ASTM E2315-03(2008)
(Guide)Standard Guide for Assessment of Antimicrobial Activity Using a Time-Kill Procedure
Standard Guide for Assessment of Antimicrobial Activity Using a Time-Kill Procedure
SIGNIFICANCE AND USE
This procedure may be used to assess the in vitro reduction of a microbial population of test organisms after exposure to a test material.
SCOPE
1.1 This guide covers examples of a basic method to measure the changes of a population of aerobic microorganisms within a specified sampling time when tested against antimicrobial test materials in vitro. Several options for organism selection and growth, inoculum preparation, sampling times and temperatures are provided. When the basic technique is performed as a specific test method it is critical when evaluating the results to ensure that such 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 under different circumstances. As an example, test results of microorganisms requiring growth supplements, or special incubation conditions, may not be directly comparable to more robust organisms under the conditions of a single procedure.
1.2 Knowledge of microbiological techniques is required for this test.
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.4 This standard may involve hazardous materials, operations and equipment. 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 requirements prior to use.
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Designation: E2315 − 03(Reapproved 2008)
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 E1054 Test Methods for Evaluation of Inactivators of Anti-
microbial Agents
1.1 This guide covers examples of a basic method to
measure the changes of a population of aerobic microorgan-
3. Terminology
isms within a specified sampling time when tested against
3.1 Definitions:
antimicrobial test materials in vitro. Several options for organ-
3.1.1 inoculum suspension, n—the initial suspension of test
ism selection and growth, inoculum preparation, sampling
organism used to inoculate the test material This may also be
timesandtemperaturesareprovided.Whenthebasictechnique
known as the organism inoculum (see 8.2).
is performed as a specific test method it is critical when
evaluating the results to ensure that such variables have been
3.1.2 microbial population, n—the microbial count (cfu/
standardized. Antimicrobial activity of specific materials, as
mL)inthefinalvolumeoftestmaterial(see9.4).Thismayalso
measured by this technique, may vary significantly depending
be known as the “initial population” or “numbers control.”
on variables selected. It is important to understand the limita-
3.1.3 neutralization, n—a process which results in the inac-
tions of in vitro tests, especially comparisons of results from
tivation or quenching of the antimicrobial activity of a test
tests performed under different circumstances.As an example,
material. This may be achieved through dilution of the test
test results of microorganisms requiring growth supplements,
material(s) or with the use of chemical agents, called
or special incubation conditions, may not be directly compa-
neutralizers, to reduce or quench the antimicrobial activity.
rable to more robust organisms under the conditions of a single
3.1.4 neutralizer, n—a procedure or chemical agent used to
procedure.
inactivate,neutralize,orquenchthemicrobiocidalpropertiesof
1.2 Knowledge of microbiological techniques is required
an antimicrobial agent.
for this test.
3.1.5 total test volume, n—the volume of test material plus
1.3 The values stated in SI units are to be regarded as
the volume of inoculum suspension.
standard. No other units of measurement are included in this
standard.
4. Summary of a Basic Test Method
1.4 This standard may involve hazardous materials, opera-
4.1 The test material or a dilution of the test material is
tions and equipment. This standard does not purport to address
brought into contact with a known population of microorgan-
all of the safety concerns, if any, associated with its use. It is
isms for a specified period of time at a specified temperature.
the responsibility of the user of this standard to establish
The activity of the test material is quenched at specified
appropriate safety and health practices and determine the
sampling intervals (for example, 30 s, 60 s, or any range
applicability of regulatory requirements prior to use.
covering several minutes or hours) with an appropriate neu-
tralization technique. The test material is neutralized at the
2. Referenced Documents
sampling time and the surviving microorganisms enumerated.
2.1 ASTM Standards:
The percent or log reduction, or both, from either an initial
D1193 Specification for Reagent Water
microbial population, or test blank is calculated.
This guide is under the jurisdiction of ASTM Committee E35 on Pesticides,
5. Significance and Use
Antimicrobials, and Alternative Control Agentsand is the direct responsibility of
Subcommittee E35.15 on Antimicrobial Agents.
5.1 This procedure may be used to assess the in vitro
Current edition approved April 1, 2008. Published May 2008. Originally
reduction of a microbial population of test organisms after
approved in 2003. Last previous edition approved in 2003 as E2315 – 03. DOI:
exposure to a test material.
10.1520/E2315-03R08.
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
6. Apparatus
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. 6.1 Sterile Vials or Test Tubes, or equivalent.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2315 − 03 (2008)
6.2 Timer (Stop-clock), one that displays minutes and sec- 8.1.2.1 Alternatively, the transfers may be made onto agar
onds. plates or slants and the inoculum suspension may be prepared
by washing the organism from the slant with an appropriate
6.3 Shaking Water Bath or Controlled Temperature
broth or diluent.
Chamber, or equivalent capable of maintaining test system at
the specified exposure temperature 6 2°C. NOTE 1—Reports in the published literature have noted differences in
microbial kill or antimicrobial resistance as a result of cell protection in
6.4 Colony Counter, any of several manual or automated
broth or as a result of washing cells. It is recommended that tests be
types may be used.
conducted with either all cells prepared in broth dilutions or with all cells
prepared by washing.
6.5 Incubator, any incubator capable of maintaining a speci-
8.2 Inoculum Suspension Preparation and Determination of
fied temperature 62°C may be used.
the Microbial Population or Numbers Control:
6.6 Sterilizer, any suitable steam sterilizer capable of pro-
8.2.1 To prepare inoculum suspension directly from broth, a
ducing the conditions of sterilization.
dilution in sterile broth (the same as that used for growth
6.7 Vortex Mixer, Magnetic Stirrer, or equivalent. medium) may be performed to reduce the concentration of the
microorganisms to the appropriate level.
6.8 Spiral Plating System, (optional).
8.2.1.1 To prepare inoculum suspension in dilute broth, a
6.9 Sterile Bacteriological Pipettes, for viscous test
1:10 dilution of the suspension into Butterfield’s buffered
materials, positive displacement pipettes or syringes may be
phosphate diluent or equivalent may be performed to reduce
necessary.
the concentration of the growth medium.
8.2.1.2 Inoculum suspensions grown from broth may be
6.10 Water Dilution Bottles, any sterilizable container hav-
dilutedtoappropriateconcentrationortheymaybecentrifuged
ing appropriate capacity and tight closures may be used.
and reconstituted in Butterfield’s buffered phosphate diluent,
broth, saline, or equivalent, to the appropriate concentration.
7. Reagents and Materials
8.2.2 Topreparetheinoculumsuspensionfromanagarplate
7.1 Dilution Fluid or Diluent, sterile water, 0.65 % saline,
or slant, wash microbial growth from the agar surface with
sterile Butterfield’s buffered phosphate diluent or equivalent.
Butterfield’s buffered phosphate diluent, saline, or equivalent.
7.2 Broth Growth Medium, soybean-casein digest broth, or
NOTE 2—Antimicrobials sensitive to organic material (for example,
equivalent and other liquid media appropriate to support
alcohol and iodine) may have reduced activity by even the slightest
growth of the test organism(s), with appropriate neutralizers, if organic load and therefore thoroughly washed inoculum suspensions only,
whether grown initially in broth or from solid media, should be used.
required (see 3.1).
8.2.3 The inoculum suspension should be prepared to
7.3 Solid Growth and Plating Medium, soybean-casein di-
4 achieve a minimum of 10 cfu/mL microbial population (see
gest agar, or equivalent, and other solid media appropriate to
9.4). Results of tests where the initial microbial populations
support growth of the test organism(s), with appropriate
differ from the test population by greater than 2log should be
neutralizers, if required (see 3.1.3 and 3.1.4).
interpreted with care because of the exponential nature of the
7.4 Sterile Deionized Water, or equivalent (Specification
populations. The final inoculum suspension should be well
D1193, Type III).
mixed prior to addition to test materials (see 9.5).
8.2.4 The inoculum suspension should be enumerated in
8. Test Methods
duplicate by standard microbiological procedures at the initia-
tion and completion of testing. Appropriate dilutions are
8.1 Test Organisms:
prepared and enumerated by standard microbiological proce-
8.1.1 The test organisms selected may be representative of
dures (Spread or pour plating, microbial filtration, or spiral
the microbial flora encountered under the conditions of use, or
plating). The initial and final count of the inoculum should be
may represent standardized strains. The organism should be
within 60.5 log for a valid test.
capable of providing reproducible results under specific test
8.2.4.1 Toperformthepopulationquantitationofthecontrol
conditions.
blank, a volume of inoculum suspension equivalent to that
8.1.2 Organism Preparation—Transfer culture(s) from
inoculated into the test material is added to a dilution blank
stock twice (once every 18 to 24 h or as appropriate for the test
containing the same volume as used for the test material. The
organism) into appropriate growth media. The second transfer
initial and final count of the population in the blank must be
may be made into a volume of growth medium to produce
within 60.5 log for a valid test.
sufficient microbial suspension to inoculate. Volumes used
8.2.5 Incubate plates at the specified temperature 62°C for
should permit testing of multiple samples or time points.
24 to 48 h or as appropriate for the test organism(s).
8.2.6 Count colonies and record raw data as cfu/plate to
determine surviving organisms. Average duplicate plates (2
Horowitz, W., Ed., Offıcial Methods of Analysis of the AOAC, 17th Ed., ch. 17,
p. 4, sec. 17.2.01, A(m), Association of Official Analytical Chemists, Washington,
DC, 2000; (As cited in, Butterfield’s Phosphate Buffer, Journal of theAssociation of
Official Analytical Chemists. Vol 22, No. 635, 1939.) Brown, M. R. W., Gilbert P., Microbiological Quality Assurance: A Guide
U.S. Pharmacopoeia, 24th Revision, The United States Pharmacopoeia Towards Relevance and Reproducibility of Inocula, CRC Press, New York, NY,
Convention, Inc. Rockville, MD, 2000. 1995.
E2315 − 03 (2008)
platesfromeachreplicatedilution)andmultiplybythedilution plating, microbial filtration, spiral plating, or other valid
factor to calculate (cfu/mL) microbial population of both the microbial recovery methods. See Fig. 1.
control blank and test system.
9.8 Incubateplatesatthespecifiedtemperature 62°Cfor24
to48horasappropriateforeachorganismselected.Incubation
9. Basic Procedure
time should allow for growth of surviving organisms, without
overgrowth
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