1、Designation: E2800 11 (Reapproved 2017)Standard 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 revi
2、sion, 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
3、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
4、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 t
5、emperature, 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 temperature
6、s.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 differe
7、ntlaboratories.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 advanc
8、ed 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 resultsobtai
9、ned 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
10、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
11、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 B
12、acillus 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 th
13、e 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
14、jurisdiction of ASTM Committee E54 on HomelandSecurity Applications and is the direct responsibility of Subcommittee E54.01 onCBRNE Sensors and Detectors.Current edition approved Sept. 1, 2017. Published October 2017. Originallyapproved in 2011. Last previous edition approved in 2011 as E2800 11. DO
15、I:10.1520/E2800-11R17.2The 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 StatesThis international standard was developed in accordance with internatio
16、nally recognized principles on standardization established in the Decision on Principles for theDevelopment of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.11.6 The values stated in SI units are to be regarded
17、asstandard. No other units of measurement are included in thisstandard.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, Guides and Recom
18、-mendations issued by the World Trade Organization TechnicalBarriers to Trade (TBT) Committee.2. Referenced Documents2.1 ASTM Standards:3D1129 Terminology Relating to WaterD4455 Test Method for Enumeration of Aquatic Bacteria byEpifluorescence Microscopy Counting ProcedureD6974 Practice for Enumerat
19、ion of Viable Bacteria andFungi in Liquid FuelsFiltration and Culture ProceduresE1873 Guide for Detection of Nucleic Acid Sequences bythe Polymerase Chain Reaction Technique (Withdrawn2014)4E2197 Quantitative Disk Carrier Test Method for Determin-ing Bactericidal, Virucidal, Fungicidal, Mycobacteric
20、idal,and Sporicidal Activities of ChemicalsE2414 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)4E2458 Practices for Bulk Sample Collection and SwabSample Co
21、llection of Visible Powders Suspected of BeingBiological Agents and Toxins from Nonporous Surfaces2.2 Standard Methods for the Examination of Water andWastewater:5Method 9218 Aerobic Endospores (2007)Method 9215 Heterotrophic Plate Count (2004)Environmental Protection Agency Standard Procedure forEn
22、umeration of Bacterial Inocula on Carriers (CarrierCounts) for the Germicidal Spray Products as Disinfec-tants Test, Disinfectant Towelette Test, and the Tubercu-locidal Activity of Disinfectants Test SOP Number: MD-04-05 Date Revised: 01-13-0962.3 ISO Standards:7ISO 4833:2003 Microbiology of food a
23、nd animal feedingstuffs - Horizontal method for the enumeration of micro-organisms Colony-count technique at 30 degrees CISO 21528-1:2004 Microbiology of food and animal feedingstuffs - Horizontal methods for the detection and enu-meration of Enterobacteriaceae Part 1: Detection andenumeration by MP
24、N technique with pre-enrichment2.4 United States Pharmacopeia Standards:USP. 2006 Microbiological Best Laboratory Practices. USP29 Suppl 2 pp. 3804-3807USP. 2003 Good Microbiological Laboratory Practices.Pharmacopeial Forum 29(3):842-850.USP. 2004 Microbiological Best Laboratory Practices. Phar-maco
25、peial Forum 29(3):1713-17213. Terminology3.1 Definitions:3.1.1 colony forming unit (CFU), nunits for the number ofviable particles present in a solution. A CFU can result from asingle viable bacterial cell or from a clump of cells. (D1129)3.1.2 vortex mixing, vapplying a tube containing a liquidsamp
26、le to a special laboratory mixer that establishes a vigorouscircular motion in the bottom of the tube.3.1.2.1 DiscussionThe circular mixing motion results in avortex in the tube that ensures the complete suspension of theentire tube contents.4. Summary of Practice4.1 Viable Spore Concentration by Pl
27、ating on NutrientAgarPlating bacteria on nutrient media is a well-establishedmethod for detection of bacteria in water (3). Suspensions ofspores are first mixed well by vortex mixing or pipetting up anddown vigorously to insure homogeneity of the spore suspen-sion and then serially diluted using an
28、appropriate buffer. Threeserial dilutions are prepared from a spore reference sample.Analiquot of the diluted spore suspension is placed on a nutrientagar plate and spread using aseptic techniques. Afterincubation, the colonies on the plates are counted and thenumbers of viable spores are referred t
29、o as colony formingunits (CFU). The average number of colonies obtained fromthe diluted suspension are used to calculate the concentrationof the original stock solution and reported as CFU/mL. In orderto obtain consistent results, careful attention must be paid todetail to ensure adequate dispersion
30、 of spores and avoidinglosses during the process.4.2 Spore Quality and Homogeneity Determined by PhaseContrast MicroscopyAn inexpensive, rapid and effectivemethod to determine quality and homogeneity of spore prepa-rations. A drop of the spore sample is placed on a microscopeslide and covered with a
31、 coverslip. The spore preparation isexamined using a high power objective (typically 100) with aphase contrast microscope. Phase bright spores, phase darkspores, phase dark vegetative cells, and spore clumps arecounted either manually, using automated counting devices orby digital imaging and comput
32、er software techniques (4). Thepercentage of phase bright spores in the sample is calculatedand reported.5. Significance and Use5.1 Standard practices for the characterization of sporesused as reference materials are important to ensure a uniform3For referenced ASTM standards, visit the ASTM website
33、, 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.4The last approved version of this historical standard is referenced onwww.astm.org.5Available from American Public
34、 Health Association, Standard Methods for theExamination of Water and Wastewater, Washington, DC 20001, http:/www.standardmethods.org/.6Available from United States Environmental Protection Agency (EPA), ArielRios Bldg., 1200 Pennsylvania Ave., NW, Washington, DC 20460, http:/www.epa.gov.7Available
35、from International Organization for Standardization (ISO), 1, ch. dela Voie-Creuse, Case postale 56, CH-1211, Geneva 20, Switzerland, http:/www.iso.ch.E2800 11 (2017)2basis for testing the performance of detection devices andlaboratory instruments. Bacillus spore suspensions can be usedfor a large v
36、ariety of purposes including testing environmentalsampling techniques, inactivation methods, decontaminationmethods and basic research.5.2 The practice is intended for both manufacturers and endusers of Bacillus spore suspensions. The results of the charac-terization measurements are presented in a
37、report of analysis(ROA). The ROA should provide sufficient detail about themeasurement technique to enable the customers to replicate themeasurements, allowing them to determine if the properties ofthe spore suspension changed during shipping and storage.5.3 The enumeration of the viable spores and
38、determinationof homogeneity by microscopic analysis are two basic mea-surements required for the minimal characterization of refer-ence materials. Phase contrast microscopy does not requirestaining to distinguish the “phase bright” dormant spores fromphase dark spores, dark vegetative cells and clum
39、ps. Whenspores germinate they appear phase dark under phase contrastimaging (5). Germinated spores in a reference sample will soondie due to lack of nutrients. It is important in storing samplesto prevent the premature germination of the spores. Thisstandard practice includes the important steps for
40、 these mea-surements and includes guidance for advanced measurements.Additional guidance is given for advanced techniques tocharacterize spore suspensions that may be used to provide ahigher level of characterized Bacillus spore reference samples.5.4 The specific properties of the spores used for th
41、eirintended application, such as susceptibility to disinfectantprocesses, should be determined in addition to the basicmeasurements covered in this practice. Additional informationon the measurement of spore properties is located in theappendix.6. Apparatus6.1 Pipettes, fixed volume or adjustable. T
42、he performanceof the pipettes should be checked to determine correct dispens-ing volume. (pipettors should be tested for proper performancefrequently).6.2 Sterile Pipette Tips.6.3 Vortex Mixer.6.4 Incubator, capable of maintaining 30 to 70 6 2C.6.5 Autoclave, for preparing sterile media and steriliz
43、ingwaste.6.6 Plate Spinner (optional).6.7 Bunsen Burner or Alcohol Lamp (optional).6.8 Sterile Glass or Sterile Plastic Disposable SpreaderRods.6.9 Phase Contrast MicroscopeLow power (10 to 20)and high power phase (40, 60, or 100) objectives arepreferred.6.10 Disposable Plastic (or Glass) Petri Dish
44、es, typically100 mm in diameter and 15 mm deep, sterile.6.11 Dilution Tubes, sterile.6.12 Glass Microscope Slides, precleaned, typically 25 by75 mm.6.13 Glass Coverslips.6.14 Immersion Oil, as recommended by microscope manu-facturer suitable for objective used and coverslips.7. Reagents7.1 Purity of
45、 WaterWater used for preparation of solutionsshould be sterile and high purity; either reverse osmosis,de-ionized or distilled.7.2 Phosphate Buffered Saline (PBS)A typical composi-tion is composed of 0.137 M NaCl, 0.0027 M KCl, 0.01 Msodium phosphate, pH 7.4. Other similar formulations may beused. T
46、he solution should be sterilized by autoclaving orfiltration.7.3 Triton X100 Stock Solution (10 % vol./vol.), preparedin sterile water in a sterile container.7.4 Nutrient Agar PlatesMay be prepared in the labora-tory or purchased. Plates should be stored and used withinexpiration date. Typically, la
47、boratory prepared plates are storedat 4C and used within 2 weeks. Specific media such as 5 %(vol./vol.) sheep blood agar plates may provide importantcolony morphology that can assist in the conformation ofbacteria.7.5 Bleach, freshly diluted, 10 % (vol./vol.). Confirm thatthe stock solution (commerc
48、ial bleach, sodium hypochlorite)has not expired.7.6 Ethanol, 70 % (vol./vol.), prepared in sterile water.8. Hazards8.1 Considerations for safe handling of spore suspensions.Some Bacillus spp. cause disease in human and animals. Priorto using these materials, the user must fully investigate thesafety
49、 hazards associated with the particular Bacillus spp. andfollow the appropriate safety guidelines.The correct training ofpersonnel and the proper use of personal protective equipment(PPE) is essential. A good source for the information onlaboratory safety is the publication “Biosafety in Microbiologi-cal and Biomedical laboratories” (2). Route of infection andinfectious dose and toxin production impact potential forinfection/toxicity.8.2 Use of good microbiology practices is important forsafely working with the pathogenic Bacillus species. Interna-tional gui
