1、Designation: E2800 11Standard 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
2、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 condition
3、s. 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 i
4、ndustrial 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 th
5、e 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 r
6、eference 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.
7、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 forcha
8、racterization 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 char
9、acterization 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 req
10、uirements 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
11、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
12、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 sta
13、ndard 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 AS
14、TM 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 of
15、this standard.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.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:3D112
16、9 Terminology Relating to WaterD4455 Test Method for Enumeration ofAquatic 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 Poly
17、merase 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 Determine Sporicidal Efficacy of Liq-uids, L
18、iquid Sprays, and Vapor or Gases on ContaminatedCarrier SurfacesE2458 Practices for Bulk Sample Collection and SwabSample Collection of Visible Powders Suspected of BeingBiothreat Agents from Nonporous Surfaces2.2 Standard Methods for the Examination of Water andWastewater:4Method 9218 Aerobic Endos
19、pores (2007)Method 9215 Heterotrophic Plate Count (2004)Environmental Protection Agency Standard Procedure forEnumeration of Bacterial Inocula on Carriers (CarrierCounts) for the Germicidal Spray Products as Disinfec-tants Test, Disinfectant Towelette Test, and the Tubercu-locidal Activity of Disinf
20、ectants Test SOP Number: MD-04-05 Date Revised: 01-13-0952.3 ISO Standards:6ISO 4833:2003 Microbiology of food and 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 feed-ing stuffs -
21、Horizontal methods for the detection andenumeration of Enterobacteriaceae Part 1: Detection andenumeration by MPN technique with pre-enrichment2.4 United States Pharmacopeia Standards:USP. 2006 Microbiological Best Laboratory Practices. USP29 Suppl 2 pp. 3804-3807USP. 2003 Good Microbiological Labor
22、atory Practices.Pharmacopeial Forum 29(3):842-850.USP. 2004 Microbiological Best Laboratory Practices. Phar-macopeial Forum 29(3):1713-17213. Terminology3.13.1.1 colony forming unit (CFU), nunits for the numberof viable particles present in a solution. A CFU can result froma single viable bacterial
23、cell or from a clump of cells.(D1129)3.1.2 vortex mixing, vapplying a tube containing a liquidsample 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 complet
24、e suspension of theentire tube contents.4. Summary of Practice4.1 Viable Spore Concentration by Plating 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 and
25、down vigorously to insure homogeneity of the spore suspen-sion and then serially diluted using an 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. Aft
26、er incuba-tion, the colonies on the plates are counted and the numbers ofviable spores are referred to as colony forming units (CFU).The average number of colonies obtained from the dilutedsuspension are used to calculate the concentration of theoriginal stock solution and reported as CFU/mL. In ord
27、er toobtain consistent results, careful attention must be paid todetail to ensure adequate dispersion 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
28、 of spore prepa-rations. A drop of the spore sample is placed on a microscopeslide and covered with a coverslip. The spore preparation isexamined using a high power objective (typically 1003) witha phase contrast microscope. Phase bright spores, phase darkspores, phase dark vegetative cells, and spo
29、re clumps arecounted either manually, using automated counting devices orby digital imaging and computer 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 refer
30、ence materials are important to ensure a uniformbasis for testing the performance of detection devices andlaboratory instruments. Bacillus spore suspensions can be usedfor a large variety of purposes including testing environmentalsampling techniques, inactivation methods, decontaminationmethods and
31、 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 report of analysis3For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service
32、 at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.4Available from American Public Health Association, Standard Methods for theExamination of Water and Wastewater, Washington, DC 20001, http:/www.standardmethods.o
33、rg/.5Available from United States Environmental Protection Agency (EPA), ArielRios Bldg., 1200 Pennsylvania Ave., NW, Washington, DC 20460, http:/www.epa.gov.6Available from International Organization for Standardization (ISO), 1, ch. dela Voie-Creuse, Case postale 56, CH-1211, Geneva 20, Switzerlan
34、d, http:/www.iso.ch.E2800 112(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
35、spores and 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 cel
36、ls and clumps. 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 importan
37、t steps for 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
38、used for theirintended 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 ad
39、justable. The 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 a
40、nd sterilizingwaste.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 203)and high power phase (40, 60, or 1003) objectives arepreferred.6.10 Disposable Plastic (or Glas
41、s) Petri Dishes, 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. Reagents
42、7.1 Purity of WaterWater used for preparation of solu-tions should 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 formulation
43、s may beused. The solution should be sterilized by autoclaving orfiltration.7.3 Triton X100y 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 dat
44、e. Typically, laboratory 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 s
45、olution (commercial 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 inves
46、tigate thesafety 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 “Biosafe
47、ty 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 guidelines for microbi
48、ological safety should be followedwhen appropriate (6).9. Sample Storage9.1 Appropriate storage conditions are essential to preservethe properties of the spores. Preservation of spore viability(prevention of germination), sterility, and lack of clumping arethe goals. Traditionally, spores have been
49、stored in solutions ofsterile water, 20 to 70 % (vol./vol.) ethanol or 1 % (wt./vol.)phenol in water at 4C to prevent bacterial growth. Spores canbe frozen, but the effect of freezing and thawing on theproperties of the spores has to be determined.9.2 It is essential to prevent premature germination ofspores, clumping, and loss of spores during storage. One studyused borosilicate glass vials with PTFE lined caps to storespores in a number of solutions including sterile deionizedwater, 20 % ethanol and 1 % phenol for periods up to severalyears at 4C and found the
