ASTM E2800-11
(Practice)Standard Practice for Characterization of Bacillus Spore Suspensions for Reference Materials
Standard Practice for Characterization of <span class="italic">Bacillus</span> Spore Suspensions for Reference Materials
SIGNIFICANCE AND USE
Standard practices for the characterization of spores used as reference materials are important to ensure a uniform basis for testing the performance of detection devices and laboratory instruments. Bacillus spore suspensions can be used for a large variety of purposes including testing environmental sampling techniques, inactivation methods, decontamination methods and basic research.
The practice is intended for both manufacturers and end users of Bacillus spore suspensions. The results of the characterization measurements are presented in a report of analysis (ROA). The ROA should provide sufficient detail about the measurement technique to enable the customers to replicate the measurements, allowing them to determine if the properties of the spore suspension changed during shipping and storage.
The enumeration of the viable spores and determination of homogeneity by microscopic analysis are two basic measurements required for the minimal characterization of reference materials. Phase contrast microscopy does not require staining to distinguish the “phase bright” dormant spores from phase dark spores, dark vegetative cells and clumps. When spores germinate they appear phase dark under phase contrast imaging (5). Germinated spores in a reference sample will soon die due to lack of nutrients. It is important in storing samples to prevent the premature germination of the spores. This standard practice includes the important steps for these measurements and includes guidance for advanced measurements. Additional guidance is given for advanced techniques to characterize spore suspensions that may be used to provide a higher level of characterized Bacillus spore reference samples.
The specific properties of the spores used for their intended application, such as susceptibility to disinfectant processes, should be determined in addition to the basic measurements covered in this practice. Additional information on the measurement of spore properties is located in t...
SCOPE
1.1 This practice is focused on two basic measurements to characterize Bacillus reference materials, the enumeration of spores using growth of colonies on nutrient media and using phase contrast microscopy to determine spore quality and homogeneity. Additional information on advanced methods for characterization is provided in Appendix X1.
1.2 This document will provide the user with recommendations for measurement methods, and the details and conditions that should be employed to ensure reliable and high-quality data are obtained. The practice will help ensure that results obtained from the characterization are reported in a uniform manner. This will allow others to replicate the measurements and facilitate the comparison of different lots of Bacillus spore suspensions used as reference materials. It is important to note that the Bacillus species are a heterogeneous group and their specific requirements for growth and sporulation may vary. Users of this practice are encouraged to consult the literature for specific information on the species of Bacillus bacteria they are using (1).
1.3 This standard practice does not provide guidance for the identification of unknown species of bacteria. The identification of Bacillus species has been traditionally based on colony morphology, growth on selective media, and biochemical tests, but more recently nucleic acid technologies have enabled the phylogenetic analysis of this group based on 16S DNA sequence similarities (1).
1.4 Some Bacillus spp. are pathogenic to humans and animals and the user is advised to adhere to safe laboratory procedures and practices for handling spores from these species (2).This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to ...
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Designation: E2800 − 11
Standard Practice for
Characterization of Bacillus Spore Suspensions for
1
Reference Materials
This standard is issued under the fixed designation E2800; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
INTRODUCTION
Bacillus spp. are aerobic, rod-shaped, Gram positive bacteria that produce endospores under
nutrient limiting conditions. The endospores are designed to persist in extreme environments and
consequently are highly resistant to inactivation by heat, chemicals and irradiation.Afew species of
Bacillus are medically important because of their impact on human and animal health while others
haveimportantagriculturalandindustrialapplications.Measurementofviable Bacillussporespresent
inasuspensioncanbeperformedusingclassicalmicrobiologytechniques,suchasgrowthonnutrient
medium.The spore suspension is diluted, an aliquot spread on solid nutrient medium, incubated at an
appropriate temperature, and the resulting colonies counted. The selection of the type of growth
medium and incubation temperature for the optimal growth of a particular Bacillus species should be
determined by consultation of relevant literature or by comparison of different growth media and
incubation temperatures.
Bacillus spore reference materials have many important applications in agriculture, basic research,
medical diagnosis, detector validation, and sterility testing. Uniform methods for the characterization
of spores will improve the comparison of different lots of materials and results between different
laboratories.
1. Scope Users of this practice are encouraged to consult the literature
forspecificinformationonthespeciesof Bacillusbacteriathey
1.1 This practice is focused on two basic measurements to
2
are using (1).
characterize Bacillus reference materials, the enumeration of
1.3 Thisstandardpracticedoesnotprovideguidanceforthe
spores using growth of colonies on nutrient media and using
identification of unknown species of bacteria. The identifica-
phase contrast microscopy to determine spore quality and
tion of Bacillus species has been traditionally based on colony
homogeneity.Additionalinformationonadvancedmethodsfor
morphology,growthonselectivemedia,andbiochemicaltests,
characterization is provided in Appendix X1.
but more recently nucleic acid technologies have enabled the
1.2 This document will provide the user with recommenda-
phylogenetic analysis of this group based on 16S DNA
tions for measurement methods, and the details and conditions
sequence similarities (1).
that should be employed to ensure reliable and high-quality
1.4 Some Bacillus spp. are pathogenic to humans and
data are obtained. The practice will help ensure that results
animals and the user is advised to adhere to safe laboratory
obtained from the characterization are reported in a uniform
procedures and practices for handling spores from these
manner. This will allow others to replicate the measurements
species (2).This standard does not purport to address all of the
and facilitate the comparison of different lots of Bacillus spore
safety concerns, if any, associated with its use. It is the
suspensions used as reference materials. It is important to note
responsibility of the user of this standard to establish appro-
that the Bacillus species are a heterogeneous group and their
priate safety and health practices and determine the applicabil-
specific requirements for growth and sporulation may vary.
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.
1
This practice is under the jurisdiction ofASTM Committee E54 on Homeland
Security Applications and is the direct responsibility of Subcommittee E54.01 on
CBRNE Sensors and Detectors.
2
Current edition approved Feb. 15, 2011. Published April 2011. DOI: 10.1520/ The boldface numbers in parentheses refer to a list of references at the end of
E2800-11. this standard.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
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E2800 − 11
1.6 The values stated in SI units are to be regarded as USP. 2004Microbiological Best Laboratory Practices. Phar-
standard. No other units of measurement are included in this macopeial Forum 29(3):1713-1721
standard.
3. Terminology
2. Referenced Documents
3.1 Definitions:
3
2.1 ASTM Standards:
3.1.1 colonyformingunit(CFU),n—unitsforthenumberof
D1129Terminology Relating to Water
viable particles present in a solution.ACFU can
...
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