ASTM E2895-19
(Practice)Standard Practice for Producing High Titers of Viable and Semi-Purified Spores of Clostridium difficile using a Liquid Medium
Standard Practice for Producing High Titers of Viable and Semi-Purified Spores of Clostridium difficile using a Liquid Medium
SIGNIFICANCE AND USE
5.1 The quantity and quality of the spores produced by this practice may be used to assess environmental surface disinfectants for sporicidal activity (4). The method is applicable to standard as well as clinically isolated toxigenic and non-toxigenic strains of C. difficile.
SCOPE
1.1 This practice describes the production and semipurification of C. difficile spores (also called endospores) primarily for use in testing the sporicidal activities of environmental surface disinfectants (Test Methods E2111and E2197); such spores can also be used to study their structure, chemistry and germination.
1.2 While the practice described is based on the use of 500-mL volumes of the liquid culture medium in an anaerobic incubator, anaerobic jars with smaller volumes of the same medium can also be used.
1.3 It is the responsibility of the investigator to determine whether Good Laboratory Practice (GLP) regulations are required and to follow them when appropriate (40 CFR, Part 160 for EPA submissions and 21 CFR; Part 58 for FDA submissions).
1.4 Warning—This standard may involve hazardous materials, chemicals, and microorganisms and should be performed only by persons with formal training in microbiology.
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.6 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.
1.7 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.
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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:E2895 −19
Standard Practice for
Producing High Titers of Viable and Semi-Purified Spores of
1
Clostridium difficile using a Liquid Medium
This standard is issued under the fixed designation E2895; 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
Clostridium diffıcile (C. diffıcile), an anaerobic spore-former, can cause acute and potentially fatal
2
gastroenteritisinhealthcareandothersettings (1). Thefrequentepisodesofdiarrheacancontaminate
the indoor environment widely with persistent (2) and microbicide-resistant (3) spores. Disinfectants
wishing to claim activity against C. diffıcile now require carrier testing using spores of high purity
(>90%) that show a >6 log reduction in spore viability (4). While the use of a semi-solid medium
10
for C. diffıcile spore production has been reported (5) (Test Method E2839), this standard describes a
liquid medium and an enzyme-based semipurification process. Appendix X1 describes an alternative
to enzyme purification.
1. Scope 1.6 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the
1.1 This practice describes the production and semipurifi-
responsibility of the user of this standard to establish appro-
cation of C. diffıcile spores (also called endospores) primarily
priate safety, health, and environmental practices and deter-
for use in testing the sporicidal activities of environmental
mine the applicability of regulatory limitations prior to use.
surface disinfectants (Test Methods E2111and E2197); such
1.7 This international standard was developed in accor-
spores can also be used to study their structure, chemistry and
dance with internationally recognized principles on standard-
germination.
ization established in the Decision on Principles for the
1.2 While the practice described is based on the use of
Development of International Standards, Guides and Recom-
500-mLvolumes of the liquid culture medium in an anaerobic
mendations issued by the World Trade Organization Technical
incubator, anaerobic jars with smaller volumes of the same
Barriers to Trade (TBT) Committee.
medium can also be used.
2. Referenced Documents
1.3 It is the responsibility of the investigator to determine
3
whether Good Laboratory Practice (GLP) regulations are
2.1 ASTM Standards:
required and to follow them when appropriate (40 CFR, Part
D1129Terminology Relating to Water
160 for EPA submissions and 21 CFR; Part 58 for FDA
E2111Quantitative Carrier Test Method to Evaluate the
submissions).
Bactericidal,Fungicidal,Mycobactericidal,andSporicidal
1.4 Warning—This standard may involve hazardous Potencies of Liquid Chemicals
E2197Quantitative Disk Carrier Test Method for Determin-
materials, chemicals, and microorganisms and should be per-
formed only by persons with formal training in microbiology. ing Bactericidal, Virucidal, Fungicidal, Mycobactericidal,
and Sporicidal Activities of Chemicals
1.5 The values stated in SI units are to be regarded as
E2756Terminology Relating toAntimicrobial andAntiviral
standard. No other units of measurement are included in this
Agents
standard.
E2839Test Method for Production of Clostridium difficile
Spores for Use in Efficacy Evaluation of Antimicrobial
1 Agents
This practice is under the jurisdiction ofASTM Committee E35 on Pesticides,
Antimicrobials, and Alternative Control Agents and is the direct responsibility of
Subcommittee E35.15 on Antimicrobial Agents.
Current edition approved April 1, 2019. Published May 2019. Originally
3
approved 2019. Last previous edition approved 2013 as E2895–13. DOI: 10.1520/ For referenced ASTM standards, visit the ASTM website, www.astm.org, or
E2895-19 contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
2
The boldface numbers in parentheses refer to a list of references at the end of Standards volume information, refer to the standard’s Document Summary page on
this standard. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1
---------------------- Page: 1 ----------------------
E2895−19
4
2.2 Federal Standards: 6.1.8 Disodium Hydrogen Phosphate (Na HPO ).
2 4
21 CFR, Part 58Good Laboratory Practice for Nonclinical 6.1.9 Sodium Dihydrogen Phosphate (NaH PO ·H O).
2 4 2
Laboratory Studies 6.1.10 Potassium Hydroxide (KOH).
40 CFR, Part 160Good Laboratory Practice Standar
...
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: E2895 − 13 E2895 − 19
Standard Test Method Practice for
Producing High Titers of Viable and Semi-Purified Spores of
1
Clostridium difficile using a Liquid Medium
This standard is issued under the fixed designation E2895; 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.
INTRODUCTION
Clostridium diffıcile (C. diffıcile), an anaerobic spore-former, can cause acute and potentially fatal
2
gastroenteritis in healthcare and other settings (1). The frequent episodes of diarrhea can contaminate
the indoor environment widely with persistent (2) and microbicide-resistant (3) spores. Disinfectants
wishing to claim activity against C. diffıcile now require carrier testing using spores of high purity
(>90%) that show a >6 log reduction in spore viability (4). While the use of a semi-solid medium
10
for C. diffıcile spore production has been reported (5) (Test Method E2839), this standard describes a
liquid medium and an enzyme-based semipurification process for the purpose. process. Appendix X1
describes an alternative to enzyme purification.
1. Scope
1.1 This test method describes the production and semipurification of C. diffıcile spores (also called endospores) primarily for
use in testing the sporicidal activities of environmental surface disinfectants (Test Methods E2111and E2197); such spores can also
be used to study their structure, chemistry and germination.
1.2 While the test method described is based on the use of 500-mL volumes of the liquid culture medium in an anaerobic
incubator, anaerobic jars with smaller volumes of the same medium can also be used.
1.3 It is the responsibility of the investigator to determine whether Good Laboratory Practice (GLP) regulations are required and
to follow them when appropriate (40 CFR, Part 160 for EPA submissions and 21 CFR; Part 58 for FDA submissions).
1.4 Warning—This standard may involve hazardous materials, chemicals, and microorganisms and should be performed only
by persons with formal training in microbiology.
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.6 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
3
2.1 ASTM Standards:
D1129 Terminology Relating to Water
E2111 Quantitative Carrier Test Method to Evaluate the Bactericidal, Fungicidal, Mycobactericidal, and Sporicidal Potencies of
Liquid Chemicals
E2197 Quantitative Disk Carrier Test Method for Determining Bactericidal, Virucidal, Fungicidal, Mycobactericidal, and
Sporicidal Activities of Chemicals
E2756 Terminology Relating to Antimicrobial and Antiviral Agents
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 Oct. 1, 2013April 1, 2019. Published December 2013May 2019. Originally approved 2019. Last previous edition approved 2013 as E2895–13.
DOI: 10.1520/E2895-1310.1520/E2895-19
2
The boldface numbers in parentheses refer to a list of references at the end of this standard.
3
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1
---------------------- Page: 1 ----------------------
E2895 − 19
E2839 Test Method for Production of Clostridium difficile Spores for Use in Efficacy Evaluation of Antimicrobial Agents
4
2.2 Federal Standards:
21 CFR, Part 58 Good Laboratory Practice for Nonclinical Laboratory Studies
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 Terminologies D1129 and E2756.
3.2 Definitions of Terms Specific to This Standard:
3.2.1 anaerobe, n—an organism that cannot grow or p
...
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