ASTM E2839-11
(Test Method)Standard Test Method for Production of Clostridium difficile Spores for Use in Efficacy Evaluation of Antimicrobial Agents
Standard Test Method for Production of <i>Clostridium difficile</i> Spores for Use in Efficacy Evaluation of Antimicrobial Agents
SIGNIFICANCE AND USE
This test method describes a procedure for preparing a spore suspension of C. difficile strain ATCC 43598 that meets specific criteria necessary for efficacy testing of antimicrobials designed to eliminate C. difficile contamination from environmental surfaces. The acceptability criteria for the spore suspension are: (1) a viability titer of >8 log10/mL, (2) purity of ≥95 %, and (3) that spores be resistant to 10 min of exposure to 2.5 M HCl.
SCOPE
1.1 This test method is for producing C. difficile spores to evaluate antimicrobial formulations for their sporicidal activity.
1.2 It is the responsibility of the investigator to determine whether Good Laboratory Practices (GLP) are required and to follow them when appropriate.
1.3 This standard may involve hazardous materials, chemicals, and microorganisms and should be performed only by persons with formal training in microbiology.
1.4 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.
General Information
Relations
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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.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: E2839 − 11
Standard Test Method for
Production of Clostridium difficile Spores 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.
INTRODUCTION
SporulationinClostridiumdiffıcileisnotasrapidorasefficientasinotherspeciesanditisgenerally
2
difficult to produce C. diffıcile spores of high titer in the laboratory (1, 2). Although quantitative test
methods are available for testing sporicidal products, a standardized method for generating spore
suspensions of C. diffıcile of high titer (>8 log /mL) and purity (≥95% spores) is not available and
10
wouldbenecessaryinordertoconductperformancetestingrequiredforregistrationpurposes (3).The
spore suspensions resulting from practice of this test method are appropriate for use in accepted test
methods for measuring the sporicidal efficacy of antimicrobial formulations (4).
1. Scope medium) in spread plates, as an indication of the total number
of viable spores/microorganisms in a sample.
1.1 This test method is for producing C. diffıcile spores to
2.1.2 QC, adj/n—quality control (QC) is the application of
evaluate antimicrobial formulations for their sporicidal activ-
procedures, products, or services to meet a laboratory’s speci-
ity.
fied standards of quality.
1.2 It is the responsibility of the investigator to determine
2.1.3 pre-reduced medium, adj/n—an agar or broth manu-
whether Good Laboratory Practices (GLP) are required and to
factured and sterilized in an oxygen-free environment, and
follow them when appropriate.
packaged individually in air-tight sealed pouches or bags.
1.3 This standard may involve hazardous materials,
2.1.4 density gradient medium, adj/n—HistoDenz (trade-
chemicals, and microorganisms and should be performed only
3
marked) is a non-ionic gradient medium used here to separate
by persons with formal training in microbiology.
spores from vegetative cells and cell fragments on the basis of
1.4 The values stated in SI units are to be regarded as
density.
standard. No other units of measurement are included in this
2.1.5 purified spores, adj/n—when spore concentration
standard.
reaches ≥95% as vegetative cells and cell fragments are
1.5 This standard does not purport to address all of the
separated by the density gradient medium.
safety concerns, if any, associated with its use. It is the
2.1.6 toxigenic strain, adj/n—possesses either toxinAgene
responsibility of the user of this standard to establish appro-
(tcdA+) or toxin B gene (tcdB+) or both.
priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use.
3. Summary of Test Method
3.1 This test method provides detailed instructions for the
2. Terminology
culture,maintenanceandsporulationofC.diffıcileonaspecific
2.1 Definitions:
agarmediumincubatedinananaerobicenvironmentfor7to10
2.1.1 CFU, adj/n—colony-forming units; the number of
days. Monitoring is performed by phase-contrast microscopy
spores or microorganisms that can form colonies (clusters of
to ensure sporulation is underway and to determine when the
microorganisms visibly growing on the surface of a solid agar
spore concentration reaches ≥90%, the optimal time of har-
vest. Upon harvesting, spores are washed several times with
1
This test method is under the jurisdiction of ASTM Committee E35 on
Pesticides, Antimicrobials, and Alternative Control Agents and is the direct
3
responsibility of Subcommittee E35.15 on Antimicrobial Agents. The sole source of supply of HistoDenz (trademark) (Cat. No. D2158) known
Current edition approved Aug. 1, 2011. Published September 2011. DOI: to the committee at this time is Sigma-Aldrich, St. Louis, MO. If you are aware of
10.1520/E2839-11. alternative suppliers, please provide this information to ASTM International
2
The boldface numbers in parentheses refer to a list of references at the end of Headquarters.Your comments will receive careful consideration at a meeting of the
1
this standard. responsible technical committee, which you may attend.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1
---------------------- Page: 1 ----------------------
E2839 − 11
saline-Tween 80, treated with heat to inactivate any remaining 5.16 Microcentrifuge tubes—Sterile 1.5-mL low-retention
viable vegetative cells, and purified using a density gradient (siliconized) microcentrifuge tubes.
medium to remove inactivated vegetative cells and cell frag-
5.17 Cryovials—Sterile 2.0
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.