ASTM E2315-23

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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: E2315 − 16 E2315 − 23
Standard Guide for
Assessment of Antimicrobial Activity Using a Time-Kill
Procedure
This standard is issued under the fixed designation E2315; 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 guide covers an example of a method that measures the changes in a population of aerobic microorganisms within a
specified sampling time when antimicrobial test materials are present. Several options for organism selection and growth, inoculum
preparation, sampling times and temperatures are provided. When the technique is performed as a specific test method, it is critical
that the above mentioned variables have been standardized. Antimicrobial activity of specific materials, as measured by this
technique, may vary significantly depending on variables selected. It is important to understand the limitations of in vitro tests,
especially comparisons of results from tests performed with different parameters. As an example, test results of microorganisms
requiring growth supplements or special incubation conditions may not be directly comparable to organisms evaluated without
those stated conditions.
1.1.1 Several options for organism selection and growth, inoculum preparation, sampling times and temperatures are provided.
1.1.2 When the technique is performed as a specific test method, it is critical that the above mentioned variables have been
standardized.
1.1.3 Antimicrobial activity of specific materials, as measured by this technique, can vary significantly depending on variables
selected.
1.1.4 Test Method E2783 may be referenced as an example of using fixed conditions and set variables to evaluate antimicrobial
efficacy of water-miscible compounds.
1.1.5 This guide serves as a general teaching document for evaluating the antimicrobial activity using a variety of conditions to
offer the flexibility needed in test conditions to cover a broad range of microorganisms and test substances.
1.1.6 It is important to understand the limitations of in vitro tests, especially comparisons of results from tests performed with
different parameters. As an example, test results of microorganisms requiring growth supplements or special incubation conditions
may not be directly comparable to organisms evaluated without those stated conditions.
1.2 Knowledge of microbiological techniques is required for this procedure.
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
This guide 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 Jan. 15, 2016April 1, 2023. Published March 2016May 2023. Originally approved in 2003. Last previous edition approved in 20082016 as
E2315 – 03E2315 – 16.(2008). DOI: 10.1520/E2315-16.10.1520/E2315-23.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2315 − 23
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 healthsafety, health, and environmental practices and determine
the applicability of regulatory requirementslimitations 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
2.1 ASTM Standards:
D1193 Specification for Reagent Water
E1054 Practices for Evaluation of Inactivators of Antimicrobial Agents
E2756 Terminology Relating to Antimicrobial and Antiviral Agents
E2783 Test Method for Assessment of Antimicrobial Activity for Water Miscible Compounds Using a Time-Kill Procedure
3. Terminology
3.1 Definitions:
3.1.1 For definitions of standard terms relating to antimicrobial agents used in this guide, refer to Terminology E2756.
3.2 Definitions:Definitions of Terms Specific to This Standard:
3.2.1 inoculum suspension, n—the initial suspension of test organism used to inoculate the test material. This may also be known
as the organism inoculum (see 8.3).
3.2.2 microbial population, n—the microbial count (cfu/mL) in the final volume of test material (see 9.4). This may also be known
as the “numbers control.” The measurement may be taken at time zero which may be termed “Initial Population.” Alternatively,
the measurement may be taken at each exposure time or the longest exposure time used during testing to simulate the test procedure
which may be termed “Final Population.”
3.2.3 neutralization, n—the process for inactivating or quenching the activity of a test material. This may be achieved through
physical means (for example, filtration, dilution) and/or the addition of chemical agents, called neutralizers.
3.2.4 neutralizer, n—a chemical agent used to inactivate, neutralize, or quench the microbicidal properties of an antimicrobial
agent.
3.2.5 total test volume, n—the volume of test material plus the volume of inoculum suspension.
4. Summary of a Basic Test Method
4.1 The test material or a dilution of the test material is brought into contact with a known population of microorganisms for a
specified period of time at a specified temperature. An appropriate and specified neutralization technique is applied to quench the
antimicrobial activity of the test material at specified sampling intervals (for example, 30 s, 60 s, or any range covering several
minutes or hours), and the surviving microorganisms are enumerated. The percent and/or log reduction is calculated by
comparison with the microbial population.
5. Significance and Use
5.1 This procedure may be used to assess the in vitro reduction of a microbial population of test organisms after exposure to a
test material.
6. Apparatus
6.1 Sterile Vials or Test Tubes, or equivalent.
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.
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6.2 Timer (Stop-clock) that displays minutes and seconds.
6.3 Water Bath, Controlled Temperature Chamber, or equivalent capable of maintaining test system at the specified exposure
temperature 62°C.62 °C.
6.4 Colony Counter, any of several manual or automated types may be used.
6.5 Incubator, any capable of maintaining a specified temperature 62°C62 °C may be used.
6.6 Sterilizer, any steam sterilizer capable of producing the conditions of sterilization.
6.7 Vortex Mixer, Magnetic Stirrer, or equivalent.
6.8 Spiral Plating System, (optional).
6.9 Sterile Bacteriological Pipettes, for viscous test materials, positive displacement pipettes or syringes may be necessary.
6.10 Water Dilution Bottles, any sterilizable container having appropriate capacity and tight closures may be used.
6.11 Sterile Cotton Applicator Swabs.
7. Reagents and Materials
7.1 Dilution Fluid or Diluent—sterile water, 0.9 % (w/v) saline, sterile Butterfield’s buffered phosphate diluent, or equivalent.
7.2 Broth Growth Medium—soybean-casein digest broth or equivalent liquid media appropriate to supporting growth of the test
organism(s), with appropriate neutralizers, if required (see 3.13.2).
7.3 Solid Growth and Plating Medium—soybean-casein digest agar or equivalent solid media appropriate to support growth of
the test organism(s), with appropriate neutralizers, if required (see 3.1.33.2.3 and 3.1.43.2.4).
7.4 Sterile Deionized Water, or equivalent (Specification D1193, Type III).
8. Test Organism Preparation
8.1 The test organism selected may be representative of the microbial flora encountered under the conditions of use of a test
material or may be standardized strains.
8.2 Organism Preparation—Transfer culture(s) from stock twice (once every 18 to 24 h or as appropriate for the test organism)
into appropriate growth medium to maintain the organism in growth phase. The second transfer may be made into a volume of
growth medium that will provide a microbial suspension sufficient to conduct testing and controls. Consider that additional volume
may be needed to permit testing of multiple samples or time points.
8.2.1 Alternatively, the transfers may be made onto agar plates or slants, and the inoculum suspension prepared by washing the
organism from the slant with an appropriate broth or diluent.
NOTE 1—Reports in the published literature have noted differences in microbial kill or susceptibility as a result of different propagation methods. It is
recommended that tests be conducted using a consistent procedure for organism propagation.
Horowitz, W., Ed., Offıcial Methods of Analysis of the AOAC, 18th Ed., Association of Official Analytical Chemists, Washington, DC, 2000; Journal of the Association
of Official Analytical Chemists. Vol 22, No. 635, 1939.
U.S. Pharmacopeia, 38–NF33, The United States Pharmacopeial Convention, Inc. Rockville, MD, 2000.
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8.3 Inoculum Suspension Preparation and Determination of the Microbial Population:
8.3.1 To prepare inoculum suspension directly from broth growth medium, a dilution in sterile broth (diluent is same as that used
for broth growth medium) may be performed to achieve the desired concentration.
8.3.2 To prepare inoculum suspension in dilute broth, an up to 1:10 dilution of the suspension into Butterfield’s buffered phosphate
diluent or equivalent may be performed to reduce the concentration of the growth medium.
8.3.3 Inoculum suspensions in broth may be diluted or concentrated to achieve the desired concentration or they may be
centrifuged and reconstituted in Butterfield’s buffered phosphate diluent, broth, saline, or equivalent, to achieve the desired
concentration.
8.3.4 To prepare the inoculum suspension from an agar plate or slant, wash microbial growth or transfer the growth aseptically
using a sterile swab from the agar surface with Butterfield’s buffered phosphate diluent, saline, or equivalent.
NOTE 2—Because certain antimicrobials (for example, alcohol and iodine) are sensitive to organic material and may have activity reduced by even the
slightest organic load, washed inoculum suspensions, whether established initially in broth or from solid media, may be used.
8.3.5 The inoculum suspension should be prepared to achieve a minimum population concentration of 10 cfu/mL (see 9.4). The
final inoculum suspension should be well-mixed prior to transfer to test material (see 9.5).
8.3.6 The inoculum suspension should be plated in duplicate by standard microbiological procedures at the initiation and
completion of testing. Appropriate dilutions should be prepared and enumerated by standard microbiological procedures (spread-
or pour-plating, microbial filtration, or spiral-plating). The initial and final titer of the inoculum should be within 60.5 log for
a valid test. This step may be omitted where Microbial Population enumeration is conducted.
8.4 To perform the Microbial Population (3.1.23.2.2) quantitation, a volume of inoculum suspension equivalent to that inoculated
into the test material is added to a dilution blank containing the same volume as used for the test material. To simulate the test
recovery fluid, the inoculated dilution blank is serially diluted and neutralized in the same manner as the test material. The Initial
Population and Final Population counts must be within 60.5 log for a valid test.
NOTE 3—Depending on the microorganism being tested and the duration of the experiment, verifying the Final Population may not be necessary. It is
recommended to confirm that there is not a loss of viability over the amount of time required for a given experiment (for example, 1 to 2 h) for a particular
organism, but not necessary to perform each time for repeat experiment using the same conditions.
8.4.1 Incubate plates at the specified t
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