1、Designation:E289513Standard Test Method forProducing High Titers of Viable and Semi-Purified Spores ofClostridium difficile using a Liquid Medium1This standard is issued under the fixed designation E2895;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.INTRODUCTIONClostridium diffcile(C.diffcile),an anaerobic spore-former,can cause acute and potentially
3、fatalgastroenteritis in healthcare and other settings(1).2The frequent episodes of diarrhea can contaminatethe indoor environment widely with persistent(2)and microbicide-resistant(3)spores.Disinfectantswishing to claim activity against C.diffcile now require carrier testing using spores of high pur
4、ity(90%)that show a 6 log10reduction in spore viability(4).While the use of a semi-solid mediumfor C.diffcile spore production has been reported(5)(Test Method E2839),this standard describes aliquid medium and an enzyme-based semipurification process for the purpose.Appendix X1 describesan alternati
5、ve to enzyme purification.1.Scope1.1 This test method describes the production and semipu-rification of C.diffcile spores(also called endospores)primar-ily for use in testing the sporicidal activities of environmentalsurface disinfectants(Test Methods E2111and E2197);suchspores can also be used to s
6、tudy their structure,chemistry andgermination.1.2 While the test method described is based on the use of500-mL volumes of the liquid culture medium in an anaerobicincubator,anaerobic jars with smaller volumes of the samemedium can also be used.1.3 It is the responsibility of the investigator to dete
7、rminewhether Good Laboratory Practice(GLP)regulations arerequired and to follow them when appropriate(40 CFR,Part160 for EPA submissions and 21 CFR;Part 58 for FDAsubmissions).1.4 WarningThis standard may involve hazardousmaterials,chemicals,and microorganisms and should be per-formed only by person
8、s with formal training in microbiology.1.5 The values stated in SI units are to be regarded asstandard.No other units of measurement are included in thisstandard.1.6 This standard does not purport to address all of thesafety concerns,if any,associated with its use.It is theresponsibility of the user
9、 of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2.Referenced Documents2.1 ASTM Standards:3D1129 Terminology Relating to WaterE2111 Quantitative Carrier Test Method to Evaluate theBactericidal,Fungicidal,M
10、ycobactericidal,and SporicidalPotencies of Liquid ChemicalsE2197 Quantitative Disk Carrier Test Method for Determin-ing Bactericidal,Virucidal,Fungicidal,Mycobactericidal,and Sporicidal Activities of ChemicalsE2756 Terminology Relating to Antimicrobial and AntiviralAgentsE2839 Test Method for Produc
11、tion of Clostridium difficileSpores for Use in Efficacy Evaluation of AntimicrobialAgents2.2 Federal Standards:421 CFR,Part 58 Good Laboratory Practice for NonclinicalLaboratory Studies40 CFR,Part 160 Good Laboratory Practice Standards1This test method is under the jurisdiction of ASTM Committee E35
12、 onPesticides,Antimicrobials,and Alternative Control Agents and is the directresponsibility of Subcommittee E35.15 on Antimicrobial Agents.Current edition approved Oct.1,2013.Published December 2013.DOI:10.1520/E2895-132The boldface numbers in parentheses refer to a list of references at the end oft
13、his standard.3For referenced ASTM standards,visit the ASTM website,www.astm.org,orcontact ASTM Customer Service at serviceastm.org.For Annual Book of ASTMStandards volume information,refer to the standards Document Summary page onthe ASTM website.4Available from Superintendent of Documents,U.S.S Gov
14、ernment PrintingOffice,Washington DC,20402.http:/www.gpo.gov/Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 3.Terminology3.1 DefinitionsFor definitions of general terms used in thistest method,refer to Terminologies D1129 and E2756.3.2 D
15、efinitions of Terms Specific to This Standard:3.2.1 anaerobe,nan organism that cannot grow or prolif-erate in the presence of free oxygen.3.2.2 enzymatic treatment,nuse of one or more enzymesto digest remnants of cells in spore suspensions.3.2.3 germination,nwhen a spore re-emerges into itsvegetativ
16、e form for active growth and replication.3.2.4 spore,nphase of dormancy in the reproductive cycleof certain types of microorganisms where individual cellsbecome condensed in a relatively impermeable coat,enablingprolonged survival and greater resistance to deleterious envi-ronmental factors.3.2.4.1 DiscussionAspore can germinate into a vegetativecell under favorable conditions to reinitiate the cycle ofreplication.4.Summary of Test Method4.1 This standard relates to a new liquid culture mediumto
