1、Designation: E2895 13Standard 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, i
2、n 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.INTRODUCTIONClostridium diffcile (C. diffcile), an anaerobic spore-former, can cause acute and
3、 potentially 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 usin
4、g spores of high purity(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. Appen
5、dix X1 describesan alternative 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)
6、; suchspores can also be used to study 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 respon
7、sibility of the investigator to determinewhether 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 microorgani
8、sms and should be per-formed only by persons 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 wi
9、th 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. Referenced Documents2.1 ASTM Standards:3D1129 Terminology Relating to WaterE2111 Quantitative Carrier Test
10、 Method to Evaluate theBactericidal, Fungicidal, Mycobactericidal, 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 Antimic
11、robial and AntiviralAgentsE2839 Test Method for Production 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 tes
12、t 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 Oct. 1, 2013. Published December 2013. DOI:10.1520/E2895-132The boldface numbe
13、rs in parentheses refer to a list of references at the end ofthis 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
14、 ASTM website.4Available from Superintendent of Documents, U.S.S Government PrintingOffice, Washington DC, 20402. http:/www.gpo.gov/Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States13. Terminology3.1 DefinitionsFor definitions of genera
15、l terms used in thistest method, refer to Terminologies D1129 and E2756.3.2 Definitions 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
16、 spore suspensions.3.2.3 germination, nwhen a spore re-emerges into itsvegetative 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, enablingprolon
17、ged 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 mediumtogether with a sem
18、ipurification process (2) designed to pro-duce high titers (109CFU/mL) of viable spores of C. diffcilewith a purity of 90%. In this method, the semipurification ofthe spores is achieved by enzyme treatment. A surcose densitygradient method (Appendix X1) is also described as analternative to enzyme t
19、reatment.5. Significance and Use5.1 The quantity and quality of the spores produced by thismethod meet the current requirements of the U.S. Environmen-tal Protection Agency (EPA) to assess environmental surfacedisinfectants for label claims of sporicidal activity (4). Themethod is applicable to stan
20、dard as well as clinically isolatedtoxigenic and non-toxigenic strains of C. diffcile.6. Reagents, Materials, and Equipment56.1 Chemicals and Reagents:6.1.1 Columbia Broth (CB) Powder.6.1.2 Special Peptone Mix (SPM).6.1.3 Brain-Heart Infusion (BHI) Broth Powder.6.1.4 Yeast Extract Powder.6.1.5 Bacte
21、riological Agar.6.1.6 Lysozyme (egg white).6.1.7 Trypsin (porcine pancreas) lyophilized.6.1.8 Disodium Hydrogen Phosphate (Na2HPO4).6.1.9 Sodium Dihydrogen Phosphate (NaH2PO4H2O).6.1.10 Potassium Hydroxide (KOH).6.1.11 Sodium Hydroxide (NaOH).6.1.12 Hydrochloric Acid (HCl).6.1.13 Potassium Dihydroge
22、n Phosphate (KH2PO4).6.1.14 Potassium Carbonate (K2CO3).6.1.15 Calcium Chloride (CaCl22H2O).6.1.16 Ammonium Sulfate (NH4)2SO4).6.1.17 Magnesium Sulfate (MgSO4).6.1.18 Taurocholic Acid Sodium Salt Hydrate.6.1.19 L-Cysteine.6.1.20 Phosphate Buffered Saline (PBS)0.85% NaCl in0.3 mM phosphate buffer (pH
23、 7.2 6 0.1).6.1.21 PBS Tween680 (PBS-T)0.85% NaCl and 0.1%(v/v) Tween 80 in 0.3 mM phosphate buffer (pH 7.2 6 0.1).6.1.22 0.1 M Sodium Phosphate BufferpH 7.0 6 0.2.6.1.23 Deionized Wateror water of equivalent purity.6.1.24 Tryptic Soy Agar Plates (TSA)to test the final sporesuspension for contaminat
24、ion.6.2 Equipment:6.2.1 Anaerobic Incubatorwith gas supply: 5% CO2, 10%H2, 85% N2, capable of maintaining 36 6 1C.NOTE 1Anaerobic jars with suitable gas packs may also be used.6.2.2 Analytical Balance.6.2.3 Disposable or Reusable Membrane Filter Holders, for47mm diameter membrane filters.6.2.4 Bench
25、-top Centrifuge.6.2.5 Centrifuge with Fixed-angle and Swinging-bucketRotors, to process spore suspensions.6.2.6 Forceps, straight or curved with smooth tips to handlemembrane filters.6.2.7 Freezers, one at -20 6 2C to store media andadditives, and another at -70C or lower to store stocks ofmicroorga
26、nisms.6.2.8 Hot Plate with Stirrerto prepare culture media andreagents.6.2.9 Laminar-flow Biological Safety CabinetClass II,Type Aprocedures for proper maintenance and use of suchcabinets are given in the reference section (6).6.2.10 Light Microscope, with an oil-immersion objective toobserve and co
27、unt spores and vegetative cells in stained slidesto assess the level of spore purity.6.2.11 Standard Glass Microscope Slides and Coverslips6.2.12 pH Meter, with electrodes and standard solutions.6.2.13 Pipettors, in the following sizes: 1-20 L, 20-100 L,and 200-1000 L.6.2.14 Positive-displacement Pi
28、pette, 2-20 L.6.2.15 Refrigerator, capable of maintaining 4 6 1C forstorage of culture media, culture plates, and reagents.6.2.16 Sterile Serological Pipettes, 1, 5, 10, and 25-mLcapacity.6.2.17 Sonicator Bath, 40 KHz; recommended to breakdown spore clusters.6.2.18 Sterilizer, any steam sterilizer s
29、uitable for processingculture media, reagents, and labware.6.2.19 Vacuum Source, a vacuum pump, access to an in-house vacuum line, or a water faucet vacuum apparatus, to pullsamples through membrane filters.5Reagent Chemicals, American Chemical Society Specifications, AmericanChemical Society, Washi
30、ngton, DC. For Suggestions on the testing of reagents notlisted by the American Chemical Society, see Annual Standards for LaboratoryChemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeiaand National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,MD.6Trademark
31、 ICI Americas.E2895 1326.2.20 Vortex Mixers, one for use outside and another toremain inside the anaerobic chamber.6.2.21 Waterbath, for heat-inactivation of vegetative cells inspore suspensions at 69 6 1C.6.3 Labware(unless otherwise indicated, all labware mustbe sterile):6.3.1 Plastic Transfer Pip
32、ettes, Pasteur pipettes.6.3.2 Polypropylene Tubes, 15 and 50-mL screw-cap.6.3.3 Glass Culture Flasks, 1L.6.3.4 Pipette Tips, 20, 200, and 1000 L.6.3.5 Polycarbonate Centrifuge Tubes, 40 and 250 mL.6.3.6 Cryovials, 2 mL.6.3.7 Membrane Filters, 47-mm diameter; 0.22-m porediameter.6.3.8 Syringe-driven
33、Filter Units, 20-mm diameter;0.22-m pore diameter; low protein-binding.6.3.9 Petri Dishes, 100 mm in diameter, for spore recoverymedium.6.4 C. diffcile Strain:6.4.1 While much of this method is based on work withATCC 43598, the procedures described are suitable for otherstandard strains, and also cl
34、inical isolates of the organism.7. Methods7.1 Preparation of Media:7.1.1 Columbia Broth (CB):7.1.1.1 Prepare a 1 solution of CB following the manu-facturers instructions.7.1.1.2 Sterilize by autoclaving and store refrigerated.7.1.2 Liquid Sporulation Medium:7.1.2.1 Prepare 1 Lof the medium in a 2-LE
35、rlenmeyer flaskby adding the following in the order given:Deionized water 700 mLSPM 10.0 gKH2PO4 2.60 g(NH4)2SO40.60 gCaCl22H2O0.08Yeast extract powder 10.0 gK2CO33.48 gMgSO40.12 gDeionized water to 1 L7.1.2.2 The pH of the medium should be 7.9 6 0.2 beforeautoclave sterilization; if needed, adjust
36、with 1 M KOH.7.1.2.3 Put 500 mL of the medium into two 1-L flasks andautoclave for 20 min at 121C.7.1.2.4 Wait about 3 h for the temperature to drop to5060C.7.1.2.5 Place the flasks in an anaerobic incubator (36 61C) for 18-24 h to prereduce the medium; during this periodthe pH of the medium should
37、rise to 8.2 6 0.3.7.1.2.6 The liquid culture medium must be prepared the daybefore its use so that it is already prereduced on the day it is tobe inoculated.7.1.3 Agar Medium for Spore Recovery (BHIYT-L):7.1.3.1 Add the following to 1 L of deionized water:BHI 37.0 gYeast extract powder 5.0 gL-Cystei
38、ne 1.0 gSodium taurocholate 1.0 gBacteriological agar 15 g7.1.3.2 Boil the medium for 1 min to dissolve the ingredi-ents and then autoclave it for 20 min at 121C; let the mediumcool down to 60 6 2C.7.1.3.3 Dissolve 200 000 units of lysozyme in 10 mL ofdeionized water; put the enzyme solution in a sy
39、ringe-drivenmembrane filter and add it directly to the medium.7.1.3.4 Pour the medium immediately into culture plates.Such plates can be stored refrigerated for no longer than sixmonths. No prereduction of the medium in the plates isnecessary when used for spore recovery.NOTE 2As shown in a three-la
40、boratory collaborative (7), certain typesof commercially available horse-blood containing recovery media may beused instead.7.2 Preparation of Reagents:7.2.1 1M Sodium Phosphate Buffer (pH 7.0):7.2.1.1 Dissolve in 800 mL of deionized water: 8.1934 g ofanhydrous Na2HPO4and 5.8374 g of NaH2PO4H2Oina2-
41、Lflask.7.2.1.2 Adjust pH to 7.0 6 0.2 with 1 M NaOH or 1 M HCl.7.2.1.3 Add more deionized water to give a total volume of1L.7.2.1.4 Autoclave for 20 min at 121C.7.2.2 Enzyme Mixture:7.2.2.1 First determine the wet weight of the spore pellet(see 8.4). Then add 800 units of lysozyme and 250 units oftr
42、ypsin per mg of pellet wet weight to 25 mL of 0.1 Mphosphate buffer (pH 7.0).7.2.2.2 Proceed as described in 8.4.8. Procedure8.1 Inoculum Preparation:8.1.1 Take a loopful from the spore stock and streak it ontoa BHIYT-L plate.8.1.2 Incubate the plate anaerobically at 36 6 1C for 48 64h.8.1.3 At the
43、same time, keep in the anaerobic chamber forpre-reduction (a) one 15-mL plastic tube containing 5 mL ofCB to prepare the pre-inoculum and (b) the same number of50-mL conical plastic tubes, each with 20 mL of CB, as thenumber of culture flasks to be inoculated.8.1.4 Pick an isolated colony from the i
44、noculated BHIYT-Lplate and suspend it in the tube containing 5 mL of prereducedCB; incubate the tube anaerobically at 36 6 1C for 24 to 36h.8.1.5 Inoculate 50 L of the 24 to 36-h culture from 8.1.3into each 50-mL conical tube containing 20 mL of prereducedCB; incubate anaerobically at 36 6 1C for 18
45、 6 2h.8.1.6 Prepare the required volume of liquid sporulationmedium as described in 7.1.2.1.8.2 Culture Flask Inoculation and Incubation Time:8.2.1 Inside the anaerobic chamber, pour the entire inocu-lum from a 20-mL tube into a 500-mL culture flask with theliquid sporulation medium.8.2.2 Incubate t
46、he flask(s) anaerobically in a stationary statefor 5 days at 36 6 1C.8.3 Harvesting:E2895 1338.3.1 Divide the sporulated culture from each flask equallyinto two 250-mL centrifuge tubes and centrifuge the suspen-sions at 4 000xg for 10 min.8.3.2 Discard the supernatant as biohazardous waste.8.3.3 Res
47、uspend the sediment from each 250-mL tube into50 mL of deionized water and pool the resuspended materialinto one 250-mL centrifuge tube.Adjust the volume to 250 mLwith deionized water and centrifuge for 10 min at 4000xg.Wash the pellet by centrifugation two more times with 250 mLof deionized water i
48、n each wash.8.3.4 After the final wash, resuspend the pellet in 15 mL ofPBS-T and transfer the suspension into a preweighed 40-mLcentrifuge tube. Rinse the original centrifuge tube once with 10mL PBS-T and add the wash to the spore suspension to makethe final volume 35 mL.8.3.5 Centrifuge the suspen
49、sion at 4 000xg for 10 min, andwash the pellet twice with 35 mL of PBS-T. Discard allsupernatants as biohazardous waste.8.3.6 Weigh the tube with the pellet and refrigerate (4 61C), for no longer than 48 h, until the next step.8.3.7 Use the wet weight of the pellet to determine thequantities of the enzymes needed in the solution for semipuri-fication of the spore suspension.8.4 Semipurification of the Spore Suspension by EnzymeTreatment:8.4.1 Resuspend the pellet (1-700 mg of wet weight) in 10mL of 0.1 M sodium phosphate
