ASTM E1327-2007(2018) Standard Test Method for Evaluation of Antimicrobial Handwash Formulations by Utilizing Fingernail Regions.pdf

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1、Designation: E1327 07 (Reapproved 2018)Standard Test Method forEvaluation of Antimicrobial Handwash Formulations byUtilizing Fingernail Regions1This standard is issued under the fixed designation E1327; the number immediately following the designation indicates the year oforiginal adoption or, in th

2、e 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 can be used to determine the effective-ness of antimicrobial handwash

3、ing agents (including handrubs)in the reduction of transient bacterial flora with particularemphasis on the fingernail region.1.2 A knowledge of microbiological techniques is requiredfor these procedures.1.3 This standard does not purport to address all of thesafety concerns, if any, associated with

4、 its use. It is theresponsibility of the user of this standard to establish appro-priate safety, health, and environmental practices and deter-mine the applicability of regulatory limitations prior to use.For more specific hazard statements, see 7.5.1.4 This international standard was developed in a

5、ccor-dance with internationally recognized principles on standard-ization established in the Decision on Principles for theDevelopment of International Standards, Guides and Recom-mendations issued by the World Trade Organization TechnicalBarriers to Trade (TBT) Committee.2. Referenced Documents2.1

6、ASTM Standards:2E1054 Test Methods for Evaluation of Inactivators of Anti-microbial AgentsE2276 Test Method for Determining the Bacteria-Eliminating Effectiveness of Hygienic Handwash andHandrub Agents Using the Fingerpads of AdultsE1838 Test Method for Determining the Virus-EliminatingEffectiveness

7、 of Hygienic Handwash and Handrub AgentsUsing the Fingerpads of Adults3. Summary of Test Method3.1 This test method, involving an improved method ofrecovering bacteria from hands, is used to study the effects ofantimicrobial handwashes including health care personnelhandwash products. The group of v

8、olunteer panelists need notrefrain from using topical antimicrobials (such as deodorantsoaps) before participating in the study. All subjects wash theirhands with a nonantimicrobial hand soap prior to testing toremove any residual hand lotions and to lower the numbers ofresident skin flora. Activity

9、 of products is measured bycomparing the numbers of marker bacteria recovered fromartificially contaminated fingernail regions after use of thehandwashing formulations to the numbers recovered from theartificially contaminated but unwashed fingernail regions.Broth cultures of Serratia marcescens (a

10、red pigmented bac-terial species) and Escherichia coli (which produces fluores-cent colonies on a special agar medium) are used as testbacteria. A spore suspension of Bacillus subtilis may beutilized to study (1) degree of physical removal by handwash-ing techniques, and (2) the recovery and precisi

11、on aspects ofthe test method.4. Significance and Use4.1 The procedure should be used to test the degermingeffectiveness of antimicrobial hand washing products used byhealth care personnel that are intended for frequent use, andthat are intended to reduce the level of contamination acquiredthrough co

12、ntact with contaminated objects or people.4.2 Performance of these procedures requires the knowl-edge of regulations pertaining to the protection of humansubjects (Ref 1).35. Apparatus5.1 Colony CounterAny of several types may be used, forexample, Quebec Colony Counter.5.2 IncubatorsOne incubator ca

13、pable of maintaining atemperature of 25 6 2 C (this temperature is required toensure pigment production of Serratia); a second incubator1This test method is under the jurisdiction of ASTM Committee E35 onPesticides, Antimicrobials, and Alternative Control Agents and is the directresponsibility of Su

14、bcommittee E35.15 on Antimicrobial Agents.Current edition approved Sept. 1, 2018. Published November 2012. Originallyapproved in 1990. Last previous edition approved in 2012 as E1327 07(2012)1.DOI: 10.1520/E1327-07R18.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact AS

15、TM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3The boldface numbers in parentheses refer to a list of references at the end ofthis standard.Copyright ASTM International, 100 Barr Harbor Dri

16、ve, PO Box C700, West Conshohocken, PA 19428-2959. United StatesThis international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for theDevelopment of International Standards, Guides and Recommendations is

17、sued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.1capable of maintaining 37 6 2 C used for E. coli and B.subtilis incubation is acceptable.5.3 Water BathCapable of maintaining temperature of 806 2 C for heat shocking of B. subtilis spores is needed.5.4 SterilizerAny s

18、uitable steam sterilizer capable of pro-ducing the conditions of sterilization is acceptable.5.5 TimerAny stop-watch that can be read in minutes andseconds is required.5.6 Handwashing SinkA sink of sufficient size to permitpanelists to wash without touching hands to sink surface orother panelists is

19、 needed.5.6.1 Water Faucet(s), to be located above the sink at aheight that permits the hands to be held higher than the elbowduring the washing procedure.5.6.2 Tap Water Temperature Regulator and TemperatureMonitor, to monitor and regulate water temperature of 40 62 C.5.7 Quad Petri plates, 100 by

20、15 mm, plastic, sterile,disposable.45.8 Small Petri Plates, 60 by 15 mm, glass.5.9 Large Petri Plates, 150 by 15 mm, glass.5.10 Tooth Brushes:5.10.1 Young Size.5.10.2 Battery Operated.5.11 Ultraviolet Lamp, having separate short wave and longwave bulbs.5.12 Germicidal Lamp Monitor Strips.5.13 Inocul

21、ating Loops or Needles, sterile.5.14 Plate Spreaders or Hockey Sticks, sterile.6. Reagents and Materials6.1 Bacteriological Pipettes, 10.0 mL, sterile.6.2 Pipettors and Pipette Tips, Eppendorf, MLA or similartypes.6.3 Disposable Analyzer Cups, 2 mL, plastic, not sterile.6.4 Sampling SolutionDissolve

22、 0.4 g KH2PO4, 10.1 gNa2PO4and 1.0 g isooctylphenoxypolyethoxyethanol5in1Ldistilled water. Adjust pH to 7.8 with 0.1 N HCl or 0.1 NNaOH. Dispense in 100 mL-volumes and sterile for 20 min at121 C.6.5 Dilution FluidThe sampling fluid may be used fordilutions or use Butterfields sterile phosphate buffe

23、red water(2) adjusted to pH 7.2 with suitable inactivator for theantimicrobial. Adjust pH with 0.1 N HCl or 0.1 N NaOH (seePractices E1054).6.6 Agar, Tryptic soy agar or equivalent. Include the appro-priate inactivator if needed.6.7 Agar with MUGTryptic soy agar with 60 to 80 g/mL4-methylumbellifery

24、l-D-glucuronide (MUG) is required.6.8 Test FormulationsDirections for use of test formula-tion should be included if available. If these are not available,liquid antimicrobial soap formulations are tested by sameroutine as the nonantimicrobial control (10.5); alcoholic lotiontype formulations are ru

25、bbed to dryness and then sampled forsurvivors (10.7).6.9 Nonantimicrobial Control Soap, a liquid castile soap orother liquid soap containing no antimicrobials.6.10 BrothTryptic soy broth or equivalent is required.7. Test Organisms7.1 Serratia marcescens American Type Culture Collection,ATCC No. 1475

26、6 is to be used as a marker organism. This isa strain having stable pigmentation. Grow in tryptic soy brothat 25 6 2 C.7.2 Escherichia coli, ATCC No. 11229 is used as anotherGram-negative marker organism. Grow in tryptic soy broth at35 6 2 C.7.3 Bacillus subtilis, ATCC No. 19659. Grow in tryptic soy

27、broth at 35 6 2 C.7.4 Preparation of Spore SuspensionInoculate each sur-face of two tryptic soy agar plates (30 mLagar in 150-mm petriplates) with 1 mL of B. subtilis tryptic soy broth culture.Spread over the entire surface of the agar. Incubate for 5 to 10days at 35 6 2 C. Suspend the growth in 20

28、mL of 0.1 %tryptone water6by rubbing the agar surface with a sterilerubber policeman. Add ethanol to the suspension to a finalconcentration of 80 % (wt/wt) and store in a refrigerator.7.5 Other bacteria containing adequate markers to enabledistinction from normal flora and of known safety may also b

29、eused for testing purposes. (WarningThe application ofmicroorganisms to the skin may involve a health risk. Prior toapplying S. marcescens or other bacteria to the skin, theantibiotic susceptibility profile of the strain should be deter-mined. If the Serratia strain is not sensitive to Gentamicin, i

30、tshould not be used. If an infection occurs, the antibioticsusceptibility profile should be made available to an attendingclinician. Following the panelists contamination and testingfor the day, the panelists hands should be decontaminated witha 70-% ethanol solution. Care should be taken to deconta

31、mi-nate around the fingernail regions.)7.6 Preparation of Marker Culture SuspensionInoculate a10-mL tryptic soy broth tube with each of the test bacteria andincubate each tube at the temperature indicated to yield inoculaof 108109CFU/mL. When studying mixed inocula, mix equalvolumes of the cultures

32、into a sterile test tube; an equivalent4Presterilized disposable quad plastic petri plates, the two sizes of glass petriplates and other equipment are available from most local laboratory supply houses.5The sole source of supply of the apparatus (Triton X-100) known to thecommittee at this time is R

33、ohm and Haas Co., Philadelphia, PA. If you are aware ofalternative suppliers, please provide this information to ASTM InternationalHeadquarters. Your comments will receive careful consideration at a meeting of theresponsible technical committee,1which you may attend.6The sole source of supply of the

34、 Bacto Tryptone (Difco) water known to thecommittee at this time is Difco Laboratories, Detroit, MI. If you are aware ofalternative suppliers, please provide this information to ASTM InternationalHeadquarters. Your comments will receive careful consideration at a meeting of theresponsible technical

35、committee,1which you may attend.E1327 07 (2018)2volume of B. subtilis spore suspension (that is prepared bycentrifuging the alcoholic suspension and resuspending cells inwater) may be added for bacterial physical removal determi-nations. Keep mixed suspension on ice during the days testing.8. Paneli

36、sts8.1 Recruit a sufficient number of healthy adult humanvolunteers who have no clinical evidence of dermatoses, openwounds, hangnails, or other skin disorders. The number ofpeople needed for a trial is dependent on the number oftreatments within a study.8.2 Volunteers are asked to maintain their no

37、rmal use ofsoaps, shampoos, and so forth. They are asked to refrain fromthe use of acids, bases, solvents on the hands during the testperiod. Gloves should be provided for use where exposure tothese agents is unavoidable.9. Experimental Design9.1 Each fingernail of a volunteer may be assayed sepa-ra

38、tely; therefore, 10 test determinations (replicates) may beobtained from one volunteer. For the comparison of severalproducts during a single study, a design such as a Latin SquareDesign may be utilized (3). For example, to compare 5antimicrobial test products, one nonantimicrobial product andunwash

39、ed hand control (7 total variables), 7 volunteers, (ormultiples of 7) should be recruited. Each person performs onetesting of product or other variable on each of 7 test days,according to schedule such as the following; the num-bers = day for testing that variable (see Table 1).9.1.1 Example: Volunt

40、eer A tests Treatment 1 on Day 1,then Treatment 2 on Day 2; Volunteer B tests Treatment 2 onDay 1, Treatment 3 on Day 2, and so forth.9.1.2 Each product or variable is tested once on each day,unless multiple numbers of volunteers are in the study.9.1.3 The number of fingers, which are inoculated and

41、 thenassayed after using the product, should be kept standardthroughout. Although the number can be as high as 10, threefingers on one hand is a more convenient and cost savingsapproach. The ring, middle, and index fingers of the left handhave been selected for several studies; however, an operatorm

42、ay select the number and particular fingers to assay as long asthey are held constant throughout.10. Procedure10.1 Before tests for the day, sterilize the analyzer cups byplacing in suitable rack (24-well culture plates with lids areconvenient) and placing the open cups under short-waveultraviolet l

43、amp for 15 to 30 min. To each sterile disposableanalyzer cup, add 0.9 mL of sterile diluent: set up sufficientcups only for each days testing.10.2 Place 7 mL of sampling solution into each of 21 smallpetri plates.10.3 Place 0.02 mL of marker culture suspension on theregion surrounding the cuticle an

44、d under the fingernails ofthree fingers of the left hand of a volunteer. The volunteer thenholds the hand in front of an electric fan for 5 min for completedrying of the suspension.10.4 For unwashed hand determinations, proceed directly to10.8.10.5 When testing nonantimicrobial soap (controls), wetb

45、oth hands under flowing warm tap water (40 6 2 C).Add 2.5to 3.0 mL of the liquid soap to hands, rub hands together innormal washing manner for 15 s (no additional water), thenrinse under the flowing water for 15 s to remove suds. Do notdry hands, proceed directly to 10.8.10.6 For testing liquid anti

46、microbial soap formulations,follow the use directions on the label or follow the routine of10.5. After washing, proceed to 10.8 without drying hands.10.7 Alcoholic formulations are tested by placing the rec-ommended volume on the hands and then rubbing the handstogether until the alcohol has evapora

47、ted. Proceed to 10.8.10.8 After performing the procedure for the day designatedin the Latin Square Design, the technician scrubs with atoothbrush for 1 min each fingernail into a separate petri platecontaining 7 mL of sampling solution.NOTE 1Although manual toothbrushes may be used for this purpose,

48、greater uniformity between scrubbings may be obtained with less operatorfatigue if an electric toothbrush such as the GE model TB-9 or anothertype is used. A brush which operates parallel with the handle is preferredbecause of less splashing.10.9 After each scrubbing, the brushes are dropped into ab

49、eaker containing 70 % ethyl or isopropyl alcohol and allowedto stand for at least 10 min. The brushes are then rinsed insterile distilled water and allowed to dry. The brushes are notsterilized.10.10 Perform serial 10-fold dilutions. Place 0.1 mLamounts of the appropriate dilutions onto the surface of agarsections of quad plates. These drops of liquid are spread withsterile inoculation loops, needle spreaders, or hockey sticks tocompletely cover the quads.Allow drops to completely absorb.10.11 Incubate inverted plates at 35 6 2 C for 12 to 18 h.Cou