ASTM_E_2839_-_11.pdf

Designation:E283911Standard Test Method forProduction of Clostridium difficile Spores for Use inEfficacy Evaluation of Antimicrobial Agents1This standard is issued under the fixed designation E2839;the number immediately following the designation indicates the year 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()indicates an editorial change since the last revision or reapproval.INTRODUCTIONSporulation in Clostridium diffcile is not as rapid or as efficient as in other species and it is generallydifficult to produce C.diffcile spores of high titer in the laboratory(1,2).2Although quantitative testmethods are available for testing sporicidal products,a standardized method for generating sporesuspensions of C.diffcile of high titer(8 log10/mL)and purity($95%spores)is not available andwould be necessary in order to conduct performance testing required for registration purposes(3).Thespore suspensions resulting from practice of this test method are appropriate for use in accepted testmethods for measuring the sporicidal efficacy of antimicrobial formulations(4).1.Scope1.1 This test method is for producing C.diffcile spores toevaluate antimicrobial formulations for their sporicidal activ-ity.1.2 It is the responsibility of the investigator to determinewhether Good Laboratory Practices(GLP)are required and tofollow them when appropriate.1.3 This standard may involve hazardous materials,chemi-cals,and microorganisms and should be performed only bypersons with formal training in microbiology.1.4 The 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-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2.Terminology2.1 Definitions:2.1.1 CFU,adj/ncolony-forming units;the number ofspores or microorganisms that can form colonies(clusters ofmicroorganisms visibly growing on the surface of a solid agarmedium)in spread plates,as an indication of the total numberof viable spores/microorganisms in a sample.2.1.2 QC,adj/nquality control(QC)is the application ofprocedures,products,or services to meet a laboratorys speci-fied standards of quality.2.1.3 pre-reduced medium,adj/nan agar or broth manu-factured and sterilized in an oxygen-free environment,andpackaged individually in air-tight sealed pouches or bags.2.1.4 density gradient medium,adj/nHistoDenz(trade-marked)3is a non-ionic gradient medium used here to separatespores from vegetative cells and cell fragments on the basis ofdensity.2.1.5 purified spores,adj/nwhen spore concentrationreaches$95%as vegetative cells and cell fragments areseparated by the density gradient medium.2.1.6 toxigenic strain,adj/npossesses either toxin A gene(tcdA+)or toxin B gene(tcdB+)or both.3.Summary of Test Method3.1 This test method provides detailed instructions for theculture,maintenance and sporulation of C.diffcile on a specificagar medium incubated in an anaerobic environment for 7 to 10days.Monitoring is performed by phase-contrast microscopyto ensure sporulation is underway and to determine when thespore concentration reaches$90%,the optimal time ofharvest.Upon harvesting,spores are washed several times with1This test method is under the jurisdiction of ASTM Committee E35 onPesticides,Antimicrobials,and Alternative Control Agents and is the directresponsibility of Subcommittee E35.15 on Antimicrobial Agents.Current edition approved Aug.1,2011.Published September 2011.DOI:10.1520/E2839-11.2The boldface numbers in parentheses refer to a list of references at the end ofthis standard.3The sole source of supply of HistoDenz(trademark)(Cat.No.D2158)knownto the committee at this time is Sigma-Aldrich,St.Louis,MO.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.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 saline-Tween 80,treated with heat to inactivate any remainingviable vegetative cells,and purified using a density gradientmedium to remove inactivated vegetative cells and cell frag-ments,with a target spore-purity of$95%.Purified spores areenumerated on specific agar-based recovery medium for titerdetermination and assessed for quality using a quantitativeacid-resistance test.4.Significance and Use4.1 This test method describes a procedure for preparing aspore suspension of C.diffcile strain ATCC 43598 that meetsspecific criteria necessary for efficacy testing of antimicrobialsdesigned to eliminate C.diffcile contamination from environ-mental surfaces.The a