1、Designation:E301115Standard Test Method forIn vitro production of Clostridium Difficile Spores1This standard is issued under the fixed designation E3011;the number immediately following the designation indicates the year oforiginal adoption or,in the case of revision,the year of last revision.A numb
2、er 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 propagate spores ofClostridium diffcile using liver broth.1.2 It is the responsibility of the user of this test
3、 method todetermine whether Good Laboratory Practices are required andfollow when appropriate.1.3 This test method should only be performed by thosetrained in microbiological techniques.1.4 UnitsThe values stated in SI units are to be regardedas standard.No other units of measurement are included in
4、 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.Re
5、ferenced Documents2.1 ASTM Standards:2E2839 Test Method for Production of Clostridium difficileSpores for Use in Efficacy Evaluation of AntimicrobialAgentsE2895 Test Method for Producing High Titers of Viable andSemi-Purified Spores of Clostridium difficile using aLiquid MediumE2756 Terminology Rela
6、ting to Antimicrobial and AntiviralAgents2.2 Federal Standard:340 CFR,Part 160 Good Laboratory Practice Standards3.Terminology3.1 Definitions:For definitions of general terms used in thistest method,refer to Terminology E2756.3.2 Definitions of Terms Specific to This Standard:3.2.1 frozen stock cult
7、ure,na culture of vegetative bacte-ria propagated,and prepared for storage at 70C in a liquidbroth medium containing a cryoprotectant such as glycerol.3.2.2 spore suspension,nharvested spores suspended in aliquid medium,sterile deionized water.4.Summary of Test Method4.1 This standard outlines a pro
8、cedure for producing high-titer spore suspensions of C.diffcile using a commerciallyavailable liquid medium with 7 to 10 d of incubation underanaerobic conditions.Once adequate levels of spores arepresent in the liquid medium,the spores are harvested andwashed several times in cold sterile deionized
9、 water.The sporesuspension is enumerated,the spore purity is assessed and theacid resistance is verified.5.Significance and Use5.1 This test method describes a procedure for producingspore suspensions of C.diffcile ATCC 700792,C.diffcileATCC 43598,or C.diffcile ATCC 43599.The spore suspen-sions may
10、be used in antimicrobial efficacy testing,or otherlaboratory testing requiring C.diffcile spores.A spore crop isconsidered acceptable if the titer is 8 log10spores/mL,purityof 95%,and is resistant to 2.5M HCl after 10 min of exposure(see Test Method E2839).6.Apparatus6.1 Anaerobe jarAny airtight jar
11、 or container that can beused in combination with gas packs to obtain an anaerobicenvironment.An anaerobic chamber and anaerobic incubatormay be substituted for an anaerobe jar.6.2 Biological safety cabinetTo help maintain an asepticwork space.6.3 CentrifugeAny type or model capable of centrifugingu
12、p to 40 mL of liquid at 7500 g.6.4 Centrifuge tubesAny type of sterile centrifuge tubewith a 50 mL capacity.6.5 IncubatorAn incubator capable of maintaining3661C.6.6 Laboratory glassware with closuresGlassware to holdup to 1L of media,and withstands autoclave temperatures.1This test method is under
13、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 May 1,2015.Published September 2015.DOI:10.1520/E3011152For referenced ASTM standards,visit the AS
14、TM 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.3Federal Insecticide,Fungicide and Rodenticide Act(FIFRA)40 CFR Part 160,Good 185 Laboratory Practice Standar
15、ds;Final Rule.1989.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 Closures must be able to cover opening of glassware to protectmedia from environmental contamination.6.7 Microcentrifuge tubesSterile 1.5 mL volume micro-centrifuge tubes.
16、6.8 MicropipettorsAny suitable models capable of pi-petting 10 L or up to 1000 L,or both.Calibrated micropi-pettors are preferred.6.9 Micropipette tips,sterileAny sterile micropipette tipsfor use with Micropipettors in 6.8.6.10 MicroscopeAny microscope capable of 1000 mag-nification with phase contrast options.6.11 Plate spreaderAny sterile spreader for spreadinginocula on agar plates.6.12 Serological pipettesAny sterile,single use pipettesthat are capable of pipetting 1.0 mL,10 mL,or 50 mLvolum