ASTM_E_2800_-_11.pdf

Designation:E280011Standard Practice forCharacterization of Bacillus Spore Suspensions forReference Materials1This standard is issued under the fixed designation E2800;the number immediately following the designation indicates the 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.INTRODUCTIONBacillus spp.are aerobic,rod-shaped,Gram positive bacteria that produce endospores undernutrient limiting conditions.The endospores are designed to persist in extreme environments andconsequently are highly resistant to inactivation by heat,chemicals and irradiation.A few species ofBacillus are medically important because of their impact on human and animal health while othershave important agricultural and industrial applications.Measurement of viable Bacillus spores presentin a suspension can be performed using classical microbiology techniques,such as growth on nutrientmedium.The spore suspension is diluted,an aliquot spread on solid nutrient medium,incubated at anappropriate temperature,and the resulting colonies counted.The selection of the type of growthmedium and incubation temperature for the optimal growth of a particular Bacillus species should bedetermined by consultation of relevant literature or by comparison of different growth media andincubation temperatures.Bacillus spore reference materials have many important applications in agriculture,basic research,medical diagnosis,detector validation,and sterility testing.Uniform methods for the characterizationof spores will improve the comparison of different lots of materials and results between differentlaboratories.1.Scope1.1 This practice is focused on two basic measurements tocharacterize Bacillus reference materials,the enumeration ofspores using growth of colonies on nutrient media and usingphase contrast microscopy to determine spore quality andhomogeneity.Additional information on advanced methods forcharacterization is provided in Appendix X1.1.2 This document will provide the user with recommenda-tions for measurement methods,and the details and conditionsthat should be employed to ensure reliable and high-qualitydata are obtained.The practice will help ensure that resultsobtained from the characterization are reported in a uniformmanner.This will allow others to replicate the measurementsand facilitate the comparison of different lots of Bacillus sporesuspensions used as reference materials.It is important to notethat the Bacillus species are a heterogeneous group and theirspecific requirements for growth and sporulation may vary.Users of this practice are encouraged to consult the literaturefor specific information on the species of Bacillus bacteria theyare using(1).21.3 This standard practice does not provide guidance for theidentification of unknown species of bacteria.The identifica-tion of Bacillus species has been traditionally based on colonymorphology,growth on selective media,and biochemical tests,but more recently nucleic acid technologies have enabled thephylogenetic analysis of this group based on 16S DNAsequence similarities(1).1.4 Some Bacillus spp.are pathogenic to humans andanimals and the user is advised to adhere to safe laboratoryprocedures and practices for handling spores from thesespecies(2).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 and health practices and determine the applicabil-ity of regulatory limitations prior to use(2).1.5 This practice assumes a basic knowledge of microbiol-ogy and molecular biology and access to the cited references.1This practice is under the jurisdiction of ASTM Committee E54 on HomelandSecurity Applications and is the direct responsibility of Subcommittee E54.01 onCBRNE Sensors and Detectors.Current edition approved Feb.15,2011.Published April 2011.DOI:10.1520/E2800-11.2The boldface numbers in parentheses refer to a list of references at the end ofthis standard.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 1.6 The values stated in SI units are to be regarded asstandard.No other units of measurement are included in thisstandard.2.Referenced Documents2.1 ASTM Standards:3D1129 Terminology Relating to WaterD4455 Test Method for Enumeration of Aquatic Bacteria byEpifluorescence Microscopy Counting ProcedureD6974 Practice for Enumeration of Viable Bacteria andFungi in Liquid FuelsFiltration and Culture ProceduresE1873 Guide for Detection of Nucleic Acid Sequences bythe Polymerase Chain Reaction TechniqueE2197 Quantitative Disk Carrier Test Method for Determin-ing Bactericidal,Virucidal,Fungicidal,Mycobactericidal,and Sporicidal Activities of ChemicalsE2414 Test Method for Quantitative Sporicidal Three-StepMethod(TSM)to Dete