1、Designation: E3011 15Standard 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.
2、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 propagate spores ofClostridium diffcile using liver broth.1.2 It is the responsibility of the user of
3、 this test 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
4、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 pri
5、or to use.2. Referenced 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 T
6、erminology Relating 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.
7、1 frozen stock culture, 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
8、 standard outlines a procedure 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 time
9、s in cold sterile deionized 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 ATC
10、C 43599. The spore suspen-sions may 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.
11、 Apparatus6.1 Anaerobe jarAny airtight jar 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 Centrifug
12、eAny type or model capable of centrifugingup 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 autocl
13、ave temperatures.1This 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 May 1, 2015. Published September 2015. DOI:10.1520/E
14、3011152For 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.3Federal Insecticide, Fungicide and Rodenticide Act (FI
15、FRA) 40 CFR Part 160,Good 185 Laboratory Practice Standards; Final Rule. 1989.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1Closures must be able to cover opening of glassware to protectmedia from environmental contamination.6.7 Mic
16、rocentrifuge tubesSterile 1.5 mL volume micro-centrifuge tubes.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 Micros
17、copeAny 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 mLvolumes.6.13 Sterile cheeseclothTwo
18、layers of cheesecloth isplaced inside an appropriately sized funnel and sterilized.6.14 Vortex mixer.7. Reagents and Materials7.1 Liver Broth4Used to sporulate C. difficile.7.2 Butterfields Phosphate Buffer Stock Solution, 0.25M(PBSS)Dissolve 34.0 g of monobasic potassium phosphate in500 mL of deion
19、ized water. Adjust pH to 7.2 with 10N NaOH,and dilute to 1 L.7.3 Butterfields Phosphate Buffered Dilution Water(PBDW)Add 1.25 mL of 0.25M PBSS to 1 L deionizedwater. Dispense into 9 mL or 99 mL portions.Autoclave for 20min at 121C.7.4 pH adjusted Phosphate Buffered Dilution Water (pHadjusted PBDW)PB
20、DW with the pH adjusted with sterile 1M Sodium hydroxide (NaOH) to a pH that neutralizes 2.5 MHydrochloric Acid (HCl) (pH 11-12 is suggested). The pH ofPBDW may also be adjusted with non-sterile 1 M NaOH priorto autoclave sterilization, provided the pH after sterilization isadequate to neutralize 2.
21、5 M HCl.7.5 Recovery Medium for Enumeration of SporeSuspensionBrain Heart Infusion Agar with yeast extract (5g/L), horse blood (70 mL/L) and sodium taurocholate (1 g/L)(BHIY-HT) , pre-reduced.7.6 Hydrochloric acid (HCl)2.5 M HCl is prepared from5 M HCl.7.7 WaterSterile deionized water.8. Hazards8.1
22、C. diffcile is a Biosafety Level 2 organism. Appropriatesafety procedures, as recommended by the US Centers forDisease Control and Prevention/National Institutes of Health5or other local agency, should be used with this organism.8.2 Consult Material Safety Data Sheets (MSDS) for thechemicals used in
23、 this method to determine the appropriatepersonal protective equipment required for handling eachchemical.9. Test Organism9.1 Frozen stock cultures of C. diffcile ATCC 700792, C.diffcileATCC 43598, or C. diffcileATCC 43599. Frozen stockcultures may be prepared from cultures obtained from areputable
24、vendor or culture collection agency.9.2 Other strains of C. diffcile may be sporulated using thismethod.10. Procedure10.1 Sporulation of Clostridium diffcile in Liquid Medium:10.1.1 Inoculate 1 L of Liver Broth with 0.25 mL to 0.5 mLof frozen stock culture of C. diffcile. Other volumes may beused as
25、 long as the ratio of Liver Broth to frozen stock cultureis the same as previously stated.10.1.2 Incubate Liver Broth under anaerobic conditions for3661C for 7-10 d, or until at least 95% spores are present.Some strains of C. diffcile may need more than 10 d to achievethe desired level of sporulatio
26、n.10.1.3 Use phase contrast microscopy, at 1000magnification, to determine percent of spores present in theLiver Broth. Stir Liver Broth prior to preparing slide for phasecontrast microscopy. Count spores and vegetative cells in fivefields of view. The broth is ready to be harvested when thepercent
27、spores is 95% as determined using equation in 11.1.Checking the spore purity beginning aroundd7isrecom-mended.10.1.4 Stir Liver Broth to resuspend any spores that havesettled to the bottom. Filter the entire volume of Liver Broththrough sterile cheesecloth, and collect in sterile centrifugetubes.10.
28、1.5 Centrifuge filtered broth at 7500 g for 20 mins at20 6 2C. Dispose of the supernate and resuspend with filteredbroth until all broth has been centrifuged. Resuspend the finalpellets in 20 to 30 mL of sterile deionized water.10.1.6 Wash the final pellets four times by centrifuging at7500 x g for
29、20 mins at 20 6 2C and discard supernate.Resuspend the pellet in sterile cold (2 - 8C) deionized water.10.1.7 Resuspend the final pellet in 10 to 30 mL of sterilecold deionized water to achieve the desired concentration ofspores. Store spore suspension at 2 - 8C for up to 6 months.10.1.8 Use phase c
30、ontrast microscopy, at 1000magnification, to determine percent of purity of the sporesuspension. Mix the spore suspension well. Count spores andvegetative cells in five fields of view. The spore suspensionshould have 95% spores as determined using equation in 11.1.10.1.9 Serially dilute the spore su
31、spension in PBDW.Spread plate appropriate dilutions in duplicate on BHIY-HT.4The sole source of supply for the Liver Broth (Cat. No. M928-500G) known tothe committee at this time is HiMedia Laboratories, Marg, Mumbai, India. If you areaware of alternative suppliers, please provide this information t
32、o ASTM Interna-tional Headquarters. Composition of media available at accessed on February 23, 2015.5Centers for Disease Control and Prevention, and National Institutes of Health,Biosafety in Microbiological and Biomedical Laboratories, 5th ed., United StatesDepartment of Health and Human Services,
33、 Washington, DC, December 2009.E3011 152Incubate inverted plates at 3661C for 72 6 4 h underanaerobic conditions. Determine the log10of the averageCFU/mL of the spore suspension.10.1.10 The spore suspension may be diluted or concen-trated as needed for efficacy testing.10.2 Quantitative Acid Resista
34、nce TestHCl Resistance(See Test Method E2839.)10.2.1 Place 990 L of 2.5 M HCl into three microcentri-fuge tubes. Place 990 L of sterile deionized water into onemicrocentrifuge tube.10.2.2 Using a micropipettor, preferably a positive displace-ment pipette, add 10 L of the high titer (8 log10spores pe
35、rmL) spore suspension to each microcentrifuge tube prepared in10.2.1. Vortex each tube. The microcentrifuge tubes should beheld at ambient temperature during the exposure time.10.2.3 At the end of the 10 min exposure time for the HClmicrocentrifuge tubes, transfer 0.1 mL from one tube to amicrocentr
36、ifuge tube with 900 L of pH adjusted PBDW toneutralize. 0.1 mL from the control microcentrifuge tube istransferred to a tube of 900 L of pH adjusted PBDW after a10 min exposure time.10.2.4 Serially dilute each neutralized spore suspension inPBDW and spread plate 0.1 mL from appropriate dilutions ind
37、uplicate onto BHIY-HT. Incubate inverted plates at 3661Cfor 72 6 4 hours under anaerobic conditions.10.2.5 A spore suspension is considered to be acid resistantif the Log10reduction is between 0 and 2 at 10 mins ofexposure as compared to the control.11. Calculation or Interpretation of Results11.1 P
38、ercent Spores Present:Average Spore CountAverage Spore Count1Average Vegetative Cell Count100! (1)Precent Spores Present=11.2 Log10of the Average CFU/mL of the Spore Suspension:Mean CFUper plate!Reciprocal of dilution!Volume Plated100! (2)Average CFU/mL=11.3 Log10Reduction of HCl treatment:Log Reduc
39、tion = LC LHLC = Log10of viable spores after control treatmentLH = Log10of viable spores after HCl treatments12. Precision and Bias12.1 A precision and bias statement cannot be made for thistest method at this time.13. Keywords13.1 C. diffcile; Clostridium; liver broth; propagation;spores; spore cro
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