ASTM E2315-03
(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
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 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.
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
Standard Guide for
Assessment of Antimicrobial Activity Using a Time-Kill
Procedure
This standard is issued under the fixed designation E 2315; 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 (e) indicates an editorial change since the last revision or reapproval.
1. Scope 3. Terminology
1.1 This guide covers examples of a basic method to 3.1 Definitions:
measure the changes of a population of aerobic microorgan- 3.1.1 inoculum suspension, n—the initial suspension of test
isms within a specified sampling time when tested against organism used to inoculate the test material This may also be
antimicrobial test materials in vitro. Several options for organ- known as the organism inoculum (see 8.2).
ism selection and growth, inoculum preparation, sampling 3.1.2 microbial population, n—the microbial count (cfu/
timesandtemperaturesareprovided.Whenthebasictechnique mL)inthefinalvolumeoftestmaterial(see9.4).Thismayalso
is performed as a specific test method it is critical when be known as the “initial population” or “numbers control.”
evaluating the results to ensure that such variables have been 3.1.3 neutralization, n—a process which results in the
standardized. Antimicrobial activity of specific materials, as inactivation or quenching of the antimicrobial activity of a test
measured by this technique, may vary significantly depending material. This may be achieved through dilution of the test
on variables selected. It is important to understand the limita- material(s) or with the use of chemical agents, called neutral-
tions of in vitro tests, especially comparisons of results from izers, to reduce or quench the antimicrobial activity.
tests performed under different circumstances. As an example, 3.1.4 neutralizer, n—a procedure or chemical agent used to
test results of microorganisms requiring growth supplements, inactivate,neutralize,orquenchthemicrobiocidalpropertiesof
or special incubation conditions, may not be directly compa- an antimicrobial agent.
rable to more robust organisms under the conditions of a single 3.1.5 total test volume, n—the volume of test material plus
procedure. the volume of inoculum suspension.
1.2 Knowledge of microbiological techniques is required
4. Summary of a Basic Test Method
for this test.
1.3 The values stated in SI units are to be regarded as 4.1 The test material or a dilution of the test material is
brought into contact with a known population of microorgan-
standard.
1.4 This standard may involve hazardous materials, opera- isms for a specified period of time at a specified temperature.
The activity of the test material is quenched at specified
tions and equipment. This standard does not purport to address
all of the safety concerns, if any, associated with its use. It is sampling intervals (for example, 30 s, 60 s, or any range
covering several minutes or hours) with an appropriate neu-
the responsibility of the user of this standard to establish
appropriate safety and health practices and determine the tralization technique. The test material is neutralized at the
sampling time and the surviving microorganisms enumerated.
applicability of regulatory requirements prior to use.
The percent or log reduction, or both, from either an initial
2. Referenced Documents
microbial population, or test blank is calculated.
2.1 ASTM Standards:
5. Significance and Use
D 1193 Specification for Reagent Water
5.1 This procedure may be used to assess the in vitro
E 1054 Practices for Evaluating Inactivators of Antimicro-
bial Agents Used in Disinfectant, Sanitizer, Antiseptic or reduction of a microbial population of test organisms after
exposure to a test material.
Preserved Products
6. Apparatus
This guide is under the jurisdiction ofASTM Committee E35 on Pesticides and
6.1 Sterile Vials or Test Tubes, or equivalent.
Alternative ControlAgents and is the direct responsibility of Subcommittee E35.15
6.2 Timer (Stop-clock), one that displays minutes and sec-
on Antimicrobial Agents.
onds.
Current edition approved Oct. 1, 2003. Published November 2003.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
E2315–03
conducted with either all cells prepared in broth dilutions or with all cells
6.3 Shaking Water Bath or Controlled Temperature Cham-
prepared by washing.
ber, or equivalent capable of maintaining test system at the
specified exposure temperature 6 2°C.
8.2 Inoculum Suspension Preparation and Determination of
6.4 Colony Counter, any of several manual or automated 4
the Microbial Population or Numbers Control:
types may be used.
8.2.1 To prepare inoculum suspension directly from broth, a
6.5 Incubator, any incubator capable of maintaining a
dilution in sterile broth (the same as that used for growth
specified temperature 6 2°C may be used.
medium) may be performed to reduce the concentration of the
6.6 Sterilizer, any suitable steam sterilizer capable of pro-
microorganisms to the appropriate level.
ducing the conditions of sterilization.
8.2.1.1 To prepare inoculum suspension in dilute broth, a
6.7 Vortex Mixer, Magnetic Stirrer, or equivalent.
1:10 dilution of the suspension into Butterfield’s buffered
6.8 Spiral Plating System, (optional).
phosphate diluent or equivalent may be performed to reduce
6.9 Sterile Bacteriological Pipettes, for viscous test materi-
the concentration of the growth medium.
als, positive displacement pipettes or syringes may be neces-
sary.
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,
required (see 3.1).
whether grown initially in broth or from solid media, should be used.
7.3 Solid Growth and Plating Medium, soybean-casein di-
gest agar, or equivalent, and other solid media appropriate to
8.2.3 The inoculum suspension should be prepared to
support growth of the test organism(s), with appropriate
achieve a minimum of 10 cfu/mL microbial population (see
neutralizers, if required (see 3.1.3 and 3.1.4).
9.4). Results of tests where the initial microbial populations
7.4 Sterile Deionized Water, or equivalent (Specification
differ from the test population by greater than 2log should be
D 1193, Type III).
interpreted with care because of the exponential nature of the
populations. The final inoculum suspension should be well
8. Test Methods
mixed prior to addition to test materials (see 9.5).
8.1 Test Organisms:
8.2.4 The inoculum suspension should be enumerated in
8.1.1 The test organisms selected may be representative of
duplicate by standard microbiological procedures at the initia-
the microbial flora encountered under the conditions of use, or
tion and completion of testing. Appropriate dilutions are
may represent standardized strains. The organism should be
prepared and enumerated by standard microbiological proce-
capable of providing reproducible results under specific test
dures (Spread or pour plating, microbial filtration, or spiral
conditions.
plating). The initial and final count of the inoculum should be
8.1.2 Organism Preparation—Transfer culture(s) from
within 6 0.5 log for a valid test.
stock twice (once every 18 to 24 h or as appropriate for the test
8.2.4.1 Toperformthepopulationquantitationofthecontrol
organism) into appropriate growth media. The second transfer
blank, a volume of inoculum suspension equivalent to that
may be made into a volume of growth medium to produce
inoculated into the test material is added to a dilution blank
sufficient microbial suspension to inoculate. Volumes used
containing the same volume as used for the test material. The
should permit testing of multiple samples or time points.
initial and final count of the population in the blank must be
8.1.2.1 Alternatively, the transfers may be made onto agar
within 6 0.5 log for a valid test.
plates or slants and the inoculum suspension may be prepared
8.2.5 Incubate plates at the specified temperature 6 2°C for
by washing the organism from the slant with an appropriate
24 to 48 h or as appropriate for the test organism(s).
broth or diluent.
8.2.6 Count colonies and record raw data as cfu/plate to
NOTE 1—Reports in the published literature have noted differences in
determine surviving organisms. Average duplicate plates (2
microbial kill or antimicrobial resistance as a result of cell protection in
broth or as a result of washing cells. It is recommended that tests be platesfromeachreplicatedilution)andmultiplybythedilution
factor to calculate (cfu/mL) microbial population of both the
control blank and test system.
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 Con- Towards Relevance and Reproducibility of Inocula, CRC Press, New York, NY,
vention, Inc. Rockville, MD, 2000. 1995.
E2315–03
9. Basic Procedure time should allow for growth of surviving organisms, without
overgrowth of colonies, making enumeration difficult.
9.1 Select the test concentrations of the test material. The
9.9 To determine surviving organisms, count colonies and
concentrations selected may reflect the anticipated concentra-
record raw data as cfu/plate.Average duplicate plates (2 plates
tionofthetestmaterial
...
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