1、Designation: E2894 13Standard Test Method forApplying Aerosolized Bacillus Spores as Dry Inocula toInanimate Surfaces1This standard is issued under the fixed designation E2894; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the ye
2、ar 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.INTRODUCTIONBacillus anthracis, the causative agent of anthrax, poses a formidable threat as an infectiousbioagent.Acombinat
3、ion of significant environmental stability, ease of production,2and high mortalitythrough the inhalational route3makes B. anthracis an ideal bioagent. Aerosolized powders areexpected to be the primary mechanism for disseminating B. anthracis spores, likely delivered as anaerosol cloud from either a
4、line-source, for example, low-flying aircraft, or point-source, for example,spray device. The characteristics of the aerosol (particle size and composition) will influence itsbehavior, thereby affecting its potency as a threat agent. This test method is designed to performsurface deposition of aeros
5、olized spores with the primary objective of evaluating the utility ofself-sanitizing antimicrobial materials (SSAMs). However, it could be used for other types of research,for example, decontamination, reaerosolization, fate and transport, contamination avoidance, collec-tive protection, and individ
6、ual protection, as well. We are aware of other consensus standards forapplying spores to surfaces (Test Method E2149, AATCC Test Method 100, Test Method E2180), butthese methods are based on liquid inocula. SSAMs are more active in the presence of moisture,4andthus standard test methods using water-
7、based inocula may overestimate their effectiveness. Protocolsfor the preparation, aerosolization, extraction, viable enumeration, and calculation of spores for testingand determining the effectiveness of SSAMs are described.1. Scope1.1 This test method is designed to uniformly apply fluid-ized spore
8、s to surfaces as an aerosol under defined conditions.1.2 This test method is specific to B. anthracis Delta()Sterne (BAS), but could be adapted for work with othertypes of Bacillus species.1.3 This test method is suitable for working with any type ofenvironmental surface.1.4 This test method should
9、be performed only by thosetrained in aerobiology, microbiology, or a combination thereof.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, associ
10、ated 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. Referenced Documents2.1 ASTM Standards:5E1054 Test Methods for Evaluation of Inactivators of Anti-m
11、icrobial AgentsE2149 Test Method for Determining the Antimicrobial Ac-tivity of Antimicrobial Agents Under Dynamic ContactConditions1This test method is under the jurisdiction of ASTM Committee E35 onPesticides, Antimicrobials, and Alternative Control Agents and is the directresponsibility of Subcom
12、mittee E35.15 on Antimicrobial Agents.Current edition approved Oct. 1, 2013. Published March 2014. Originallyapproved in 2012. Last previous edition approved in 2012 as E289412. DOI:10.1520/E2894-13.2Woods, J. B., Darling, R. G., Dembek, Z. F., Carr, B. K., Cieslak, T. J., andLawler, J. V., et al.,U
13、SAMRIIDs Medical Management of Biological CasualtiesHandbook, 6th edition, U.S. Army Medical Research Institute of InfectiousDiseases, Fort Detrick, Frederick, MD, 2005.3LeClaire, R. D., and Pitt, M. L. M., “Biological Weapons Defense: EffectLevels,” Biological Weapons Defense: Infectious Diseases a
14、nd Counterterrorism,Humana Press Inc., Totowa, NJ, 2005, pp.4161.4Prugh, A., and Calomiris, J. J., “Inactivation of Bacillus anthracis SporesDelivered as Liquid Suspension or Aerosol to Self-Decontaminating Fabric,”Defense Technical Information Center (DTIC), AFRL-HE-WP-TP-2006-0060,2006.5For refere
15、nced 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.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West C
16、onshohocken, PA 19428-2959. United States1E2180 Test Method for Determining the Activity of Incor-porated Antimicrobial Agent(s) In Polymeric or Hydro-phobic MaterialsE2756 Terminology Relating to Antimicrobial and AntiviralAgents2.2 AATCC Standards:6AATCC Test Method 100 Antimicrobial Finishes on T
17、extileMaterials: Assessment of3. Terminology3.1 DefinitionsFor definitions of general terms used in thistest method, refer to Terminology E2756.3.2 Definitions of Terms Specific to This Standard:3.2.1 aerosol, na suspension of solid or liquid particles ina gaseous medium.3.2.2 anthrax, nan infectiou
18、s disease of warm-bloodedanimals caused by the gram-positive, endospore-formingbacterium, Bacillus anthracis.3.2.3 biological aerosol, na suspension of particles con-taining biological agents which have been dispersed in agaseous medium.3.2.4 carrier, nsubstrate onto which aerosolized sporesare depo
19、sited.3.2.5 count median diameter (CMD), nthe calculateddiameter in a population of particles in a gas or liquid phaseabove which there are as many particles with larger diametersas there are particles below it with smaller diameters.3.2.6 fluidizing agent, nan additive used to reduce ag-glomeration
20、 of particles.3.2.7 line source, na source of air, noise, watercontamination, or electromagnetic radiation that emanatesfrom a linear geometry.3.2.8 lyophilize, vto freeze-dry.3.2.9 point source, na source of air, noise, watercontamination, or electromagnetic radiation that emanatesfrom a single poi
21、nt.3.2.10 self-sanitizing antimicrobial material (SSAM), namaterial containing an antimicrobial component that collec-tively acts as a microbicide.4. Summary of Test Method4.1 The test method describes the steps required to depositspores onto surfaces and quantitatively assess loading distri-bution
22、and recovery.4.2 The test method describes the protocols for evaluatingthe antimicrobial activity of SSAMs.4.2.1 Using an aerosol device capable of meeting the dataquality objectives set forth in this test method, BAS sporesare aerosolized and allowed to deposit onto surfaces.4.2.2 Spores are recove
23、red from the test and control carriers,and viability is assessed by dilution plating (CFU/mL).4.2.3 Loading efficiency is determined as colony-formingunits (CFU) per cm2obtained from the viability assay.4.2.4 Loading variability is determined by calculating thecoefficient of variation (CV) of the me
24、an loading density fromreplicate samples.4.2.5 Antimicrobial efficiency is calculated as the log10reduction of viable spores between the test and control groups,and its significance is determined by a statistical comparison ofthe two populations.5. Significance and Use5.1 This test method provides d
25、efined procedures for creat-ing fluidized spore aerosols with particular emphasis onparticle size distribution and spore preparation.5.2 The efficacy of disinfection technologies can be evalu-ated on finished items, as well as those under development.5.3 This test method defines procedures for the v
26、alidation ofthe aerosol generator, preparation of the test specimen, appli-cation of the aerosolized spores, enumeration of viable spores,assessing data quality, and determining effectiveness ofSSAMs.5.4 Safety concerns associated with aerosolizing microbialagents are not addressed as part of this t
27、est method. Individualusers should consult their safety authority, and a detailedbiological aerosol safety plan and risk assessment must beestablished prior to using this method. Users are strongly urgedto consult Biosafety in Microbiological and Biomedical Labo-ratories.76. Apparatus6.1 Biological
28、aerosol generatorused to load microorgan-isms onto a substrate (see Annex A1 for detailed information).6.2 Autoclavecapable of maintaining 121 to 123C and 15to 17 psig.6.3 Shaking Incubatorcapable of maintaining 36 6 1Cand 300 r/min.6.4 Incubatorcapable of maintaining 36 6 1C.6.5 Phase Contrast Micr
29、oscopecapable of 400 and1000 magnification.6.6 Centrifugecapable of maintaining 3000 g.6.7 Water Bathcapable of achieving 65C.6.8 Single-tube Vortex Mixer.6.9 Multi-tube Vortex Mixer.6.10 Sonicating Water Bath50 to 60 Hz.6.11 Analytical Balancecapable of weighing 0.001 g.6.12 Refrigeratorcapable of
30、maintaining 2 to 8C.6.13 Stopwatch or Electronic Timer.6.14 Pipettorwith a precision of 0.1 mL.6Available from American Association of Textile Chemists and Colorists(AATCC), PO Box 12215, Research Triangle Park, NC 27709-2215. http:/www.aatcc.org.7CDC-NIH, Biosafety in Microbiological and Biomedical
31、 Laboratories, 5thEdition, U.S. Department of Health and Human Services, Washington, D.C.,December 2009.E2894 1327. Reagents and Materials7.1 Reagents:7.1.1 Bacillus anthracisSterne strain spores, preparedaccording to procedures outlined in Annex A2.7.1.2 Trypticase Soy Agar (TSA).7.1.3 Trypticase S
32、oy Broth (TSB).7.1.4 Sporulation Broth2.5 % nutrient broth, 200 mMglutamate, and salts, as defined in Table 1, pH 7 (see AnnexA2.2.2 for preparation instructions).87.1.5 Extraction BufferpH 7, as defined in Table 2.7.1.6 Hydrophobic Fumed SilicaAerosil (Trademarked) R812 S97.1.7 Nutrient Agar.7.1.8
33、Phosphate-Buffered Saline (PBS).7.1.9 Glycine.7.1.10 Polysorbate 20.7.1.11 Hydrochloric Acid (HCl)2.5N.7.1.12 Sodium Hydroxide (NaOH).7.1.13 Deionized (DI) Water.7.1.14 Ethanollaboratory grade.7.1.15 Sodium Hypochlorite10 %.7.1.16 Nitrogen Gas (N2)dry.7.2 Materials:7.2.1 Sterile Inoculating Loops.7.
34、2.2 Baffled Flasks1000 mL.7.2.3 Erlenmeyer Flasks250 mL.7.2.4 Glass Microscope Slides.7.2.5 Petri Dishes.7.2.6 Test Tube Racks.7.2.7 Pipettes1, 5, 10, and 25 mL.7.2.8 Micropipettescapable of delivering 0.1 and 1 mLaccurately and consistently.7.2.9 Sterile Centrifuge Tubes15 and 50 mL.7.2.10 Sterile
35、Round-bottom Tubes15 mL.7.2.11 Borosilicate Solid Glass Beads3-mm diameter,sterile.7.2.12 Parafilm.7.2.13 L-shaped Sterile Spreaders.7.2.14 Test Specimens.8. Sampling, Test Specimens, and Test Units8.1 Cut test specimens from finished products or thoseunder development.NOTE 1The size dimensions of t
36、he test specimens can vary, but willneed to fit into a 50-mL centrifuge tube for extraction.8.2 The coupon size used to validate the method was a2.6-cm diameter circle and is recommended for this method.8.3 For SSAMs, place the specimens in the aerosol chamberwith the reactive side facing upward.8.4
37、 For non-reactive materials (if used), the sample sidefacing upward needs to be consistent throughout the samplegroup.9. Experimental Design9.1 Non-reactive MaterialsA minimum of three indepen-dent samples per specimen is required for statistical analysis ofthe data.9.2 SSAMsGenerally, most SSAMs re
38、quire incubationunder specific environmental conditions (that is, temperature,relative humidity, time) for their antimicrobial activity to beeffective. Following aerosol deposition, the samples will beincubated under the specific conditions as specified for aparticular SSAM. This period is referred
39、to as the incubationperiod. Aerosol deposition requires a significant period of timefor the spores to settle onto surfaces and is exclusive from theincubation period. For this purpose, two sample sets will beincluded in the test plana conditioned (test) group and anunconditioned (control) group. A t
40、raditional control that doesnot include the antimicrobial treatment is not needed for thismethod. The unconditioned control samples will be incubatedfor the same duration of time as the conditioned test samples,but will be held at room temperature and 80 % phase-bright spores.A2.2.3 Spore Harvesting
41、 and PurificationDivide thespore solution into (2) 50-mL centrifuge tubes and centrifugefor 10 min at 3000 g at room temperature. Discard thesupernatant, resuspend the spore pellet in 20 mL refrigerated(4C) sterile DI water, and centrifuge for 10 min at 3000 g atroom temperature. Discard, resuspend,
42、 and centrifuge in simi-lar fashion twice more.After the third centrifuge cycle, discardthe supernatant, and resuspend the pellet in 10 mL of sterile DIwater. Using a water bath, heat-treat the spores for 30 min at65C. Centrifuge the solution again for 10 min at 3000 g atroom temperature. Discard th
43、e supernatant and resuspend thespore pellet in 20 mL 4C sterile DI water. Repeatcentrifugation, discard, and resuspension steps until the sus-pension contains 85 % phase-bright spores when viewedunder the phase-contrast microscope. Suspend the final sporeE2894 135pellet in 10 mL sterile 0.05 % Polys
44、orbate 20 solution. Soni-cate the spore suspension for 1 min in a sonicating water bath.Vortex the sample for 30 s. Prepare microscope slides of thespore suspension and allow spores to settle so they will be inthe same visual field. Take photos of the four quadrants andperform a manual count under p
45、hase-contrast microscopy todetermine the ratio of phase-bright spores to phase-dark spores,vegetative cells, and debris.A2.2.4 Lyophilization of the SporesCentrifuge the sporestock suspensions at 3000 g for 10 min. Discard thesupernatant and resuspend the spore pellet in an equal volumeof sterile DI
46、 water as the discarded supernatant. Lyophilize thespores according to the specifications provided by the manu-facturer of the lyophilizing device. Aliquot the spores into15-mL conical tubes and store at 80C. Perform a viabilityassay on the lyophilized spore powder to determine the density(CFU/g). T
47、he spore concentration must be -1010viable sporesper gram for suitable use.A2.2.5 Final spore preparation shall be 80 % pure byphase-contrast microscopy with a concentration that exceeds1010CFU/gram of lyophilized spore powder.APPENDIX(Nonmandatory Information)X1. EXAMPLE DEVICE: THE BIOLOGICAL DISP
48、ERSING SYSTEM (BDS)X1.1 Diagram of the Biological Dispersing System (BDS)X1.1.1 See Fig. X1.1.X1.2 System ParametersX1.2.1 The BDS consists of a glass-walled chamber, afluidized bed aerosol generator,10an aerodynamic particle sizer(APS), and a computer (not shown) for data acquisition andcontrol. Th
49、e chamber consists of a 0.6-m2base by 1.2-m highenclosure with four primarily glass walls. Two doors areincluded for easy access. The left door opens the entire side,while the right door provides access to the lower portion of thechamber. A turntable for holding test samples is provided onthe interior floor. The turntable has a variable speed control,providing rotation speeds up to 7 r/min. Rotation of theturntable is not used during spore loading, but is required forsample placement and retrieval. The top an
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