ASTM E2783-10

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Designation:E2783–10
Standard Test Method for
Assessment of Antimicrobial Activity for Water Miscible
Compounds Using a Time-Kill Procedure
This standard is issued under the fixed designation E2783; 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 An-
timicrobial Agents
1.1 This test method measures the changes of a population
E2315 Guide for Assessment of Antimicrobial Activity
of aerobic and anaerobic microorganisms within a specific
Using a Time-Kill Procedure
sampling time when tested against antimicrobial test materials
in vitro. The organisms used are standardized as to growth
3. Terminology
requirements and inoculum preparation and must grow under
3.1 Definitions:
the conditions of the test. The primary purpose of this test
3.1.1 antimicrobial, n—describes an agent that kills or
method is to provide a set of standardized conditions and test
inactivates microorganisms or suppresses their growth or
organisms to facilitate comparative assessments of antimicro-
reproduction.
bial materials miscible in aqueous systems.
3.1.2 drug, n—articles intended for use in the diagnosis,
1.2 Thistestmethodallowstheoptionofusingatestsample
cure, mitigation, treatment, or prevention of disease in man or
size of 10 mL or 100 mL.
other animals. Drugs are intended to affect the structure or any
1.3 Knowledge of microbiological techniques is required
function of the body of man or other animals.
for this procedure.
3.1.3 initial microbial population, n—bacterial count (CFU/
1.4 Aseptic technique should be practiced at all times.
mL) in the final volume of test material.Also known as initial
1.5 In this test method, SI units are used for all applications,
bacterial population, numbers control or control.
except for distance in which case inches are used and SI units
3.1.4 inoculum, n—the viable microorganisms used to con-
follow in parentheses.
taminate a sample, device or surface, often expressed as to
1.6 This standard does not purport to address all of the
number and type.
safety concerns, if any, associated with its use. It is the
3.1.5 microbiocide, n—a physical or chemical agent that
responsibility of the user of this standard to establish appro-
kills microorganisms.
priate safety and health practices and determine the applica-
3.1.6 neutralization, n—the process for inactivating or
bility of regulatory limitations prior to use.
quenching the activity of a microbiocide. Often achieved
2. Referenced Documents through chemical or physical means (for example, filtration or
2 dilution).
2.1 ASTM Standards:
3.1.7 room temperature, n—temperature in the range of 20
E691 Practice for Conducting an Interlaboratory Study to
to 30°C (68 to 85°F).
Determine the Precision of a Test Method
3.1.8 test material, n—a formulation which incorporates
antimicrobial ingredient(s). Also known as test formulation.
3.1.9 total test volume, n—the volume of test material plus
This test method is under the jurisdiction of ASTM Committee E35 on
the volume of inoculum suspension.
Pesticides and Alternative Control Agents and is the direct responsibility of
Subcommittee E35.15 on Antimicrobial Agents.
4. Summary of Test Method
Current edition approved Oct. 1, 2010. Published November 2010. DOI:
10.1520/E2783–10.
4.1 A dilution/aliquot of the test material is brought into
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contactwithaknownpopulationoftestorganismsforspecified
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
periods of time, at a specified temperature. The activity of the
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. test material is quenched at specified sampling intervals
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
E2783–10
(example 15, 30, and 60 s, or any range covering several 7.4 Dilution Fluid or Diluent—SterileButterfield’sbuffered
minutes or hours) with an appropriate neutralizing technique. phosphate diluent or other suitable diluent with appropriately
The test material is neutralized at the sampling time and the validated neutralizers. Perform Test Method E1054 to deter-
surviving microorganisms enumerated. The percent and log mine what diluent or neutralizers are required. Volume is 9.0
reduction, from an initial microbial population is calculated. mL after sterilization.
7.5 Flip Top Centrifuge Tubes—Sterile. For 10 mL sample
size. Minimum of 50 mL capacity.
5. Significance and Use
5.1 This procedure may be used to assess the in vitro
NOTE 2—Pre-sterilized/ disposable flip-top centrifuge tubes are avail-
reduction of a microbial population of test organisms after able from most laboratory supply houses.
exposure to a test material.
7.6 Petri Dishes—100by15mm.Requiredtoplatesamples
and control.
6. Apparatus
NOTE 3—Pre-sterilized/disposable plastic petri dishes are available
6.1 Adjustable or Fixed Volume Pipet—Capable of dispens-
from most local laboratory supply houses.
ing 0.1 mL and 1.0 mL.
7.7 Physiological Saline—Sterile. Used to prepare inocu-
6.2 Anaerobic Jar or Incubator—Any incubator or appara-
lum.
tus in an incubator that creates an environment having a level
7.8 Pipet Tips—Sterile. 0.1 and 1.0 mL capacity.
ofoxygenthatdoesnotsupportthegrowthofoxygen-requiring
7.9 Positive Displacement Pipet Tips—Sterile. 1.0 mL ca-
microorganisms. Required only for organisms that need an
pacity.
anaerobic environment to grow.
7.10 Solid Growth and Plating Medium—Sterile soybean-
6.3 Balance—Any suitable laboratory balance with a mini-
casein digest agar (tryptic soy agar) or other solid media
mum readability of 0.01 g.
appropriate to support growth of the test organism(s) with
6.4 Beakers and Magnetic Stir Bars—For 100 mL sample
appropriately validated neutralizers. Perform Test Method
size.A250 mLbeaker containing a 51 by 8 6 2 mm magnetic
E1054 to determine what neutralizers are required. Should be
stir bar. The beaker and stir bar should be sterile.
tempered to between 40 to 50°C if using a pour plate method.
6.5 Colony Counter—Any of several types may be used.
7.11 Water—Sterile deionized or distilled water.
6.6 Incubator—Any incubator capable of maintaining a
suitable temperature 62ºC may be used.
8. Hazards
6.7 Laboratory Centrifuge—Any centrifuge capable of pro-
8.1 Alltestorganismslistedforuseinthismethodfallunder
ducing 3200 r/min (1520 RCF).
the Biosafety level 1 or 2 categories and should be handled in
6.8 Magnetic Stirring Plate—Any rotor powered magnetic
accordance with CDC and NIH guidelines.
stirrer.
6.9 Positive Displacement Pipet—1.0 mL capacity. Re-
9. Calibration and Standardization
quired for viscous test materials.
9.1 Ensure that all equipment used in this test method has
6.10 Sterilizer—Any suitable steam sterilizer capable of
been calibrated and standardized as required for that piece of
producing the conditions of sterility.
equipment.
6.11 Sterile Container—Any container of sufficient size to
dilute test material into.
10. Test Organisms
6.12 Test Tubes with Cap—Sterile. Alternate sample con-
10.1 The following list of organisms may be used in this
tainer to (7.5) for 10 mL sample size. Size of test tube should
procedure. This list is not all inclusive and other organisms
allow the thorough mixing of samples.
may be used. Organisms selected may be representative of the
6.13 Timer (Stop Clock)—One that displays minutes and
microbialfloraencounteredundertheconditionsofuse,ormay
seconds.
represent standardized strains.The organism should be capable
6.14 Vortex Mixer—Any suitable vortex mixer capable of
of providing reproducible results under the test conditions.
mixing test material and diluents.
10.2 The organisms listed shall be maintained as specified
6.15 Waterbath—Any waterbath capable of maintaining
by ATCC or other validated methods.
suitable temperature.
10.2.1 Acinetobacter species.
10.2.2 Candida albicans ATCC 10231.
7. Reagents and Materials
10.2.3 Enterobacter species.
7.1 Bacteriological Loops—Any type of disposable sterile
10.2.4 Enterococcus faecalis ATCC 29212.
or sterilizable bacteriological loop is suitable.
10.2.5 Enterococcus faecium.
7.2 Bacteriological Pipets—Sterile. 1.1, 2.0, 5.0, 10.0 mL
10.2.6 Escherichia coli ATCC 8739, 11229, or 25922.
capacity.
NOTE 1—Pre-sterilized/disposable bacteriological pipets are available
Horowitz, W., (Ed.), Offıcial Methods of Analysis of the AOAC, 17th Ed., Sec.
from most local laboratory supply houses.
6.3.03 A.(f), Chapter 6, 2000, p.10. Official Methods of Analysis of AOAC
International, Gaithersburg, MD.
7.3 Broth Growth Medium—Sterile soybean-casein digest
Richmond, J. Y. and McKinney, R. W. (eds.), 1999, Biosafety in Microbio-
broth (tryptic soy broth) or other broth media appropriate to
logical and Biomedical Laboratories, 4th ed., Washington DC, U.S. Government
support growth of the test organisms. Printing Office.
E2783–10
10.2.7 Klebsiella pneumoniae ATCC 10031 or 51504. 11.8.1 The stock culture, frozen, or lypholized should be at
10.2.8 Micrococcus luteus ATCC 7468. least one 24 h liquid growth media (7.3) transfer from the
original source.
10.2.9 Pseudomonas aeruginosa ATCC 9027, 15442, or
27853.
11.8.2 Incubate at appropriate temperature and environment
10.2.10 Proteus mirabilis ATCC 4675 or 7002.
for the organism.
10.2.11 Salmonella enterica ATCC 10708.
11.8.3 Centrifuge at conditions appropriate to sediment the
10.2.12 Serratia marcescens ATCC 14756.
culture completely.
10.2.13 Shigella species.
11.8.4 Decant or pipet supernatant and re-suspend organism
10.2.14 Staphylococcus aureus ATCC 6538, 29213, 33591,
pellet in 20 mL physiological saline (7.7).
or 33592.
11.8.5 Centrifuge at conditions appropriate to sediment the
10.2.15 Staphylococcus epidermidis ATCC 12228.
culture completely.
10.2.16 Staphylococcus haemolyticus.
11.8.6 Decant or pipet supernatant and re-suspend organism
10.2.17 Staphylococcus hominis.
in an amount of physiological saline (7.7) sufficient to achieve
10.2.18 Staphylococcus saprophyticus.
a minimum final suspension of 1.0 3 10 CFU/mL. Use
10.2.19 Streptococcus pyogenes.
McFarland Barium Sulfate Standard #2, turbidimetry, optical
10.2.20 Streptococcus pneumoniae.
density, or other technique that correlates to an aerobic plate
count.
11. Preparation of Organism
NOTE 4—Antimicrobials sensitive to organic material (for example
11.1 Allorganismsusedinthetestmethodshallbeprepared
alcohol and iodine) may have reduced activity by even the slightest
using a validated method. The same method shall be used to
organic load and therefore only use thoroughly washed inoculum suspen-
prepare all organisms for the test.
sions, whether initially grown in broth or from solid media.
11.2 A minimum starting inoculum level of 1.0 3 10
CFU/mL should be used for the test.
12. Test Conditions
11.3 Other starting inoculum levels may be used depending
12.1 Test should be performed at room temperature.
on the organisms growth potential.
12.2 Test materials and diluents should be at room tempera-
11.4 All organisms shall be no more than five passages
ture.
removed from the original source.
12.3 Test materials may require a lower or higher tempera-
11.5 The inoculum shall be used within4hof preparation.
ture than room temperature (for example, solids that require
11.6 If a validated method is not available, the following
warming to and be held at 45°C, or test material may only be
instructions shall be used to prepare the inoculum.
stable at a certain temperature). If this is a requirement, all
11.7 Inoculum Preparation Directly from Agar:
steps of the test should be run at that temperature.
11.7.1 The stock culture, frozen or lypholized should be at
12.4 This test method allows the use of either a 10 or 100
least one 24 h broth growth media (7.3) transfer from the
mL sample size. The test shall be run using the same sample
original source.
size for all test materials.
11.7.2 Inoculate a sufficient number of solid growth media
slants or plates (7.10).
13. Test (Contact) Times
11.7.3 Incubate at appropriate temperature and environment
for the organism.
13.1 For selection of contact times, a minimum time period
11.7.4 Wash each slant by adding 5 to 10 mLof physiologi-
should be selected based on the ability to reproduce the test
cal saline (7.7) to each slant.
sampling in this short time frame (for example 15, 30 and 60
11.7.5 Using bacteriological loop (7.1), gently scrape or rub
s).
surface of agar to remove growth.
13.2 Time points should reflect the intended use of the test
11.7.6 Using a sterile pipet, collect the washings in a 50 mL
material.
centrifuge tube (7.5). Repeat for all slants, adding washing to
13.3 Times may be chosen to construct a kinetic kill model.
the centrifuge tube.
11.7.7 Centrifuge at conditions appropriate to sediment the
14. Test Material Concentration
culture completely.
14.1 Select the test concentrations of the test material. The
11.7.8 Decant or pipet supernatant and re-suspend organism
concentrations selected may reflect the anticipated concentra-
pellet in 20 mL physiological saline (7.7).
tionofthetestmaterialduringuse.Eachconcentrationistested
11.7.9 Centrifuge at conditions appropriate to sediment the
in triplicate.
culture completely.
11.7.10 Decant or pipet supernatant and re-suspend organ- 14.2 If test material is to be diluted, dilute using sterile
ism in an amount of physiological saline (7.7) sufficient to
water (7.11). Dilution using other materials, such as sterile
achieve a minimum final suspension of 1.0 3 10 CFU/mL. saline or sterile buffer may be appropriate if test material is
Use McFarland Barium Sulfate Standard #2, turbidimetry,
typically diluted that way under conditions of use. Dilute all
optical density, or other technique that correlates to an aerobic test material needed at once. Use sterile container (6.11). Mix
plate count.
thoroughly.Test material can be mixed using a sterile magnetic
11.8 Inoculum Prepared Directly from Broth: stir bar and a magnetic stir plate.
E2783–10
15. Inoculum (Start) Count 16.3.6 Prepare serial ten-fold dilutions of each time point.
16.3.7 Enumerate, in duplicate, using standard plating tech-
15.1 To be done at the start and end of the test phase. The
niques. Ensure that the proper dilutions are plated in order to
start and end counts must
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