-eliminating activity.5. Significance and Use5.1 This in vivo procedure is designed to test the ability ofhygienic handwash or handrub agents to eliminate selectedtypes of bacteria from experimentally contaminated skin of thehands of adult subjects. Since the two thumbpads and all eightf

27、ingerpads can be used in any given test, it allows for theincorporation of an input control (two), control for viablebacteria remaining after the inoculum has been allowed to dry(two), bacteria eliminated after treatment with a control orreference solution (two), and up to four replicates to assess

28、thebacteria-eliminating efficiency of the product under test. Nomore than 100 L of the test bacterial suspension is required tocomplete one test. The results of testing with this test methodmay form the basis for confirmatory tests using a suitablewhole-hand test protocol, such as Test Method E1174.

29、5.2 Whereas this test method relates to testing with bacteria,it can be readily adapted to work with protozoa and bacterioph-ages. Similar methods for work with fungi (Test MethodE2613) and viruses of human origin (Test Method E1838) arealready ASTM standards.5.3 Potentially infectious microorganism

30、s left on handsafter washing can be reduced further by drying the washedhands with paper, cloth, or warm air (7). A step for the dryingof fingerpads after exposure to the control or test solution,therefore, has not been included to avoid bacterial removal bythe drying process itself.5.4 This test me

31、thod is not meant for use with surgical handscrubs (Test Method E1115) or preoperative skin preps (TestMethod E1173).5.5 The level of contamination with viable bacteria on eachfingerpad after the drying of the inoculum should be five- toten-fold higher than the product performance criterion re-quire

32、d. For example, the titer in the dried inoculum on eachfingerpad should be about 105colony forming units of the testbacterium when a 104reduction is required under the condi-tions of this test method.6. Equipment and Apparatus6.1 Colony CounterAny of several types may be used, forexample, Quebec Col

33、ony Counter.6.2 FreezersA freezer at -20 6 2C is required for thestorage of culture media. A second freezer at -70C or lower isrequired to store bacterial stocks.6.3 Handwashing SinkA sink of sufficient size to permitsubjects to wash hands without touching hands to sink surface.6.4 IncubatorAny incu

34、bator capable of maintaining atemperature of 36 6 1C. Serratia marcescens requires incu-bation at 25 6 2C for pigment formation.E2276 1026.5 Laminar Flow CabinetA Class II biological safetycabinet is required for this work. The procedures for the propermaintenance and use of such cabinets are given

35、in Ref (2).6.6 Magnetic Stirrer and MagnetsLarge enough to hold a5-L beaker or Erlenmeyer flask for preparing culture media orother solutions.6.7 Membrane Filtration SystemA membrane filtrationsystem and membranes with a pore diameter of 0.22-m arerequired to sterilize heat-sensitive media/solutions

36、 and tocapture and culture viable test bacteria in control samples andeluates.6.8 Positive Displacement PipetteA pipette and pipettetips that accurately can dispense 10-L volumes.6.9 RefrigeratorA refrigerator at 4 6 2C for storage ofprepared culture media and reagents.6.10 SterilizerAny suitable st

37、eam sterilizer capable ofproducing the conditions of sterilization is acceptable.6.11 Timer (Stop-clock)One that can be read for minutesand seconds.6.11.1 Tap Water Temperature Regulator and TemperatureMonitorto monitor and regulate water temperature at 40 62C.6.11.2 Water Faucet(s)to be located abo

38、ve the sink at aheight that permits the hands to be held higher than the elbowduring the washing procedure. Faucets with electronic sensorsor those that are wrist-, elbow-, knee-, or foot-operated arepreferred to avoid recontamination of the washed hands.7. Materials and Reagents7.1 Serological Pipe

39、ttesSterile reusable or single-use pi-pettes of 10.0, 5.0, and 1.0-mL capacity.7.2 Culture PlatesPetri plates of 100 mm diameter forculturing bacteria.NOTE 1Plastic culture ware may be purchased from most laboratorysupply houses.7.3 Culture Media and SupplementsCulture media andthe types and ratios

40、of supplements will vary depending on thetype of test bacterium being used.7.4 Soil Load:7.4.1 Fetal Bovine Serum, at a final concentration of 5 % inthe bacterial inoculum.7.4.2 Tripartite Soil Load, as an alternative to serum.7.4.2.1 Add 0.5 g of tryptone or yeast extract to 10 mL ofphosphate buffe

41、r.7.4.2.2 Add 0.5 g of bovine serum albumin (BSA) to 10 mLof phosphate buffer.7.4.2.3 Add 0.04 g of bovine mucin to 10 mL of phosphatebuffer.7.4.3 Prepare the stock solutions separately and sterilize bypassage through a 0.22 m pore diameter membrane filter,aliquot and store at either 4 6 2C or 20 6

42、2C; the stocksolution of bovine mucin can be autoclave-sterilized.7.4.4 To obtain a 500-L inoculum of the test inoculum, addto 340 L of the bacterial suspension, 35 L of tryptone oryeast extract (7.4.2.1), 25 L BSA(7.4.2.2) , and 100 L mucin(7.4.2.3) stock solutions. This mixture contains approximat

43、ely2 g of total protein/L, which is roughly equivalent to theprotein content of a 5 % solution of fetal bovine serum.7.5 Standard Hard WaterThe quality and disinfectant (forexample, chlorine) residual in tap water can vary from site tosite and also at different times at the same site. The use ofstan

44、dard hard water, therefore, is recommended here to avoidvariations in results due to differences in tap water quality.Water prepared in accordance with AOAC 960.09 E and F (8)to a standard hardness of at least 200 ppm as calcium carbonateis used for dilution of the test substance, as the control sol

45、utionto determine the baseline level of bacterial elimination, and torinse the fingerpads after exposure to the test product. Thestandard hard water and tap water (if used) must first be testedto ensure that they do not have any activity against the testbacterium. If water with a different level of

46、hardness is used formaking the use-dilution of the test formulation, this changemust be clearly justified and documented in the report.7.6 Test AgentsAt least two samples of the product shallbe tested.7.7 Diluent for Bacterial TitrationNormal saline (0.85 %NaCl) at pH 7.2-7.4, or appropriate buffer.

47、7.8 Eluent for Bacterial Recovery from FingerpadsNormal saline or another suitable eluent.7.9 Plastic VialsSterile screw-capped 2.0-mL vials withan inside diameter of about 8 mm will be required fordemarcation of the fingerpads and to hold various test solu-tions.7.10 Miscellaneous Laboratory WareAu

48、tomatic pipettes,pipette tips, plastic vials for storing stock cultures.7.11 BrothTryptose phosphate broth (TPB) or equivalent,to prepare the inoculum of the test organisms.7.12 AgarTrypticase soy agar (TSA) or equivalent, torecover and count the colonies of the test organism in controland test samp

49、les. The addition of any neutralizer(s) in suchrecovery media must first be properly validated. The use ofselective-differential media for the detection of the test bacteriain such studies is not recommended because cells stressed orinjured after germicide exposure may not form colonies onsuch agars.8. Test Bacteria8.1 Appendix X1 contains a list of suggested test bacteria.(WarningThe application of microorganisms to the skinmay involve a health risk. Prior to applying the test organismto the skin, the antibiotic sensitivit