ASTM-E2800 Standard Practice for Characterization of Bacillus Spore Suspensions for Reference Materials

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Standard Practice for Characterization of Bacillus Spore Suspensions for Reference Materials




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).2

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 use (2).

1.5 This practice assumes a basic knowledge of microbiology and molecular biology and access to the cited references.

1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.

1.7 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.


Bacillus; phase contrast microscopy; plate count; spores;; ICS Number Code 07.100.99 (Other standards related to microbiology)

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07.100.99 (Other standards related to microbiology)

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Document Number


Revision Level

2011 R17 EDITION



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Publication Date

Oct. 1, 2017

Document Type


Page Count

8 pages

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