ASTM E2276-2003e1 Standard Test Method for Determining the Bacteria-Eliminating Effectiveness of Hygienic Handwash and Handrub Agents Using the Fingerpads of Adult Subjects《用成人受验者的.pdf

1、Designation: E 2276 03e1Standard Test Method forDetermining the Bacteria-Eliminating Effectiveness ofHygienic Handwash and Handrub Agents Using theFingerpads of Adult Subjects1This standard is issued under the fixed designation E 2276; the number immediately following the designation indicates the y

2、ear oforiginal adoption or, 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.e1NOTEEditorial corrections were made throughout in July 2004.IN

3、TRODUCTIONHands can spread many types of pathogens directly (1)2or by transfer 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 ha

4、ndantisepsis is meant to reduce the load of transient microflora on hands, 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 suspe

5、nsionis placed on the thumb- and fingerpads of adults to simulate the contamination 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

6、 permits theinclusion of the required controls and several replicates of the 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 surgic

7、alhand scrubs or preoperative skin preps.1.2 Performance of this procedure 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

8、agents at biosafety level 2 (5).1.4 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 determine the applica-bility of regulatory limitati

9、ons prior to use.2. Referenced Documents2.1 ASTM Standards:4D 1129 Terminology Relating to WaterE 1115 Test Method for Evaluation of Surgical Hand ScrubFormulationE 1173 Test Method for Evaluation of a Pre-Operative SkinPreparationE 1174 Test Method for Evaluation of the Effectiveness ofHealth Care

10、Personnel or Consumer Handwash Formula-tionsE 1838 Test Method for Determining the Virus-EliminatingEffectiveness of Liquid Hygienic Handwash and HandrubAgents Using the Fingerpads of Adult Volunteers3. Terminology3.1 DefinitionsFor definitions of general terms used inthis test method, refer to Term

11、inology D 1129.3.2 Definitions of Terms Specific to This Standard:1This test method is under the jurisdiction of ASTM Committee E35 onPesticides and is the direct responsibility of Subcommittee E35.15 on AntmicrobialAgents.Current edition approved May 10, 2003. Published July 2003.2The boldface numb

12、ers 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 ASTMStandards volume information

13、, 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 or inactivation of microo

14、r-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 between hand-washings and

15、 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 personnel in hospitals, oth

16、er 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 neutralization, na process

17、which results in quench-ing the antimicrobial activity of a test material. This may beachieved through dilution of the test material(s) to reduce theantimicrobial activity, or through the use of chemical agents,called neutralizers, to eliminate antimicrobial activity.3.2.8 nonmedicated soap, na soap

18、 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 extraneous substances.3.2

19、.10 test formulation, na formulation which incorpo-rates 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 transienttopical microbial contaminant. It may also b

20、e 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 microflora, nmicroorganisms from theenvironment that contaminate but do not normally colonize theskin.4. Summary of Test Met

21、hod4.1 This test method is conducted on a group of adultsubjects who have provided informed consent and the skin ofwhose hands has been determined to be free from any apparentdamage. Panelists are to refrain from using any productscontaining antimicrobial agents for one week prior to the test.A know

22、n volume of the test bacterial suspension is placed ona demarcated area on each fingerpad and the inoculum allowedto dry. The contaminated area then is exposed to the control(standard hard water) or test formulation for the desired contacttime and organisms remaining on the fingerpad are eluted andt

23、he eluates are assayed for viable bacteria. Percent reductionsin the numbers of viable bacteria after treatment with thecontrol and test agents are then determined. The fingerpadmethod gives results that are comparable to those obtainedusing a whole-hand procedure (6). If two different formulationsa

24、re 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 products bacteria-eliminating activity.5.

25、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 eightfingerpads can be used in any given te

26、st, 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 thebacteria-eliminating efficiency of

27、 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 E 1174.5.2 Whereas, this test method relate

28、s to testing with bacte-ria, it can be readily adapted to work with fungi (7), protozoaand bacteriophages. A similar method for work with viruses ofhuman origin is already a test method, ( Test Method E 1838).5.3 Potentially infectious microorganisms left on handsafter washing can be reduced further

29、 by drying the washedhands with paper, cloth, or warm air (8). 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 method is not meant for use with surgical handscrubs

30、or preoperative skin preps.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-quired. For example, the titer in the dried inoculum on eachfingertip should be about 105colon

31、y 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 Colony Counter.6.2 FreezersA freezer at -20 6 2C is required for thestorage of culture media

32、. A second freezer at -70C or lower isrequired to store bacterial stocks.6.3 Handwashing SinkA sink of sufficient size to permitpanelists to wash hands without touching hands to sink surface.6.4 IncubatorAny incubator capable of maintaining thefollowing temperatures: Serratia marcescens (25 6 2C); t

33、histemperature is necessary to ensure pigmentation) or Escheri-chia coli, Staphylococcus aureus, Staphylococcus epidermidis(35 6 2C).E227603e126.5 Laminar Flow CabinetA Class II biological safetycabinet is required for this work. The procedures for the propermaintenance and use of such cabinets are

34、given 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/sol

35、utions 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 suita

36、ble steam 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 locat

37、ed above 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 Serologica

38、l PipettesSterile reusable or single-use pi-pettes of 10.0, 5.0, and 1.0-mL capacity.7.2 Culture Plates5Petri plates of 100 mm diameter forculturing bacteria.7.3 Culture Media and SupplementsCulture media andthe types and ratios of supplements will vary depending on thetype of test bacterium being u

39、sed.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 to 10 mL of phosphate buffer.7.4.2.2 Add 0.5 g of bovine serum albumin (BSA) to 10 mLof phosphate buffer.7.4.2.3 A

40、dd 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 -2062C.7.4.4 To obtain a 500-L inoculum of the test inoculum, addto 340 L of the bacterial s

41、uspension, 35 L of tryptone(7.4.2.1), 25 L BSA (7.4.2.2) , and 100 L mucin (7.4.2.3)stock solutions. This mixture contains approximately2goftotal protein/L, which is roughly equivalent to the proteincontent of a 5 % solution of fetal bovine serum.7.5 Standard Hard WaterThe quality and disinfectant (

42、forexample, chlorine) residual in tap water can vary from site tosite and also at different times at the same site. The use ofstandard 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

43、F (9)to a standard hardness of at least 200 ppm as calcium carbonateis used for dilution of test products, as the control solution todetermine the baseline level of bacterial elimination, and torinse the fingerpads after exposure to the test product. Thestandard hard water and tap water (if used) mu

44、st first be testedto ensure that they do not have any activity against the testbacterium. If water with a different level of 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 t

45、he product shallbe tested.7.7 Diluent for Bacterial TitrationNormal saline (0.85 %NaCl) at pH 7.2-7.4, or appropriate buffer.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

46、mm will be required fordemarcation of the fingerpads and to hold various test solu-tions.7.10 Miscellaneous Laboratory WareAutomatic 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.

47、12 AgarTrypticase soy agar (TSA) or equivalent, torecover and count the colonies of the test organism in controland test samples. 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

48、 such studies is not recommended because cells stressed orinjured after germicide exposure may not form colonies onsuch agars.8. Test Bacteria8.1 The selection of the following test bacteria is based ontheir (a) relative safety to the volunteers as well as experiment-ers, (b) ability to grow to tite

49、rs sufficiently high for testing, (c)ease of recovery and identification in the laboratory, and (d)potential to spread through contaminated hands. Other strainsor types of bacteria may be substituted provided they meet thepreceding criteria.8.1.1 Serratia marcescens (ATCC 14756). This is a strainhaving stable pigmentation at 25C.8.1.2 Escherichia coli (ATCC 11229), is an alternativeGram-negative test organism that is considered safe for theexperimental contamination of the skin of adult subjects.8.1.3 Staphylococcus aureus (ATCC