ASTM E1874-2009 Standard Test Method for Recovery of Microorganisms from Skin using the Cup Scrub Technique《用杯擦洗技术在皮肤上恢复微生物的标准试验方法》.pdf

1、Designation: E1874 09Standard Test Method forRecovery of Microorganisms From Skin using the CupScrub Technique1This standard is issued under the fixed designation E1874; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of l

2、ast 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 recover microorganismsfrom the skin of human subjects or human subject surrogates(anima

3、l skin, isolated porcine skin, human skin equivalents,and other such surfaces).1.2 Knowledge of microbiological techniques is requiredfor these procedures.1.3 It is the responsibility of the investigator to determine ifGood Laboratory Practice (GLP) and Good Clinical Practice(GCP) is required.1.4 Th

4、e values stated in SI units are to be 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-pria

5、te safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2E1054 Test Methods for Evaluation of Inactivators of An-timicrobial Agents3. Terminology3.1 Definitions of Terms Specific to This Standard:3.1.1 contralat

6、eral, adjon or relating to the opposite side(of the body).3.1.2 resident flora, nmicroorganisms that live and mul-tiply on skin, forming a permanent population.3.1.3 scrub cups, nsterile cylinders of suitable composi-tion (that is, glass, ceramic, stainless steel, plastic, etc.) used toisolate a sam

7、ple area of skin (or skin equivalent) and confine aaliquot of liquid which is used to facilitate the scrubbing of theskin and removal of microorganisms from the skin surface bypipetting.3.1.4 transient organisms, norganisms from the environ-ment that contaminate but do not normally colonize skin.4.

8、Summary of Test Method4.1 This test method describes a technique suitable for therecovery of resident and transient microorganisms from humanor animal skin; the technique may be used in situ withinclinical protocols or in vitro for studies using isolated skin orskin equivalents.4.1.1 Resident microo

9、rganisms or transient microorganisms(previously applied to a test site), are recovered from the siteby pressing a rigid cylinder against the skin with sufficientpressure to form a seal and instilling recovery liquid into thecylinder. The surface of the skin is then mechanicallyscrubbed with a glass

10、rod, rubber policeman, or some othersuitable device for a prescribed period of time. The fluid ispipetted from the cylinder into a test tube, or other suitablereceptacle, for further analysis.5. Significance and Use5.1 The procedure can be incorporated into protocols usedto evaluate test materials c

11、ontaining antibacterial ingredientsthat are intended to reduce significantly the number of organ-isms on intact skin. It also may be used to provide an indicationof residual antibacterial activity.5.2 Performance of this technique may require the knowl-edge of regulations pertaining to the protectio

12、n of humansubjects if the protocol involves application of the technique tothe skin of human subjects.1This tests 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

13、edition approved Oct. 1, 2009. Published October 2009. Originallyapproved in 1997. Last previous edition approved in 1997 as E1874 97, which waswithdrawn January 2006 and reinstated in October 2009. DOI: 10.1520/E1874-09.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact

14、 ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards 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.6. Apparatus6.1 Sterilize

15、rAny suitable steam sterilizer capable ofproducing the conditions of sterilization.7. Reagents and Materials7.1 Scrub CupsSterile cylinders of suitable composition,preferably with rod handles to facilitate stabilization, heightapproximately 2.5 cm, inside diameter of convenient size.Useful sizes ran

16、ge from approximately 1.5 to 4.0 cm.7.2 Polished Glass Rod or Rubber PolicemanCan befashioned in the laboratory or purchased.7.3 PipettorWith disposable tips to deliver appropriatevolume(s).7.4 Sterile Beakers, Test Tubes or other container, to receivethe cup scrub fluid.7.5 Appropriate Bacterial Cu

17、lturesIf this test method willbe used within a protocol targeting transient organisms.7.6 Sampling and Dilution FluidSterile Butterfieldsphosphate buffered water or other recovery fluid of suitablecomposition; this should contain an antimicrobial inactivatorspecific for any antimicrobial that might

18、be on the test site;inactivator efficacy should be determined by Test MethodE1054.8. Test Control and Baseline Skin Sites8.1 Select skin sites appropriate for target flora and theprotocol objectives; where possible, contralateral sample sitesare recommended for use as controls.9. Sample Site9.1 Subj

19、ectsThe number of subjects (human or animal)required (if the protocol is in vivo) depends on the statisticalconfidence needed for the expected test results, the variabilityencountered in the study, and the relative efficacy of anyantibacterial agent that may be evaluated. There may bemultiple sites

20、available on subjects; randomization is requiredto suppress sample bias.9.2 Isolated Skin or EquivalentsThe number of replicatesrequired to discriminate effects will depend in part on theappropriateness and design of controls within the protocol.9.2.1 The use of this technique on isolated skin or eq

21、uiva-lents is dependent on securing the test site in order toeffectively perform the procedure.10. Sampling Live Subjects (Human or Animal)10.1 Method:10.1.1 Quantitative microbial counts are obtained by thecup scrub technique.3This procedure is used at test and controlsites.10.1.2 Subjects are posi

22、tioned for site sampling.10.1.3 The area to be sampled is delineated by a sterilesampling cylinder. The cylinder is pressed firmly against theskin surface during sampling to ensure that the sampling fluiddoes not leak from the sampling site.10.1.4 A minimum 1.5-mL aliquot of sterile sampling fluid,w

23、ith or without product neutralizers, is pipetted into thecylinder. The entire area is then scrubbed with moderatepressure for 60 6 6 s using a sterile polished glass rod orpoliceman.After scrubbing, the sampling fluid is transferred bypipette into a sterile sample tube. This procedure is repeatedonc

24、e more with a fresh aliquot of sampling fluid. The samplingfluids are pooled. This procedure is repeated for each samplingsite.10.1.5 The same pipettes, cylinders, glass rods, and police-man are used for both washes of a site, but new sterileequipment is used for each site. After samples are collect

25、ed,paper toweling is used to blot the site dry.10.1.6 Care must be taken during this process to prevent thesampling fluid from spilling into an adjacent site that has notbeen sampled.10.1.7 Following all sampling, when using marker organ-isms, the sampling site should be decontaminated using 70 to90

26、 % isopropanol or equivalent, followed by a 4 % chlorhexi-dine scrub.11. Sampling Isolated Skin or Skin Equivalents11.1 Method:11.1.1 Quantitative microbial counts are obtained by the cupscrub technique.3This procedure is used for test and controlsamples.11.1.2 Samples are positioned and secured as

27、necessary toenable placement and effective use of the sampling cylinder.11.1.3 The area to be sampled is delineated by a sterilesampling cylinder. The cylinder is pressed firmly against thesample surface during sampling to ensure that the samplingfluid does not leak from the sampling site.11.1.4 A m

28、inimum 1.5-mL aliquot of sterile sampling fluid,with or without product neutralizers, is pipetted into thecylinder. The entire area is then scrubbed with moderatepressure for 60 6 6 s using a sterile polished glass rod orpoliceman.After scrubbing, the sampling fluid is transferred bypipet into a ste

29、rile sample tube. This procedure is repeated oncemore with a fresh aliquot of sampling fluid. The samplingfluids are pooled. This procedure is repeated for each samplingsite.11.1.5 The same pipettes, cylinders, glass rods, and police-man are used for both washes of a site, but new sterileequipment i

30、s used for each site.11.1.6 If there are multiple sample sites on the same piece ofisolated tissue, care must be taken during this process toprevent the sampling fluid from spilling into an adjacent sitethat has not been sampled.12. Microbial Counts12.1 Each sample is mixed thoroughly. Tenfold seria

31、l dilu-tions of each sample are prepared in dilution fluid. Duplicatequantitative pour or spread plates using soybean-casein digestagar with suitable neutralizer are prepared. Incubate platedsamples at suitable growth temperature, 62C for 24 to 72 h,or until colonies are visible on the plates.3Willi

32、amson, P., and Kligman, A. M., “A New Method for the QuantitativeInvestigation of Cutaneous Bacteria,” Journal of Investigative Dermatology,Vol46, 1965, pp. 498503.E1874 09213. Precision and Bias13.1 A precision and bias statement cannot be made for thistest method at this time.14. Keywords14.1 cup

33、scrub; resident flora; skin; skin equivalent; tran-sient organismASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentionedin this standard. Users of this standard are expressly advised that determination of the validity of any su

34、ch patent rights, and the riskof infringement of such rights, are entirely their own responsibility.This standard is subject to revision at any time by the responsible technical committee and must be reviewed every five years andif not revised, either reapproved or withdrawn. Your comments are invit

35、ed either for revision of this standard or for additional standardsand should be addressed to ASTM International Headquarters. Your comments will receive careful consideration at a meeting of theresponsible technical committee, which you may attend. If you feel that your comments have not received a

36、 fair hearing you shouldmake your views known to the ASTM Committee on Standards, at the address shown below.This standard is copyrighted by ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959,United States. Individual reprints (single or multiple copies) of this standard may be obtained by contacting ASTM at the aboveaddress or at 610-832-9585 (phone), 610-832-9555 (fax), or serviceastm.org (e-mail); or through the ASTM website(www.astm.org).E1874 093