1、Designation: E2966 14Standard Test Method forQuantitative Assessment of Sanitizing Solutions for Carpet1This standard is issued under the fixed designation E2966; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last rev
2、ision. 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 evaluate quantitativelythe antibacterial and antifungal activity of solutions for sani-tizing
3、carpets.1.2 Efficacy is reported as the log reduction in viablebacteria and fungi.1.3 The bacteria used in the test are Staphylococcus aureus,Enterobacter aerogenes, and Pseudomonas aeruginosa. Themold used is Aspergillus brasiliensis.1.4 Knowledge of microbiological techniques is requiredfor this t
4、est method.1.5 UnitsThe values stated in SI units are to be regardedas standard. 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 with its use. It is theresponsibility of the user of this standard t
5、o establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2E1054 Test Methods for Evaluation of Inactivators of Anti-microbial AgentsE2471 Test Method for Using Seeded-Agar for the Screen-ing
6、 Assessment of Antimicrobial Activity In Carpets3. Terminology3.1 Definitions:3.1.1 carpet cleaner/sanitizer, nsolution that cleans em-bedded soil from the carpet fiber and reduces biocontaminantlevels on carpet when applied at the recommended dilution andcontact time specified on the product label.
7、3.1.2 carpet sanitizer, nchemical solution that reducesbiocontaminant levels on carpet when applied at the recom-mended dilution and contact time specified on the productlabel.3.1.3 sanitizer, nchemical or physical agent(s) used toreduce the number of microorganisms to a level judged to beappropriat
8、e for a defined purpose and/or claim.3.1.3.1 DiscussionThe US EPA regulates sanitizers usedon porous and non-porous surfaces. EPA 810 Guidelinesprovide a description of each category, the required testmethod, test conditions, and performance criteria. EPA810.2400 (f) describes requirements for testi
9、ng carpet sanitiz-ers.33.1.3.2 DiscussionIn the context of this method effectivesanitization reduces the number of microorganisms to levelsconsidered safe as determined by public health codes orregulations.3.1.4 vacuum extraction unit, nmachine for deep cleaningcarpet that delivers a spray of cleani
10、ng solution, provides brushagitation of the pile, and recovers soil and cleaning solutionunder vacuum.4. Summary of Test Method4.1 In this test method, the efficacy of solutions intended tohave a sanitizing effect on carpet are quantitatively evaluated.Carpet sample coupons are cut from a larger fie
11、ld of carpet andre-embedded within the field. The carpet coupons are inocu-lated with microorganisms followed by a drying period. Afterthe inoculum-drying period, the sanitizing solution is appliedto the carpet coupons followed by scrubbing.Additional carpetcoupons are spray treated and scrubbed wit
12、h a inert solution.After the chemical contact period, carpet coupons are asepti-cally removed and placed into neutralizing broth. Each neu-tralizing broth with carpet coupon is placed into a ultrasonicbath for 1 min followed by 1 min of wrist action shaking. Serialdilutions are performed on each sam
13、ple followed by plating(pour or spread plates or other standard for enumerating viablecells). Log reduction of the viable cell counts recovered from“scrubbed-controls” versus viable cell counts recovered fromthe sanitizer-treated carpets are recorded.1This test method is under the jurisdiction of AS
14、TM Committee E35 onPesticides, Antimicrobials, and Alternative Control Agents and is the directresponsibility of Subcommittee E35.15 on Antimicrobial Agents.Current edition approved May 15, 2014. Published June 2014. DOI: 10.1520/E2966142For referenced ASTM standards, visit the ASTM website, www.ast
15、m.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.3http:/www.epa.gov/ocspp/pubs/frs/publications/Test_Guidelines/series810.htmCopyright ASTM International, 100 Barr Harbor Dr
16、ive, PO Box C700, West Conshohocken, PA 19428-2959. United States15. Significance and Use5.1 Carpet, when exposed to the environment or foot traffic,accumulates soil and biocontaminants during its in-service life.While routine vacuuming may effectively remove dry particu-late soils, it has a limited
17、 effect on removing or killingaccumulated and embedded biocontaminants. In this testmethod, steps are described to assess test substances for theability to sanitize carpet.5.2 This test method compares an inert control solution to asanitizing test solution for the ability to reduce viable bacteriaan
18、d fungi inoculated onto carpet samples.5.3 This test method provides for efficient recovery ofsurviving bacteria from inoculated carpets.6. Apparatus6.1 For broadloom-type carpets (typically a 3.7-m rollcarpet),a2cmthick plywood cut (40 by 40 cm) with the samedimension tempered hardboard attached is
19、 used to mount thecarpet. Brass brads are used to secure the carpet sample to thetempered hardboard.6.2 For carpet tile with a dimensionally stable backing, nomounting board is required.6.3 Cutting the carpet into samples can be accomplishedwith a traditional carpet knife or the use of mechanical cu
20、ttingdies and a hydraulic press. One mechanical cutting die, 20 cmby 30 cm, and another mechanical cutting die, 5 cm by 5 cm,are used.6.4 One-sided adhesive tabs are used to temporarily securethe precut 5-cm by 5-cm carpet carriers “in plane” with theremaining carpet during the scrubbing procedure.
21、Alternately,the 90 corners of the 20 cm by 30 cm section may be nailed ortacked to the mounting board.6.5 Spray DeviceAspray unit is used to atomize the carpetsanitizer.NOTE 1An atomizer may also be used. Aerosol formulated sprayproducts may be directly sprayed onto the carpet.6.6 Scrub Brushes, sur
22、gical hand brush.6.7 Extraction Bottles, wide-mouth round 500-mL polypro-pylene bottles with screw caps.NOTE 2For the procedure, each bottle will contain 100 mL of sterileneutralizer broth.6.8 Ultrasonic Bath.6.9 Wrist-action-shaker.7. Reagents and Media7.1 Sanitizer Solutions:7.1.1 Test a single lo
23、t of the candidate carpet sanitizer.Consult the appropriate regulatory 101 guidelines for lotreplication requirements for registration purposes (forexample, US EPA 810 102 Guidelines).7.1.2 If the product is to be used as a “One-step cleaner-sanitizer,” a 5% soil load (for example, animal sera) may
24、beadded to the inoculum.7.1.3 Recommended application rates (volume per unit area,that is, ml/m2) are extrapolated and reported from the proposedcarpet application rate to the sample size used for this testmethod.7.2 Carpet Specifications:7.2.1 Two carpet types should be tested. For example, carpetw
25、ith nylon face fiber or polypropylene face fiber may be usedfor this test. If the candidate product is to be used on woolcarpet, a wool carpet sample shall be included in the test.7.2.2 The test report should indicate: face fiber composition,weight of the pile fiber (kg/m), pile density, and pile he
26、ight.7.3 Media:7.3.1 Phosphate-buffered saline.7.3.2 Nutrient agar.7.3.3 Nutrient broth.7.3.4 Potato dextrose agar or Saubarouds agar (EmmonsModified).7.3.5 Appropriate neutralization media / technique must beselected that allows for immediate neutralization at the comple-tion of the contact time. F
27、or example double strength neutral-izer broth (Letheen broth + 0.7-g lecithin and 5-g polysorbate80 per litre) may be used for quaternary actives. Sodiumthioglycolate 0.1 %, and 0.01 % iso-octyl-phenoxy-polyethoxyethanol, are suggested for heavy metal and halogen-based actives. Neutralizer efficacy
28、can be confirmed using TestMethod E1054. EPA 810.2000 also outlines neutralizationrequirements.7.3.6 Neutralizing agar (Letheen agar) or Dey-Engley neu-tralizing agar.7.3.7 Sterile deionized water.8. Microorganisms8.1 Staphylococcus aureus ATCC 6538.8.2 Enterobacter aerogenes ATCC 13048.8.3 Pseudomo
29、nas aeruginosa ATCC 15442.8.4 Aspergillus brasiliensis ATCC 9642 or ATCC 16404.8.5 Maintain bacterial stocks on nutrient agar slants or asfrozen stocks. Bacterial stocks should be purchased from thesupplier every 18 months. Stock cultures should be transferredmonthly. Transfers from lyophilized stoc
30、ks are limited to sixbefore replacement is required.8.6 Maintain mold on potato dextrose agar or as suspensionsof conidia. Mold stocks should be purchased from the supplierevery 18 months. Stock cultures should be transferred monthly.Transfers from lyophilized stocks are limited to six beforereplace
31、ment is required.9. Inoculum Preparation9.1 Bacteria:9.1.1 Transfer one 4 mm loop scraping of Staphylococcus,Enterobacter and Pseudomonas bacteria from stock slants toseparate 9.0 ml tubes of sterile nutrient broth. Additional testorganisms may require different media or growth conditions.9.1.2 Grow
32、 broth cultures overnight (18-24 h) at 3762C.Incubate Enterobacter aerogenes at 25-3062C.E2966 1429.1.3 Adjust cell density to 1-5 109CFU/mL in sterilephosphate-buffered saline. This may be based onhemacytometer, McFarland standards, spectrophotometer orhistorical culture counts, or a combination th
33、ereof. Note thatconcentration by centrifugation may be required to achieve theabove titer.9.1.4 Add 5 % horse or fetal bovine serum to mimic soilload (if one-step “cleaner and sanitizer” claim is to be made)9.2 Mold:9.2.1 Grow Aspergillus brasiliensis for 7-14 d at 3062Con potato dextrose or Sabarou
34、d dextrose agar (modified) untilmature conidia are present. Multiple plates of mature Asper-gillus growth may be required along with “pooled conidiasuspensions from these plates” to achieve the specified sporedensities.9.2.2 Harvest mature conidia using phosphate-buffered sa-line and gently scraping
35、 the surface with a bent glass rod.9.2.3 Filter hyphal fragments through sterile funnels fittedwith sterile glass wool or sterile gauze.9.2.4 Standardize condia solution in phosphate bufferedsaline to 1-5 109CFU/ml using a hemocytometer.10. Procedure10.1 Carpet Preparation:10.1.1 Use a utility knife
36、 or mechanical die to cut the carpetinto 20 cm by 30 cm samples. Two of these carpet samplesshould be prepared for each test substance and carpet typetested, one carpet piece for evaluating the test substance lot andone 20 cm by 30 cm carpet piece for the scrubbed andunscrubbed control solution.10.1
37、.2 Use a utility knife or mechanical die to cut 5 cm by5 cm square carriers (two rows with three carrier squares perrow) within the 20 cm by 30 cm rectangular piece of carpet.Space the cuts leaving approximately 10 cm between thecenters of each carrier square. The test substance is evaluatedon 6 car
38、riers from a single 20 cm by 30 cm carpet piece. Froma separate 20 by 30 cm carpet section, three 5 cm by 5 cmcarriers are used as scrubbed controls and three 5 cm by 5 cmcarriers are used as un-scrubbed controls for the inert solution.10.1.3 Wrap the carpet in aluminum foil, autoclave for 15min, an
39、d then air dry. Make sure the foil seam is at the top ofthe carpet for access to the samples.10.1.4 After autoclaving, apply the adhesive tabs to thebackside of the carpet to secure the smaller square carpetcarriers within the larger rectangular carpet. Use a marking pento mark the center of each sm
40、all carpet carrier (carpet pileside).10.1.5 Only carpets with no antimicrobial activity should beused. Test Method E2471 (seeded agar overlay test) may beused to document no inherent inhibitory activity.10.2 Inoculation of Carpet:10.2.1 Place 0.1 mL of the standardized bacterial innocu-lum onto the
41、center (previously marked) of each 5 cm by 5 cmcarpet square.10.2.2 Allow inoculated carpet to dry for 1 h 6 2 min in a35-37 62C incubator with foil cover loosely in place.10.2.3 Inoculate the control solution carpet sample in thesame manner as described in 10.2.1.10.2.4 Determine “0-hr” cell densit
42、y by performing serialdilution and plating of the inoculum.10.3 Application of Sanitizer:10.3.1 Calculate the intended application rate of the carpetsanitizer per square meter and extrapolate the amount ofmaterial to be applied to a 600 cm2test piece of carpet.10.3.2 Use the spray device unit to app
43、ly the sanitizeruniformly to the inoculated carpet. Alternately, glass chroma-tography spayers may be used to apply the product in a mannerconsistent with intended application and dosing rate.10.3.3 Dip the surgical hand brush into fresh sanitizer andscrub the first inoculated square of carpet with
44、a clockwisemotion for 30 s making 30 clockwise and 30 counter-clockwisepasses with moderate pressure (slight bend of brush bristles).Repeat this process until all six inoculated samples have beenscrubbed. Report the actual total sanitizer volume applied tothe carpet by calculating the liquid holding
45、 capacity of thescrub brush. This can be accomplished by dipping and tappingthe brush (five replicates) into a weigh boat and calculatingbased on mass of the liquid.10.3.4 Allow the carpet with applied sanitizer to remain atroom temperature for 60 min.10.4 Control Carpet:10.4.1 The following steps c
46、an be performed during the60-min contact period for the sanitizer-treated carpet.10.4.2 Apply the inert control solution (sterile phosphate-buffered saline) to three of the six inoculated squares on thecontrol carpet. Use half the volume calculated for the treatedsamples. Scrub three of the six inoc
47、ulated control carriersamples as described in 10.3.3 but with brush dipped into theinert control solution. Do not scrub three of the carriersdesignated as “unscrubbed population controls”.10.4.3 Allow the scrubbed and unscrubbed control carriersto stand at room temperature for 60 61 min.10.5 Recover
48、y of Viable Cells from Carpet:10.5.1 After the 6061 min contact period, use sterileforceps or hemostats or both to lift the small carpet carriersamples from the larger carpet field.10.5.2 Transfer each carpet carrier sample to separate ex-traction bottles containing 100 ml of neutralizing broth.10.5
49、.3 Place the bottles with neutralizing solution andcarpet into a sonic bath for 1 min 6 10 s. The fluid level in thesonic bath should equal the fluid level in the recovery bottlewhen immersed.10.5.4 After sonication, pat bottles dry with paper towelsand mount them onto a wrist-action shaker. Shake the bottles atmaximum setting for 1 min 6 10 seconds. Alternately, manu-ally shake the bottles for the same time period.10.5.5 Perform serial dilutions from each neutralizing solu-tion bottle. Plate each serial dilution in duplicate (pour plates orspread plates for bac
