ASTM E2839-21

Standard Practice for Production and Storage of Spores of <emph type="bdit">C. difficile</emph > for Use in Efficacy Evaluation of Antimicrobial Agents

SIGNIFICANCE AND USE
5.1 This practice describes a procedure for preparing and storing a suspension of C. difficile spores that meets the following acceptance criteria: (1) spore titer of approximately 5.0×108 spores/mL, (2) spore purity of ≥95 %, and (3) a mean log10 reduction (LR) value >5.0 for 3 carriers exposed to 5000 ppm and a mean LR of E3218.
SCOPE
1.1 This practice specifies the procedures for producing and storing standardized suspensions of Clostridioides difficile spores for the evaluation of the sporicidal activity of antimicrobial formulations using the Quantitative Method for Testing Antimicrobial Agents against Spores of C. difficile on Hard, Non-porous Surfaces or other procedures.  
1.2 This practice may involve hazardous materials, chemicals, and microorganisms and should be performed only by persons with formal training in microbiology.  
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.4 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, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.5 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.

General Information

• Status: Published
• Publication Date: 31-May-2021
• ICS: 07.100.99 - Other standards related to microbiology
• Technical Committee: E35 - Pesticides, Antimicrobials, and Alternative Control Agents
• Drafting Committee: E35.15 - Antimicrobial Agents

Standards Content (Sample)

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.
Designation: E2839 − 21
Standard Practice for
Production and Storage of Spores of C. difficile for Use in
1
Efficacy Evaluation of Antimicrobial Agents
This standard is issued under the fixed designation E2839; 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.
1. Scope* 3. Terminology
1.1 This practice specifies the procedures for producing and 3.1 For definition of terms used in this practice refer to
storing standardized suspensions of Clostridioides diffıcile Terminology E2756.
spores for the evaluation of the sporicidal activity of antimi-
3.2 Definitions of Terms Specific to This Standard:
crobialformulationsusingtheQuantitativeMethodforTesting
3.2.1 density gradient medium, adj/n—HistoDenz (trade-
Antimicrobial Agents against Spores of C. diffıcile on Hard, 3
marked) is a non-ionic gradient medium used here to separate
Non-porous Surfaces or other procedures.
spores from vegetative cells and cell fragments on the basis of
1.2 This practice may involve hazardous materials, density.
chemicals, and microorganisms and should be performed only
3.2.2 pre-reduced medium, n—an agar or broth manufac-
by persons with formal training in microbiology.
tured in an oxygen-free environment and packaged in air-tight
sealed pouches or bags.
1.3 The values stated in SI units are to be regarded as
standard. No other units of measurement are included in this
3.2.3 purified spores, n—level of quality based on when
standard.
sporeconcentrationreaches≥95%,asvegetativecellsandcell
fragments are separated by the density gradient medium.
1.4 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the
3.3 Acronyms:
responsibility of the user of this standard to establish appro-
CDC = Centers for Disease Control and Prevention
priate safety, health, and environmental practices and deter-
CFU = colony-forming unit
mine the applicability of regulatory limitations prior to use.
VFS = vegetative frozen stock.
1.5 This international standard was developed in accor-
dance with internationally recognized principles on standard-
4. Summary of Practice
ization established in the Decision on Principles for the
4.1 This practice provides detailed instructions for the
Development of International Standards, Guides and Recom-
mendations issued by the World Trade Organization Technical culture, maintenance, sporulation, and storage of spores of C.
diffıcile. Spores are harvested from an agar medium following
Barriers to Trade (TBT) Committee.
incubation in an anaerobic environment for 10 days. Upon
2. Referenced Documents
harvesting, spores are washed several times with phosphate-
2
buffered saline with polysorbate 80, exposed to heat to inacti-
2.1 ASTM Standards:
vate any vegetative cells, and purified using a density gradient
E2756Terminology Relating toAntimicrobial andAntiviral
medium. Purified spores are enumerated and assessed for
Agents
qualityusingacarrier-basedtestemployingtwoconcentrations
E3218Test Method for Quantitative Method for Testing
of sodium hypochlorite (NaOCl). Spores of C. diffıcile are
Antimicrobial Agents against Spores of C. diffıcile on
stored for up to 90 days at -80°C 6 5°C.
Hard, Nonporous Surfaces
5. Significance and Use
1
This practice is under the jurisdiction ofASTM Committee E35 on Pesticides,
5.1 This practice describes a procedure for preparing and
Antimicrobials, and Alternative Control Agents and is the direct responsibility of
storing a suspension of C. diffıcile spores that meets the
Subcommittee E35.15 on Antimicrobial Agents.
CurrenteditionapprovedJune1,2021.PublishedJuly2021.Originallyapproved
in2011.Lastpreviouseditionapprovedin2018asE2839–18.DOI:10.1520/E2839-
3
21. The sole source of supply of HistoDenz (trademark) (Cat. No. D2158) known
2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or to the committee at this time is Sigma-Aldrich, St. Louis, MO. If you are aware of
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM alternative suppliers, please provide this information to ASTM International
Standards volume information, refer to the standard’s Document Summary page on Headquarters.Your comments will receive careful consideration at a meeting of the
1
the ASTM website. responsible technical committee, which you may attend.
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: E2839 − 18 E2839 − 21
Standard Practice for
Production and Storage of Spores of C. difficile for Use in
1
Efficacy Evaluation of Antimicrobial Agents
This standard is issued under the fixed designation E2839; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope Scope*
1.1 This practice specifies the procedures for producing and storing standardized suspensions of Clostridioides diffıcile spores for
the evaluation of the sporicidal activity of antimicrobial formulations using the Quantitative Method for Testing Antimicrobial
Agents against Spores of C. diffıcile on Hard, Non-porous Surfaces or other procedures.
1.2 This practice may involve hazardous materials, chemicals, and microorganisms and should be performed only by persons with
formal training in microbiology.
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.4 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.
1.4 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, health, and environmental practices and determine the applicability of
regulatory limitations prior to use.
1.5 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.
2. Referenced Documents
2
2.1 ASTM Standards:
E2756 Terminology Relating to Antimicrobial and Antiviral Agents
E3218 Test Method for Quantitative Method for Testing Antimicrobial Agents against Spores of C. diffıcile on Hard,
Non-porousNonporous Surfaces
3. Terminology
3.1 For definition of terms used in this practice refer to Terminology E2756.
1
This practice is under the jurisdiction of ASTM Committee E35 on Pesticides, Antimicrobials, and Alternative Control Agents and is the direct responsibility of
Subcommittee E35.15 on Antimicrobial Agents.
Current edition approved Oct. 1, 2018June 1, 2021. Published September 2019July 2021. Originally approved in 2011. Last previous edition approved in 20112018 as
E2839–11.–18. DOI: 10.1520/E2839-18.10.1520/E2839-21.
2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1

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E2839 − 21
3.2 Definitions of Terms Specific to This Standard:
3
3.2.1 density gradient medium, adj/n—HistoDenz (trademarked) is a non-ionic gradient medium used here to separate spores
from vegetative cells and cell fragments on the basis of density.
3.2.2 pre-reduced medium, n—an agar or broth manufactured in an oxygen-free environment and packaged in air-tight sealed
pouches or bags.
3.2.3 purified spores, n—level of quality based on when spore concentration reaches ≥95 %, as vegetative cells and cell fragments
are separated by the density gradient medium.
3.3 Acronyms:
CDC = Centers for Disease Control and Prevention
CFU = colony-forming unit
VFS = vegetative frozen stock.
4. Summary of Practice
4.1 This practice provides detailed instructions for the culture, maintenance, sporulation, and storage of spores of C. diffıcile.
Spores are harvested from an agar medium following incubation in an anaerobic environment for 10 days. Upon harvesting, spores
are washed several times with phosphate-buffered saline with polysorbate 80, exposed to heat to inactivate any vegetative cells,
and purified using a density gradient medium. Purified spores are enumerated and assessed for quality using a carrier-based t
...