ASTM E1327-2007 Standard Test Method for Evaluation of Antimicrobial Handwash Formulations by Utilizing Fingernail Regions《利用手指甲区域评估抗菌洗手液配方的标准试验方法》.pdf

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1、Designation: E 1327 07Standard Test Method forEvaluation of Antimicrobial Handwash Formulations byUtilizing Fingernail Regions1This standard is issued under the fixed designation E 1327; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revisi

2、on, 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 can be used to determine the effective-ness of antimicrobial handwashing agents (inc

3、luding 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 its use. It is

4、 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 specifichazard statements, see 7.5.2. Referenced Documents2.1 ASTM Standards:2E 1054 Practices for Evaluating Inacti

5、vators of Antimicro-bial Agents Used in Disinfectant, Sanitizer, Antiseptic, orPreserved Products3E 2276 Test Method for Determining the Bacteria-Eliminating Effectiveness of Hygienic Handwash and Han-drub Agents Using the Fingerpads of Adult SubjectsE 1838 Test Method for Determining the Virus-Elim

6、inatingEffectiveness of Liquid Hygienic Handwash and HandrubAgents Using the Fingerpads of Adult Volunteers3. 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 perso

7、nnelhandwash products. The group of volunteer 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 nu

8、mbers ofresident skin flora. Activity 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.Brot

9、h cultures of Serratia marcescens (a 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 techniq

10、ues, and (2) the recovery and precision 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 leve

11、l of contamination acquiredthrough contact 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).45. Apparatus5.1 Colony CounterAny of several types may be used, forexample, Quebec Colony C

12、ounter.5.2 IncubatorsOne incubator capable of maintaining atemperature of 25 6 2C (this temperature required to ensurepigment production of Serratia); a second incubator capable ofmaintaining 37 6 2C used for E. coli and B. subtilisincubation is acceptable.5.3 Water BathCapable of maintaining temper

13、ature of 806 2C for heat shocking of B. subtilis spores is needed.5.4 SterilizerAny suitable steam sterilizer capable ofproducing the conditions of sterilization is acceptable.1This test method is under the jurisdiction of ASTM Committee E35 onPesticides and Alternative Control Agents and is the dir

14、ect responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved April 1, 2007. Published June 2007. Originallyapproved in 1990. Last previous edition approved in 2000 as E 1327 90 (2000).2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Cu

15、stomer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Withdrawn.4The boldface numbers in parentheses refer to a list of references at the end ofthis standard.1Copyright ASTM International, 100 Barr Har

16、bor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.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 needed.5.6.1 Water F

17、aucet(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 62C.5.7 Quad Petri plates, 100 by 15 mm, plastic, steril

18、e,disposable.55.8 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 Inoculating Loops or Needles

19、, 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 Cusp, 2 mL, plastic, not sterile.6.4 Sampling SolutionDissolve 0.4 g KH2PO4, 10.1 gN

20、a2PO4and 1.0 g isooctylphenoxypolyethoxyethanol6in1Ldistilled 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 at121C.6.5 Dilution FluidThe sampling fluid may be used fordilutions or use Butterfields sterile phosphate buffered water(2) adjusted t

21、o pH 7.2 with suitable inactivator for theantimicrobial. Adjust pH with 0.1 N HCl or 0.1 N NaOH (seePractices E 1054).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-methylumbelliferyl-b-D-glucuronide (MUG

22、) 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 rubbed to dryness and

23、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 Collec-tion,ATCC No. 14756 is to be used as

24、a marker organism. Thisis a strain having stable pigmentation. Grow in tryptic soybroth at 25 6 2C.7.2 Escherichia coli, ATCC No. 11229 is used as anothergram negative marker organism. Grow in tryptic soy broth at35 6 2C.7.3 Bacillus subtilis, ATCC No. 19659. Grow in tryptic soybroth at 35 6 2C.7.4

25、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 2C. Suspend the growth in 20 mL of 0.1 %tryptone wat

26、er7by 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 beused for testing purpo

27、ses. (WarningThe application ofmicroorganisms to the skin may involve a health risk. Prior toapplying S. marcescens or other bacteria to the skin, theantibiotic sensitivity profile of the strain should be determined.If the Serratia strain is not sensitive to gentamycin, it shouldnot be used. If an i

28、nfection occurs, the antibiotic sensitivityprofile should be made available to an attending clinician.Following the panelists contamination and testing for the day,the panelists hands should be decontaminated with a 70 %ethanol solution. Care should be taken to decontaminatearound the fingernail reg

29、ions.)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 into a sterile test tube; an equi

30、valentvolume 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. Panelists8.1 Recruit a sufficient number of heal

31、thy adult humanvolunteers who have no clinical evidence of dermatosis, 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 normal use ofsoaps, shampoos, and so forth.

32、They are asked to refrain fromthe use of acids, bases, solvents on the hands during the test5Presterilized disposable quad plastic petri plates, the two sizes of glass petriplates and other equipment are available from most local laboratory supply houses.6The sole source of supply of the apparatus (

33、Triton X-100) known to thecommittee at this time is Rohm 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,1w

34、hich you may attend.7The sole source of supply of the 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 co

35、nsideration at a meeting of theresponsible technical committee,1which you may attend.E1327072period. 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-rately; therefore, 10 test determinations (

36、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 andunwashed hand control (7 total variables), 7 vo

37、lunteers, (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; thenumbers = day for testing that variable (see Table 1).9.1.1 Example: Volunteer A tests Treatment 1 on Day 1, thenTreat

38、ment 2 on Day 2; Volunteer B tests Treatment 2 on Day 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 thenassayed after using the product, shoul

39、d 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 operatormay select the number and particular fingers

40、 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 lamp for 15 to 30 min. To each sterile dispo

41、sableanalyzer 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 and under the fingernails ofthree fingers of

42、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), wetboth hands under flowing warm tap water (40

43、6 2C). 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 antimicrobial soap formulations,follow the use

44、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 evaporated. Proceed to 10.8.10.8 After performing

45、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,greater uniformity between scrubbings may b

46、e obtained with less operatorfatigue if an electric toothbrush such as the GE model TB-9 or anothertype is used. A brush which operates longitudinally to the handle ispreferred because of less splashing.10.9 After each scrubbing, the brushes are dropped into abeaker containing 70 % ethyl or isopropy

47、l 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 d

48、rops 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 2C for 12 to 18 h.Count the E. coli colony forming units (CFU) that fluoresceunder long wave ultraviolet li

49、ght. Transfer the plates to a 25Cincubator and incubate for another day.10.12 Count the red pigmented S. marcescens CFU. Recordthe CFU per countable sections of the plates and convert valuesto the CFU obtained per finger by multiplying by the appro-priate dilution factors.10.13 Convert each CFU per finger determination to thelog10value.10.14 Determine the mean log10CFU per finger value. Thisis the mean log10value for that variable and subject for thatday. These log10values are used for statistical comparisons.10.15 If an estimation of the degree of phys