1、Designation: E3180 18Standard Test Method forQuantification of a Bacillus subtilis Biofilm Comprised ofVegetative Cells and Spores Grown Using the ColonyBiofilm Model1This standard is issued under the fixed designation E3180; the number immediately following the designation indicates the year oforig
2、inal 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 test method specifies the operational parametersrequired t
3、o grow and quantify a Bacillus subtilis biofilmcomprised of vegetative cells and endospores (spores) usingthe colony biofilm method (CBM).2,3The resulting biofilm isrepresentative of static environments that can develop a spo-rulating biofilm rather than being representative of one particu-lar envir
4、onment.1.2 This test method utilizes a modified CBM to grow thebiofilm. The CBM uses a semipermeable membrane on an agarplate as the biofilm growth surface and nutrient source.2,3Inthis test method, membranes are inoculated and incubated fora total of 8 days to promote sporulation within the biofilm
5、.1.3 This test method describes how to sample and analyzethe biofilm for vegetative cells and spores. Biofilm populationis expressed as total colony forming units (CFU) and spores permembrane.1.4 Basic microbiology training is required to perform thistest method.1.5 The values stated in SI units are
6、 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 with its use. It is theresponsibility of the user of this standard to establish appro-priate safety, health, and environm
7、ental practices and deter-mine the applicability of regulatory limitations prior to use.1.7 This international standard was developed in accor-dance with internationally recognized principles on standard-ization established in the Decision on Principles for theDevelopment of International Standards,
8、 Guides and Recom-mendations issued by the World Trade Organization TechnicalBarriers to Trade (TBT) Committee.2. Referenced Documents2.1 ASTM Standards:4E2756 Terminology Relating to Antimicrobial and AntiviralAgents3. Terminology3.1 DefinitionsFor definitions of terms used in this TestMethod see T
9、erminology E2756.3.2 Definitions of Terms Specific to This Standard:3.2.1 biofilm, nmicroorganisms living in a self-organizedcommunity attached to surfaces, interfaces, or each other,embedded in a matrix of extracellular polymeric substances ofmicrobial origin, while exhibiting altered phenotypes wi
10、threspect to growth rate and gene transcription.3.2.2 spore, na dormant, robust, and non-metabolicallyactive structure produced by certain bacteria enabling pro-longed survival and greater resistance to deleterious environ-mental factors.4. Summary of Test Method4.1 This test method utilizes a modif
11、ied CBM to grow thebiofilm. The CBM uses an inoculated semipermeable mem-brane on an agar plate as the biofilm growth surface andnutrient source.2In the published method, the inoculatedmembranes are transferred to fresh agar plates regularly(usually every 8-24 h) to provide new nutrients to themembr
12、ane-grown cells and are incubated for a total of approxi-mately 48 h before sampling or subjecting the biofilm to furthertests.3In this test method, membranes are inoculated andincubated for 24 h before transferring the membrane to a newmedia plate. This initial growth step is similar to the publish
13、edCBM and allows the colony biofilm to grow and spread across1This 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 Oct. 1,
14、2018. Published January 2019. DOI: 10.1520/E3180182Anderl, J. N., Franklin, M. J., “r”istheinterval representing the critical difference between two testresults for the same material, obtained by the same operatorusing the same equipment on the same day in the samelaboratory. Additionally, r = 2.8 s
15、r, where sris the repeatabilitystandard deviation.10.1.1 Preliminary repeatability was determined based onten replicate determinations from each of three experimentsperformed by a single operator (Appendix X1).For Log10total CFU, sr= 0.14 X and r = 0.39 X, where X isLog10total CFU/membrane.For Log10
16、spores, sr= 0.26 X and r = 0.72 X, where X is Log10spores/membrane.10.2 BiasSince there is no accepted reference materialsuitable for determining the bias of the procedure, bias cannotbe determined.11. Keywords11.1 biofilm; Bacillus subtilis; colony; membrane; sam-pling; sporeE3180 183APPENDIX(Nonma
17、ndatory Information)X1. PRELIMINARY REPEATABILITY STUDY DATAX1.1 This Interlaboratory Study (ILS) was conducted toestablish a preliminary repeatability statement for ASTME3180, Test Method for Quantification of a Bacillus subtilisBiofilm Comprised of Vegetative Cells and Spores GrownUsing the Colony
18、 Biofilm Model. A single laboratory partici-pated in this study (Baxter Healthcare Corporation) and quan-tified the Log10total CFU and spores per membrane of aBacillus subtilis biofilm prepared according to Test MethodE3180. The laboratory reported ten replicate test results fromeach of three experi
19、ments performed by a single operator. Thestatistical data summary is presented in Table X1.1 and TableX1.2.ASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentionedin this standard. Users of this standard are expressly advised th
20、at determination of the validity of any such patent rights, and the riskof infringement of such rights, are entirely their own responsibility.This standard is subject to revision at any time by the responsible technical committee and must be reviewed every five years andif not revised, either reappr
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24、Copyright Clearance Center, 222Rosewood Drive, Danvers, MA 01923, Tel: (978) 646-2600; http:/ X1.1 Repeatability Limits for Log10Total CFU per Membrane of Bacillus subtilis BiofilmTotal CFUReplicate Experiment 1 Experiment 2 Experiment 3 Average RepeatabilityStandard DeviationRepeatability Limit1 7.
25、46 7.48 7.787.57 0.14 0.392 7.61 7.67 7.623 7.70 7.58 7.554 7.57 7.59 7.545 7.90 7.51 7.456 7.57 7.62 7.217 7.56 7.58 7.248 7.68 7.57 7.519 7.54 7.67 7.4210 7.74 7.73 7.50TABLE X1.2 Repeatability Limits for Log10Spores per Membrane of Bacillus subtilis BiofilmSporesReplicate Experiment 1 Experiment 2 Experiment 3 Average RepeatabilityStandard DeviationRepeatability Limit1 5.00 5.43 5.715.60 0.26 0.722 5.48 5.41 5.183 5.61 5.60 5.784 6.03 5.34 5.265 5.71 5.80 5.766 5.54 5.60 5.157 5.89 5.58 5.298 5.98 5.64 5.809 5.48 5.70 5.6910 5.92 5.93 5.70E3180 184
