1、Designation: E3092 18Standard Practice forEvaluating Efficacy of Vaporous Decontaminants onMaterials Contaminated with Bacillus Spores and ContainedWithin 0.2m Filter-Capped Tubes1This standard is issued under the fixed designation E3092; the number immediately following the designation indicates th
2、e year oforiginal adoption or, in 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.1. Scope1.1 This practice is used to quantify the efficacy of
3、vaporousdecontaminants on Bacillus spores dried on the surface ofcoupons made from porous and non-porous materials andcontained within 0.2m filter-capped tubes.1.2 This practice should be performed only by those trainedin microbiological techniques, are familiar with antimicrobial(sporicidal) agents
4、 and with the end use of such products.1.3 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.4 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the
5、user of this standard to establish appro-priate safety, health, and environmental practices and deter-mine the applicability of regulatory limitations prior to use.1.5 This international standard was developed in accor-dance with internationally recognized principles on standard-ization established
6、in the Decision on Principles for theDevelopment of International Standards, Guides and Recom-mendations issued by the World Trade Organization TechnicalBarriers to Trade (TBT) Committee.2. Referenced Documents2.1 ASTM Standards:2E1054 Test Methods for Evaluation of Inactivators of Anti-microbial Ag
7、entsE2111 Quantitative Carrier Test 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 ChemicalsE2
8、414 Test Method for Quantitative Sporicidal Three-StepMethod (TSM) to Determine Sporicidal Efficacy ofLiquids, Liquid Sprays, and Vapor or Gases on Contami-nated Carrier Surface (Withdrawn 2014)3E2756 Terminology Relating to Antimicrobial and AntiviralAgents3. Terminology3.1 For definitions of terms
9、 used in this guide, see Termi-nology E2756.3.2 For inactivators and neutralizers of decontaminants seeTest Methods E1054.3.3 Definitions of Terms Specific to This Standard:3.3.1 decontaminant, na physical or chemical agent orprocess that destroys pathogenic or potentially pathogenicmicroorganisms i
10、n/on surfaces or objects.3.3.2 endospore, na dormant, robust and non-metabolically active structure produced by certain bacteriafrom the Firmicutes phylum.3.3.3 exosporium, nthe outermost layer of spores of Ba-cillus anthracis and its close relatives Bacillus thuringiensisand Bacillus cereus.3.3.4 m
11、acrobacillus, na Bacillus endospore that possessan exosporium.3.3.5 microbacillus, na Bacillus endospore that does notpossess an exosporium.3.3.6 vapor, na substance in the gas phase at a temperaturelower than its critical temperature, such that it can be con-densed back into a liquid by increasing
12、the pressure on itwithout reducing the temperature.3.3.7 vaporous decontaminant, nfor the purpose of thispractice, a vaporous decontaminant can be interpreted toinclude gases, vapors, fogs, mists and thermal decontaminants.1This practice is under the jurisdiction of ASTM Committee E35 on Pesticides,
13、Antimicrobials, and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved March 1, 2018. Published May 2018. DOI: 10.1520/E3092182For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer
14、Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3The last approved version of this historical standard is referenced onwww.astm.org.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West
15、Conshohocken, PA 19428-2959. United StatesThis international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for theDevelopment of International Standards, Guides and Recommendations issued by the World Trad
16、e Organization Technical Barriers to Trade (TBT) Committee.14. Summary of Practice4.1 This practice quantitatively evaluates the efficacy ofvaporous decontaminants on coupons contaminated with Ba-cillus spores (pathogenic and non-pathogenic strains). Sporesare dried on coupon surfaces, and the coupo
17、ns are thentransferred to 0.2 m filter-capped 50-ml conical tubes (1-3).44.2 Coupon material is selected according to the claims orintended use of the decontaminant. Coupons may be made ofany material hard, flexible, porous, non-porous, metallic, ornon-metallic. Flat (2 2 cm) coupons are preferred;
18、howevernon-flat coupons and smaller coupons have been tested usingthis practice.4.3 Fifty-ml conical tubes are used for extraction vessels.This allows for greater flexibility in coupon material selection,accommodating materials that are difficult to manufacture inextremely small sizes, for example,
19、concrete, asphalt, carpetand wallboard.4.4 Contaminated test coupons are subjected to decontami-nation procedures. Control coupons are subjected to identicalprocedures without the decontaminant.4.5 Solution controls (spores suspended in aqueous solu-tion) will represent the 100 % recovery reference
20、for calculat-ing spore survival after decontamination treatment and analy-sis.4.6 Spore extraction percentage will be calculated by divid-ing the number of spores recovered from each spore-inoculatedcontrol coupon by the number of spores recovered from thesolution controls.4.7 The number of survivin
21、g spores from decontaminationtests will be divided by the extraction percentage to determinethe number of surviving spores in CFU ml-1. This sporeconcentration is then multiplied by 10 ml to give a total numberof spores surviving (CFU) from each test sample. A log10transformation of the total surviv
22、ing spores will then beperformed (log10(total CFU + 1).5. Significance and Use5.1 The practice can be used to evaluate coupon materials ofany composition, insofar as the coupon can be prepared smallenough to fit inside a 50-ml conical tube.5.2 This practice defines procedures that are quantitative,s
23、calable, rapid, sensitive, safe, reduces consumables, mini-mizes labor and addresses statistical confidence (1, 2, 4).5.2.1 QuantitativeThe total number of spores per couponis determined by dilution-plating, and all spores remaining onthe coupon are assayed for activity in the extraction tube toprov
24、ide confidence that 100 % of spores were assayed.5.2.2 Statistical ConfidenceThe use of five independentpreparations of spore inoculum for a statistical N of 5.5.2.3 SensitivityAllows for complete detection of all vi-able spores inoculated on a coupon, including the spores thatremain attached to the
25、 coupon.5.2.4 SafetyInoculated coupons are contained within0.2 m filter-capped 50-ml conical tubes. The 0.2 m filterallows vaporous decontaminants to pass through while pre-venting escape of spores, thereby providing an important levelof containment when working with pathogenic strains.5.2.5 Simplic
26、ity of TestingTests and extractions are per-formed in the same 50-ml conical tube to minimize handlingsteps.5.2.6 Scalable and RapidA maximum of 36 samples canbe processed in1hbytwotechnicians; a total of 300 sampleshave been processed by six technicians in 5 h (1-3).5.2.7 Wide application for numer
27、ous Bacillus species andstrains.NOTE 1This practice differs from similar quantitative methods(E2111, E2197 and E2414) in the size and variety of coupon materialsavailable for testing, in the practical confidence of the statistics, theapplication of the decontaminant, scalability and sensitivity.6. A
28、pparatus6.1 Autoclave.6.2 Shaking Incubator.6.3 Incubator.6.4 Phase-Contrast Microscope.6.5 Centrifuge.6.6 Water Bath.6.7 Single-tube Vortex Mixer.6.8 Multi-tube Vortex Mixer.6.9 Analytical Balance.6.10 -80 C Freezer.6.11 Stopwatch or Electronic Timer.6.12 Manual or Electronic Pipettes.6.13 Bio-Safe
29、ty Cabinet (BSC).6.14 Environmental Chamber, capable of maintaining tem-perature 62 C and relative humidity 65% of target param-eters; must be capable of maintaining vapor concentration.6.15 Appropriate PPE, for example, gloves, safety glasses,lab coats, etc. (5).7. Reagents and Materials7.1 Reagent
30、s5:7.1.1 Bacillus anthracisAmes, Sterne, Sterne.7.1.2 Bacillus thuringiensisAl Hakam, cry-HD-1.7.1.3 Tryptic Soy Broth (TSB).7.1.4 Tryptic Soy Agar (TSA).7.1.5 Nutrient Broth (NB).7.1.6 Tween 80.7.1.7 L-Alanine.4The boldface numbers in parentheses refer to the list of references at the end ofthis st
31、andard.5Reagent Chemicals, American Chemical Society Specifications, AmericanChemical Society, Washington, 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 Pha
32、rmacopeiaand National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,MD.E3092 1827.1.8 Inosine.7.1.9 Sporulation Broth0.8 % (wv) Nutrient broth or2.5 % (w/v) Nutrient broth and salts, as defined in Table 1,pH7 (see Annex A1 for preparation instructions) (1, 6, 7).7.1.10 Extraction
33、BufferpH 7, as defined in Table 2 (2, 3).7.1.11 pH-adjusted Bleach0.6% (v/v) hypochlorite, 0.2%(v/v) acetic acid, pH 7.7.1.12 90 % (v/v) Ethanol.7.2 Materials:7.2.1 Sterile 50-ml conical tube.7.2.2 Sterile 50-ml conical tubes with 0.2m filter cap, forexample, TPP TP87050; Techno Plastic Products6.7.
34、2.3 Baffled flasks.7.2.4 Sterile Petri dishes.7.2.5 50-ml conical tube and microfuge tube racks.7.2.6 Pipette tips.7.2.7 Parafilm.77.2.8 L-shaped sterile spreaders.7.2.9 1.5-ml sterile microcentrifuge tubes.7.2.10 Coupon materialsAll coupon materials must be astandardized surface area, preferably fl
35、at,22cm;however,it is understood that not all materials are easily adaptable tothese size constraints.7.2.11 Sterile forceps.8. Hazards8.1 It is the responsibility of the individual user(s) of thispractice to follow all safety guidelines and to be knowledge-able about these procedures. Individual us
36、ers should consulttheir safety authority and establish detailed safety plans andrisk assessments prior to using this practice. Users are stronglyurged to consult the Biosafety in Microbiological and Biomedi-cal Laboratories (5).9. Test Organisms9.1 Specific organisms are recommended, but the choice
37、oforganism(s) should be relevant to the environment in which thedecontaminant is expected to perform.9.2 Pathogenic and non-pathogenic stains of Bacillus an-thracis Ames, Sterne, Sterne.9.3 Acrystalliferous strains of Bacillus thuringiensis AlHakam, cry-HD-1.9.4 Other macrobacillus and microbacillus
38、 strains, vegeta-tive bacteria, bacteriophage and viruses may also be testedusing this practice.10. Preparation of Inoculum10.1 Prepare five independent spore inocula from five inde-pendent spore preparations.10.2 Transfer concentrated spores from -80 C directly to a50 C water bath for at least 30 m
39、in. This temperature ismaintained during spore inoculation to mitigate the risk ofspore clumping prior to and during coupon inoculation.10.3 Vortex concentrated spores for 15-30 s.10.4 Transfer concentrated spores into pre-labeled 50-mlconical tubes containing preheated (50 C) sterile 0.1 % (v/v)Twe
40、en 80. Spores from each independent spore preparation areused to prepare its corresponding independent spore inoculum.The volume of 0.1 % Tween 80 is set to achieve a targetconcentration of 1-2 108spores ml-1. Pipette tips should berinsed by pipetting up and down twice in the 50-ml conicaltube in or
41、der to rinse spores from the plastic tips.10.5 Hold the diluted spore inoculum at 50 C until couponinoculation, which should occur within 24 h of preparing theinocula.10.6 In order to titer the spore inoculum, transfer 0.1 ml ofspore inoculum into 0.9 ml of 0.1 % Tween 80, serially diluteand plate o
42、nTSAplates. Invert plates and incubate at 35 62Cfor 16 62 h. Count and record data. The time and temperatureof plate incubation can be adjusted for strains, for example, B.thuringiensis HD-1 strains produce large colonies and thisstrain is incubated at 30 62 C for 16 62 h in order to ensurecountable
43、 plates.10.7 Optional: Spores may be mixed with inorganic debrisprior to coupon inoculation. Kaolin (Al2Si2O5(OH)4) has beenselected as a potential inorganic debris in published tests (2).Kaolin was suspended in 0.1 % Tween 80 at 100 g l-1kaolinand autoclave-sterilized for 30 min on a wet cycle. Spo
44、res weresuspended in kaolin at a final concentration of 1-2108sporesml-1, 0.1 % Tween 80, 50 g l-1kaolin.At a test concentration of1-2108spores ml-1, kaolin was 250-500 excess over sporesby weight.6The sole source of manufacturing of the apparatus known to the committee atthis time is Techno Plastic
45、 Products, Trasadingen, Switzerland. There are multiplesources for purchasing. If you are aware of alternative manufactures, please providethis information to ASTM International Headquarters. Your comments will receivecareful consideration at a meeting of the responsible technical committee,1whichyo
46、u may attend.7Trademarked by Bemis Corporate 2301 Industrial Drive Neenah, WI 54956.TABLE 1 Sporulation Broth (pH 7)Reagent AmountNutrient Broth 2.5 % (2.5 g l-1)or0.8%(0.5gl-1)KH2PO42.15 gK2HPO44.35 gCaCl22H2O015MnCl22H2O 0.016 gZnCl20.068 gFeCl36H2O 0.0003 gSterile, 18-megaohm water Add water to 1
47、000 ml final volumeTABLE 2 Extraction Buffer (pH 7)AReagent Defined Amount1 Extraction BufferTSB 10 gL-Alanine 100 mMInosine 1 mMTween 80 1 mlSterile, 18-megaohm water Add water to 1000 ml final volume2 Extraction BufferTSB 20 gL-Alanine 200 mMInosine 2 mMSterile, 18-megaohm water Add water to 1000
48、ml final volumeACan include a chemical neutralizer if necessary to neutralize any sporicidalactivity of a chemical vapor.E3092 18310.8 Optional: Spores may be mixed with organic debrisprior to coupon inoculation. Humic acid suspended in spentsporulation medium (SSM) has been selected as a potentialo
49、rganic debris in published tests (2). The SSM collected afterspore harvest (see Refs (1, 6, 7) and Annex A1) was 0.2mfilter-sterilized and then stored at -75 65 C. Humic acid wassuspended in SSM at 10 g l-1humic acid and autoclave-sterilized for 30 min on a wet cycle. Spores were thensuspended in the humic acid + SSM at a final concentration of1-2108spores ml-1, 0.05 % (v/v) Tween 80,5gl-1humicacid, 0.5 SSM. At a test concentration of 1-2 108sporesml-1, the humic acid was 25-50 excess over spores by weight.11. Preparati
