1、Designation: E1874 09E1874 14Standard 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 y
2、ear 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 is designed to recover microorganisms from the skin of human subjects or human subject surroga
3、tes(animal skin, isolated porcine skin, human skin equivalents, and other such surfaces).1.2 Knowledge of microbiological techniques is required for these procedures.1.3 It is the responsibility of the investigator to determine if Good Laboratory Practice (GLP) and Good Clinical Practice (GCP)is req
4、uired.1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibilityof the user of this standard to estab
5、lish appropriate safety and health practices and determine the applicability of regulatorylimitations prior to use.2. Referenced Documents2.1 ASTM Standards:2E1054 Test Methods for Evaluation of Inactivators of Antimicrobial Agents3. Terminology3.1 Definitions of Terms Specific to This Standard:3.1.
6、1 contralateral, adjon or relating to the opposite side (of the body).3.1.2 resident flora, nmicroorganisms that live and multiply on skin, forming a permanent population.3.1.3 scrub cups, nsterile cylinders of suitable composition (that is, glass, ceramic, stainless steel, plastic, etc.) used to is
7、olatea sample area of skin (or skin equivalent) and confine a aliquot of liquid which is used to facilitate the scrubbing of the skin andremoval of microorganisms from the skin surface by pipetting.3.1.4 transient organisms, norganisms from the environment that contaminate but do not normally coloni
8、ze skin.4. Summary of Test Method4.1 This test method describes a technique suitable for the recovery of resident and transient microorganisms from human oranimal skin; the technique may be used in situ within clinical protocols or in vitro for studies using isolated skin or skin equivalents.4.1.1 R
9、esident microorganisms or transient microorganisms (previously applied to a test site), are recovered from the site bypressing a rigid cylinder against the skin with sufficient pressure to form a seal and instilling recovery liquid into the cylinder. Thesurface of the skin is then mechanically scrub
10、bed with a glass rod, rubber policeman, or some other suitable device for aprescribed period of time. The fluid is pipetted from the cylinder into a test tube, or other suitable receptacle, for further analysis.5. Significance and Use5.1 The procedure can be incorporated into protocols used to evalu
11、ate test materials containing antibacterial ingredients that areintended to reduce significantly the number of organisms on intact skin. It also may be used to provide an indication of residual1 This tests method is under the jurisdiction of ASTM Committee E35 on Pesticides, Antimicrobials, and Alte
12、rnative Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved Oct. 1, 2009April 1, 2014. Published October 2009April 2014. Originally approved in 1997. Last previous edition approved in 19972009 asE1874 97E1875, which was withdrawn Jan
13、uary 2006 and reinstated in October 2009. DOI: 10.1520/E1874-09.09. DOI: 10.1520/E1874-14.2 For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to the standards Document Summ
14、ary page on the ASTM website.This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Becauseit may not be technically possible to adequately depict all changes accurately, ASTM recommends
15、 that users consult prior editions as appropriate. In all cases only the current versionof the standard as published by ASTM is to be considered the official document.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1antibacterial activ
16、ity. Examples of test materials, for which this method is applicable, include hand-washes, surgical scrubs, acnereduction products, and others. For each type of test material, types of resident flora or transient organisms, or a combinationthereof, may differ and should be considered (this is, aerob
17、ic bacteria, anaerobic bacteria, yeast, or mold).5.2 The procedure may be used in protocols intended to evaluate and identify resident flora from the skin.5.3 Performance of this technique may require the knowledge of regulations pertaining to the protection of human subjects ifthe protocol involves
18、 application of the technique to the skin of human subjects.6. Apparatus6.1 SterilizerAny suitable steam sterilizer capable of producing the conditions of sterilization.7. Reagents and Materials7.1 Scrub CupsSterile cylinders of suitable composition, preferably with rod handles to facilitate stabili
19、zation, heightapproximately 2.5 cm, inside diameter of convenient size. Useful sizes range from approximately 1.5 to 4.0 cm.7.2 Polished Glass Rod or Rubber PolicemanCan be fashioned in the laboratory or purchased.7.3 PipettorWith disposable tips to deliver appropriate volume(s).7.4 Sterile Beakers,
20、 Test Tubes or other container, to receive the cup scrub fluid.7.5 Appropriate Bacterial CulturesIf this test method will be used within a protocol targeting transient organisms.7.6 Sampling and Dilution FluidSterile Butterfields phosphate buffered water or other recovery fluid of suitable compositi
21、on;this should contain an antimicrobial inactivator specific for any antimicrobial that might be on the test site; inactivator efficacyshould be determined by Test Method E1054.8. Test Control and Baseline Skin Sites8.1 Select skin sites appropriate for target flora and the protocol objectives; wher
22、e possible, contralateral sample sites arerecommended for use as controls.9. Sample Site9.1 SubjectsThe number of subjects (human or animal) required (if the protocol is in vivo) depends on the statisticalconfidence needed for the expected test results, the variability encountered in the study, and
23、the relative efficacy of any antibacterialagent that may be evaluated. There may be multiple sites available on subjects; randomization is required to suppress sample bias.9.2 Isolated Skin or EquivalentsThe number of replicates required to discriminate effects will depend in part on theappropriaten
24、ess and design of controls within the protocol.9.2.1 The use of this technique on isolated skin or equivalents is dependent on securing the test site in order to effectivelyperform the procedure.10. Sampling Live Subjects (Human or Animal)10.1 Method:10.1.1 Quantitative microbial counts are obtained
25、 by the cup scrub technique.3 This procedure is used at test and control sites.10.1.2 Subjects are positioned for site sampling.10.1.3 The area to be sampled is delineated by a sterile sampling cylinder. The cylinder is pressed firmly against the skin surfaceduring sampling to ensure that the sampli
26、ng fluid does not leak from the sampling site.10.1.4 A minimum 1.5-mL aliquot of sterile sampling fluid, with or without product neutralizers, is pipetted into the cylinder.The entire area is then scrubbed with moderate pressure for 60 6 6 s using a sterile polished glass rod or policeman. Afterscru
27、bbing, the sampling fluid is transferred by pipette into a sterile sample tube. This procedure is repeated once more with a freshaliquot of sampling fluid. The sampling fluids are pooled. This procedure is repeated for each sampling site.10.1.5 The same pipettes, cylinders, glass rods, and policeman
28、 are used for both washes of a site, but new sterile equipment isused for each site. After samples are collected, paper toweling is used to blot the site dry.10.1.6 Care must be taken during this process to prevent the sampling fluid from spilling into an adjacent site that has not beensampled.10.1.
29、7 Following all sampling, when using marker organisms, the sampling site should be decontaminated using 70 to 90 %isopropanol or equivalent,(or equivalent), followed by a 4 % chlorhexidine scrub.scrub (or equivalent).3 Williamson, P., and Kligman, A. M., “A New Method for the Quantitative Investigat
30、ion of Cutaneous Bacteria,” Journal of Investigative Dermatology, Vol 46, 1965,pp. 498503.E1874 14211. Sampling Isolated Skin or Skin Equivalents11.1 Method:11.1.1 Quantitative microbial counts are obtained by the cup scrub technique.3 This procedure is used for test and controlsamples.11.1.2 Sample
31、s are positioned and secured as necessary to enable placement and effective use of the sampling cylinder.11.1.3 The area to be sampled is delineated by a sterile sampling cylinder. The cylinder is pressed firmly against the samplesurface during sampling to ensure that the sampling fluid does not lea
32、k from the sampling site.11.1.4 A minimum 1.5-mL aliquot of sterile sampling fluid, with or without product neutralizers, is pipetted into the cylinder.The entire area is then scrubbed with moderate pressure for 60 6 6 s using a sterile polished glass rod or policeman. Afterscrubbing, the sampling f
33、luid is transferred by pipet into a sterile sample tube. This procedure is repeated once more with a freshaliquot of sampling fluid. The sampling fluids are pooled. This procedure is repeated for each sampling site.11.1.5 The same pipettes, cylinders, glass rods, and policeman are used for both wash
34、es of a site, but new sterile equipment isused for each site.11.1.6 If there are multiple sample sites on the same piece of isolated tissue, care must be taken during this process to preventthe sampling fluid from spilling into an adjacent site that has not been sampled.12. Microbial Counts12.1 Each
35、 sample is mixed thoroughly. Tenfold serial dilutions of each sample are prepared in dilution fluid. Duplicatequantitative pour or spread plates using soybean-casein digest agar with suitable neutralizer are prepared. are prepared. Selectionof agar is dependent upon purpose of method execution. For
36、determination of antibacterial effectiveness or residual antimicrobialactivity, or both, agar shall contain suitable neutralizer. Type(s) of resident flora of interest in the study shall also be consideredin selecting media (that is, aerobic bacteria, anaerobic bacteria, yeast or mold). Incubate pla
37、ted samples at suitable growthtemperature, 62C for 24 to 72 h, temperature and atmosphere conditions for organism(s) of interest, or until colonies are visibleon the plates.12.2 If the intended evaluation is for identification of resident flora, the collected samples may be frozen and utilized forse
38、quencing or other methods for direct (that is, without first growing samples on agar) organism identification.13. Precision and Bias13.1 A precision and bias statement cannot be made for this test method at this time.14. Keywords14.1 cup scrub; resident flora; skin; skin equivalent; transient organi
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42、tee 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). Permission rights to photocopy the standard may also be secured from the ASTM website (www.astm.org/COPYRIGHT/).E1874 143
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