ASTM E2895-13

Standard Test Method 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 method meet the current requirements of the U.S. Environmental Protection Agency (EPA) to assess environmental surface disinfectants for label claims of 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 test method 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 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.

General Information

• Status: Historical
• Publication Date: 30-Sep-2013
• ICS: 07.100.99 - Other standards related to microbiology
• Technical Committee: E35 - Pesticides, Antimicrobials, and Alternative Control Agents
• Drafting Committee: E35.15 - Antimicrobial Agents

Relations

• Replaced By: ASTM E2895-19 - Standard Practice for Producing High Titers of Viable and Semi-Purified Spores of Clostridium difficile using a Liquid Medium
• Effective Date: 01-Apr-2019

Standards Content (Sample)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation:E2895 −13
Standard Test Method for
Producing High Titers of Viable and Semi-Purified Spores of
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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
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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
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for C. diffıcile spore production has been reported (5) (Test Method E2839), this standard describes a
liquidmediumandanenzyme-basedsemipurificationprocessforthepurpose.AppendixX1describes
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 test method describes the production and semipu-
responsibility of the user of this standard to establish appro-
rification of C. diffıcile spores (also called endospores) primar-
priate safety and health practices and determine the applica-
ily for use in testing the sporicidal activities of environmental
bility of regulatory limitations prior to use.
surface disinfectants (Test Methods E2111and E2197); such
spores can also be used to study their structure, chemistry and
2. Referenced Documents
germination.
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2.1 ASTM Standards:
1.2 While the test method described is based on the use of
D1129Terminology Relating to Water
500-mLvolumes of the liquid culture medium in an anaerobic
E2111Quantitative Carrier Test Method to Evaluate the
incubator, anaerobic jars with smaller volumes of the same
Bactericidal,Fungicidal,Mycobactericidal,andSporicidal
medium can also be used.
Potencies of Liquid Chemicals
1.3 It is the responsibility of the investigator to determine
E2197Quantitative Disk Carrier Test Method for Determin-
whether Good Laboratory Practice (GLP) regulations are
ing Bactericidal, Virucidal, Fungicidal, Mycobactericidal,
required and to follow them when appropriate (40 CFR, Part
and Sporicidal Activities of Chemicals
160 for EPA submissions and 21 CFR; Part 58 for FDA
E2756Terminology Relating toAntimicrobial andAntiviral
submissions).
Agents
E2839Test Method for Production of Clostridium difficile
1.4 Warning—This standard may involve hazardous
Spores for Use in Efficacy Evaluation of Antimicrobial
materials, chemicals, and microorganisms and should be per-
Agents
formed only by persons with formal training in microbiology.
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2.2 Federal Standards:
1.5 The values stated in SI units are to be regarded as
21 CFR, Part 58Good Laboratory Practice for Nonclinical
standard. No other units of measurement are included in this
Laboratory Studies
standard.
40 CFR, Part 160Good Laboratory Practice Standards
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This test method is under the jurisdiction of ASTM Committee E35 on
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Pesticides, Antimicrobials, and Alternative Control Agents and is the direct For referenced ASTM standards, visit the ASTM website, www.astm.org, or
responsibility of Subcommittee E35.15 on Antimicrobial Agents. contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
Current edition approved Oct. 1, 2013. Published December 2013. DOI: Standards volume information, refer to the standard’s Document Summary page on
10.1520/E2895-13 the ASTM website.
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The boldface numbers in parentheses refer to a list of references at the end of Available from Superintendent of Documents, U.S.S Government Printing
this standard. Office, Washington DC, 20402. http://www.gpo.gov/
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
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E2895−13
3. Terminology 6.1.13 Potassium Dihydrogen Phosphate (KH PO ).
2 4
6.1.14 Potassium Carbonate (K CO ).
2 3
3.1 Definitions—Fordefinitionsofgeneraltermsusedinthis
6.1.15 Calcium Chloride (CaCl ·2H O).
2 2
test method, refer to Terminologies D1129 and E2756.
6.1.16 Ammonium Sulfate (NH ) SO ).
4 2 4
3.2 Definitions of Terms Specific to This Standard:
6.1.17 Magnesium Sulfate (MgSO ).
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3.2.1 anaerobe, n—an organism that cannot grow or prolif-
6.1.18 Taurocholic Acid Sodium Salt Hydrate.
erate in the presence of free oxygen.
6.1.19 L-Cysteine.
3.2.2 enzymatic treatment
...