ASTM E3011-22

Standard Practice for In vitro production of <emph type="ital">Clostridioides difficile</emph > Spores

SIGNIFICANCE AND USE
5.1 This practice describes a procedure for producing spore suspensions of C. difficile ATCC 700792, C. difficile ATCC 43598, or C. difficile ATCC 43599. The spore suspensions may be used in antimicrobial efficacy testing, or other laboratory testing requiring C. difficile spores. A spore crop is considered acceptable if the titer is >8 log10 spores/mL, purity of 95 %, and is resistant to 2.5M HCl after 10 min of exposure.
SCOPE
1.1 This practice is designed to propagate spores of Clostridioides difficile using liver broth.  
1.2 It is the responsibility of the user of this practice to determine whether Good Laboratory Practices are required and follow when appropriate.  
1.3 This practice should only be performed by those trained in microbiological techniques.  
1.4 Units—The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.5 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.  
1.6 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: 30-Sep-2022
• 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: E3011 − 22
Standard Practice for
1
In vitro production of Clostridioides difficile Spores
This standard is issued under the fixed designation E3011; 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
3.1 Definitions: For definitions of general terms used in this
1.1 This practice is designed to propagate spores of
practice, refer to Terminology E2756.
Clostridioides diffıcile using liver broth.
3.2 Definitions of Terms Specific to This Standard:
1.2 It is the responsibility of the user of this practice to
3.2.1 frozen stock culture, n—a culture of vegetative bacte-
determinewhetherGoodLaboratoryPracticesarerequiredand
ria propagated, and prepared for storage at ≤ –70°C in a liquid
follow when appropriate.
broth medium containing a cryoprotectant such as glycerol.
1.3 This practice should only be performed by those trained
3.2.2 spore suspension, n—harvested spores suspended in a
in microbiological techniques.
liquid medium, sterile deionized water.
1.4 Units—The values stated in SI units are to be regarded
4. Summary of Practice
asstandard.Nootherunitsofmeasurementareincludedinthis
standard. 4.1 This standard outlines a procedure for producing high-
titer spore suspensions of C. diffıcile using a commercially
1.5 This standard does not purport to address all of the
available liquid medium with 7d to 10 d of incubation under
safety concerns, if any, associated with its use. It is the
anaerobic conditions. Once adequate levels of spores are
responsibility of the user of this standard to establish appro-
present in the liquid medium, the spores are harvested and
priate safety, health, and environmental practices and deter-
washedseveraltimesincoldsteriledeionizedwater.Thespore
mine the applicability of regulatory limitations prior to use.
suspension is enumerated, the spore purity is assessed and the
1.6 This international standard was developed in accor-
acid resistance is verified.
dance with internationally recognized principles on standard-
ization established in the Decision on Principles for the
5. Significance and Use
Development of International Standards, Guides and Recom-
5.1 This practice describes a procedure for producing spore
mendations issued by the World Trade Organization Technical
suspensions of C. diffıcile ATCC 700792, C. diffıcile ATCC
Barriers to Trade (TBT) Committee.
43598,or C. diffıcileATCC43599.Thesporesuspensionsmay
be used in antimicrobial efficacy testing, or other laboratory
2. Referenced Documents
testing requiring C. diffıcile spores.Aspore crop is considered
2
acceptable if the titer is >8 log spores/mL, purity of 95%,
2.1 ASTM Standards:
10
E2756Terminology Relating toAntimicrobial andAntiviral and is resistant to 2.5M HCl after 10 min of exposure.
Agents
6. Apparatus
3
2.2 Federal Standard:
6.1 Anaerobe jar—Any airtight jar or container that can be
40 CFR, Part 160Good Laboratory Practice Standards
used in combination with gas packs to obtain an anaerobic
environment. An anaerobic chamber and anaerobic incubator
may be substituted for an anaerobe jar.
1
This practice is under the jurisdiction ofASTM Committee E35 on Pesticides,
6.2 Biological safety cabinet—To help maintain an aseptic
Antimicrobials, and Alternative Control Agents and is the direct responsibility of
Subcommittee E35.15 on Antimicrobial Agents. work space.
Current edition approved Oct. 1, 2022. Published December 2022. Originally
6.3 Centrifuge—Any type or model capable of centrifuging
approved in 2015. Last previous edition approved in 2015 as E3011–15. DOI:
10.1520/E3011–22. up to 40 mL of liquid at 7500 × g.
2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
6.4 Centrifuge tubes—Any type of sterile centrifuge tube
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on with a 50 mL capacity.
the ASTM website.
3
6.5 Incubator—An incubator capable of maintaining
Federal Insecticide, Fungicide and RodenticideAct (FIFRA) 40 CFR Part 160,
Good 185 Laboratory Practice Standards; Final Rule. 1989. 36°C61°C.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1

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E3011 − 22
5
6.6 Laboratory glassware with closures—Glasswaretohold Disease Control and Prevention/National Institutes of Health
...

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: E3011 − 15 E3011 − 22
Standard Test Method Practice for
In vitro production of Clostridium DifficileClostridioides difficile
1
Spores
This standard is issued under the fixed designation E3011; 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
1.1 This test method is designed to propagate spores of Clostridium diffıcile using liver broth.
1.2 It is the responsibility of the user of this test method to determine whether Good Laboratory Practices are required and follow
when appropriate.
1.3 This test method should only be performed by those trained in microbiological techniques.
1.4 Units—The values stated in SI units are to be regarded as standard. No other units of measurement are included in this
standard.
1.5 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. Referenced Documents
2
2.1 ASTM Standards:
E2839 Practice for Production and Storage of Spores of C. difficile for Use in Efficacy Evaluation of Antimicrobial Agents
E2895 Practice for Producing High Titers of Viable and Semi-Purified Spores of Clostridium difficile using a Liquid Medium
E2756 Terminology Relating to Antimicrobial and Antiviral Agents
3
2.2 Federal Standard:
40 CFR, Part 160 Good Laboratory Practice Standards
3. Terminology
3.1 Definitions: For definitions of general terms used in this test method, refer to Terminology E2756.
3.2 Definitions of Terms Specific to This Standard:
1
This test method 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 May 1, 2015Oct. 1, 2022. Published September 2015December 2022. Originally approved in 2015. Last previous edition approved in 2015 as
E3011–15. DOI: 10.1520/E3011–1510.1520/E3011–22.
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.
3
Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) 40 CFR Part 160, Good 185 Laboratory Practice Standards; Final Rule. 1989.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1

---------------------- Page: 1 ----------------------
E3011 − 22
3.2.1 frozen stock culture, n—a culture of vegetative bacteria propagated, and prepared for storage at ≤ –70°C in a liquid broth
medium containing a cryoprotectant such as glycerol.
3.2.2 spore suspension, n—harvested spores suspended in a liquid medium, sterile deionized water.
4. Summary of Test Method
4.1 This standard outlines a procedure for producing high-titer spore suspensions of C. diffıcile using a commercially available
liquid medium with 7 to 10 d of incubation under anaerobic conditions. Once adequate levels of spores are present in the liquid
medium, the spores are harvested and washed several times in cold sterile deionized water. The spore suspension is enumerated,
the spore purity is assessed and the acid resistance is verified.
5. Significance and Use
5.1 This test method describes a procedure for producing spore suspensions of C. diffıcile ATCC 700792, C. diffıcile ATCC 43598,
or C. diffıcile ATCC 43599. The spore suspensions may be used in antimicrobial efficacy testing, or other laboratory testing
requiring C. diffıcile spores. A spore crop is considered acceptable if the titer is >8 log spores/mL, purity of 95%, and is resistant
10
to 2.5M HCl after 10 min of exposure (see Test Method E2839).
6. Apparatus
6.1 Anaerobe jar—Any airtight jar or container that can be used in combination with gas packs to obtain an anaerobic
environment. An anaerobic chamber and anaerobic incubator may be substituted for an anaerobe jar.
6.2 Biological safety cabinet—To help maintain an aseptic work space.
6.3 Centrifuge—Any type or model capable of centrifuging up to 40 mL of liquid at 7500 × g.
6.4 Centrifuge tubes—Any type of sterile centrifuge tube with a
...