ASTM E2783-22

Standard Test Method for Assessment of Antimicrobial Activity for Water Miscible Compounds Using a Time-Kill Procedure

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.
SCOPE
1.1 This test method measures the changes of a population of aerobic and anaerobic microorganisms within a specific sampling time when tested against antimicrobial test materials in vitro. The organisms used are standardized as to growth requirements and inoculum preparation and must grow under the conditions of the test. The primary purpose of this test method is to provide a set of standardized conditions and test organisms to facilitate comparative assessments of antimicrobial materials miscible in aqueous systems.  
1.2 This test method allows the option of using a test sample size of 10 mL or 100 mL.  
1.3 Knowledge of microbiological techniques is required for this procedure.  
1.4 Aseptic technique should be practiced at all times.  
1.5 In this test method, SI units are used for all applications, except for distance in which case inches are used and SI units follow in parentheses.  
1.6 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, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.7 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.

General Information

• Status: Published
• Publication Date: 31-Mar-2022
• ICS: 07.100.20 - Microbiology of water
• Technical Committee: E35 - Pesticides, Antimicrobials, and Alternative Control Agents
• Drafting Committee: E35.15 - Antimicrobial Agents

Relations

• Refers: ASTM E691-13 - Standard Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
• Effective Date: 01-May-2013
• Refers: ASTM E691-11 - Standard Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
• Effective Date: 01-Nov-2011
• Refers: ASTM E691-08 - Standard Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
• Effective Date: 01-Oct-2008
• Refers: ASTM E1054-08 - Standard Test Methods for Evaluation of Inactivators of Antimicrobial Agents
• Effective Date: 01-Apr-2008
• Refers: ASTM E691-05 - Standard Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
• Effective Date: 01-Nov-2005
• Refers: ASTM E1054-02 - Standard Test Methods for Evaluation of Inactivators of Antimicrobial Agents
• Effective Date: 10-May-2002
• Refers: ASTM E691-99 - Standard Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
• Effective Date: 10-May-1999

Overview

ASTM E2783-22 is a standard test method developed by ASTM International for the assessment of antimicrobial activity for water miscible compounds using a time-kill procedure. This standard is designed to measure the reduction of aerobic and anaerobic microorganisms over specific time intervals after exposure to a test material in vitro. The purpose of this procedure is to provide a reliable, reproducible approach for comparing the effectiveness of antimicrobial agents that are miscible in aqueous systems.

Employing standardized test organisms and controlled laboratory conditions, ASTM E2783-22 supports regulatory, research, and manufacturing activities in developing and testing antimicrobial products used across healthcare, pharmaceutical, and industrial applications.

Key Topics

• In vitro antimicrobial activity: The test evaluates reductions in microbial populations (aerobic and anaerobic) upon exposure to a test compound.
• Time-kill procedure: The test uses specific sampling times to assess microbial survival, allowing construction of kill curves and kinetic models.
• Standardized test conditions: Use of uniform inoculum preparation, growth requirements, and environmental conditions to ensure reproducibility.
• Sample size flexibility: The method accommodates sample volumes of either 10 mL or 100 mL, supporting a variety of test scenarios.
• Microbiological best practices: Emphasis on aseptic techniques and proper calibration and standardization of laboratory equipment.
• Data analysis: Results expressed in percent and log reductions of microorganisms, with strict validity criteria for inoculum counts.

Applications

ASTM E2783-22 is widely applicable in industries needing reliable antimicrobial testing of water miscible compounds, including:

• Pharmaceutical and healthcare: Evaluating disinfectants, antiseptics, and topical drugs for their antimicrobial efficacy.
• Consumer products: Testing antimicrobial agents in personal care and household cleaning products formulated for aqueous systems.
• Food and beverage: Assessing preservatives and sanitizers that interact with microbial contaminants in process water or liquid foods.
• Industrial microbiology: Comparing and validating antimicrobial agents used in water treatment and manufacturing processes.
• Research and development: Supporting formulation science and regulatory submissions with standardized efficacy data.

Microbial strains used in testing may include common bacterial and fungal species relevant to the intended application, and laboratories must adhere to appropriate biosafety levels and international best practices.

Related Standards

ASTM E2783-22 references and is complemented by several related standards, including:

• ASTM E691: Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method.
• ASTM E1054: Practices for Evaluation of Inactivators of Antimicrobial Agents, guiding the use of neutralizing agents in the test.
• International guidelines: Developed in line with WTO Technical Barriers to Trade (TBT) principles for harmonized approaches to standardization.

Other relevant standards may address specific antimicrobial efficacy testing for different product categories or formulations not directly covered by the water miscible focus of ASTM E2783-22.

Practical Value

The ASTM E2783-22 standard is crucial for organizations seeking consistent and validated measurement of antimicrobial effectiveness. By following this method, laboratories and manufacturers can:

• Ensure regulatory compliance with in vitro antimicrobial efficacy standards.
• Generate reproducible and comparable data for product development and quality assurance.
• Reduce risks associated with inadequate microbial control in products and processes.
• Support market claims regarding antimicrobial activity with scientifically valid tests.

Adopting ASTM E2783-22 enables efficient, accurate evaluation of antimicrobial agents in water miscible forms, strengthening product safety and performance in competitive industries.

Standards Content (Sample)

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.
Designation: E2783 − 22
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.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 2. Referenced Documents
2.1 ASTM Standards:
1.1 This test method measures the changes of a population
E691Practice for Conducting an Interlaboratory Study to
of aerobic and anaerobic microorganisms within a specific
Determine the Precision of a Test Method
sampling time when tested against antimicrobial test materials
E1054PracticesforEvaluationofInactivatorsofAntimicro-
in vitro. The organisms used are standardized as to growth
bial Agents
requirements and inoculum preparation and must grow under
the conditions of the test. The primary purpose of this test
3. Terminology
method is to provide a set of standardized conditions and test
organisms to facilitate comparative assessments of antimicro-
3.1 Definitions:
bial materials miscible in aqueous systems.
3.1.1 antimicrobial, adj—describes an agent that kills or
inactivates microorganisms or suppresses their growth or
1.2 Thistestmethodallowstheoptionofusingatestsample
reproduction.
size of 10 mL or 100 mL.
3.1.2 drug, n—articles intended for use in the diagnosis,
1.3 Knowledge of microbiological techniques is required
cure, mitigation, treatment, or prevention of disease in man or
for this procedure.
other animals. Drugs are intended to affect the structure or any
function of the body of man or other animals.
1.4 Aseptic technique should be practiced at all times.
3.1.3 initial microbial population, n—bacterial count (CFU/
1.5 Inthistestmethod,SIunitsareusedforallapplications,
mL) in the final volume of test material.Also known as initial
except for distance in which case inches are used and SI units
bacterial population, numbers control or control.
follow in parentheses.
3.1.4 inoculum, n—in microbiology, a specimen compro-
1.6 This standard does not purport to address all of the
mised of living spores, bacteria, yeast or the multicellular
safety concerns, if any, associated with its use. It is the
filamentous fungi, or combination of two or more types of
responsibility of the user of this standard to establish appro-
microorganisms,thatareintroducedintoatestmediumoronto
priate safety, health, and environmental practices and deter-
aspecimentobetestedinordertoinvestigatetheabilityofthe
mine the applicability of regulatory limitations prior to use.
medium or specimen to support microbial growth or to
1.7 This international standard was developed in accor-
investigate its antimicrobial properties.
dance with internationally recognized principles on standard-
3.1.5 microbiocide, n—a physical or chemical agent that
ization established in the Decision on Principles for the
kills microorganisms.
Development of International Standards, Guides and Recom-
3.1.6 neutralization, n—the process for inactivating or
mendations issued by the World Trade Organization Technical
quenching the activity of a microbiocide, often achieved
Barriers to Trade (TBT) Committee.
through physical (for example, filtration or dilution) or chemi-
cal means.
This test method 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. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Current edition approved April 1, 2022. Published June 2022. Originally contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
approved in 2010. Last previous edition approved in 2016 as E2783–11(2016). Standards volume information, refer to the standard’s Document Summary page on
DOI: 10.1520/E2783–22. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2783 − 22
3.1.7 room temperature, n—temperature in the range of 6.14 Vortex Mixer—Any suitable vortex mixer capable of
20°C to 30°C (68°F to 85°F). mixing test material and diluents.
3.1.8 test material, n—a formulation which incorporates
6.15 Waterbath—Any waterbath capable of maintaining
antimicrobial ingredient(s). Also known as test formulation.
suitable temperature.
3.1.9 total test volume, n—the volume of test material plus
the volume of inoculum suspension. 7. Reagents and Materials
7.1 Bacteriological Loops—Any type of disposable sterile
4. Summary of Test Method
or sterilizable bacteriological loop is suitable.
4.1 A dilution/aliquot of the test material is brought into
7.2 Bacteriological Pipets—Sterile. 1.1 mL, 2.0 mL, 5.0
contactwithaknownpopulationoftestorganismsforspecified
mL, 10.0 mL capacity.
periods of time, at a specified temperature. The activity of the
NOTE 1—Pre-sterilized/disposable bacteriological pipets are available
test material is quenched at specified sampling intervals
from most local laboratory supply houses.
(example 15, 30, and 60 s, or any range covering several
minutes or hours) with an appropriate neutralizing technique.
7.3 Broth Growth Medium—Sterile soybean-casein digest
The test material is neutralized at the sampling time and the
broth (tryptic soy broth) or other broth media appropriate to
surviving microorganisms enumerated. The percent and log
support growth of the test organisms.
reduction, from an initial microbial population is calculated.
7.4 Dilution Fluid or Diluent—SterileButterfield’sbuffered
phosphate diluent or other suitable diluent with appropriately
5. Significance and Use
validated neutralizers. Perform Test Method E1054 to deter-
5.1 This procedure may be used to assess the in vitro
mine what diluent or neutralizers are required. Volume is 9.0
reduction of a microbial population of test organisms after
mL after sterilization.
exposure to a test material.
7.5 Flip Top Centrifuge Tubes—Sterile. For 10 mL sample
6. Apparatus
size. Minimum of 50 mL capacity.
6.1 Adjustable or Fixed Volume Pipet—Capable of dispens- NOTE 2—Pre-sterilized/ disposable flip-top centrifuge tubes are avail-
able from most laboratory supply houses.
ing 0.1 mL and 1.0 mL.
7.6 Petri Dishes—100mm by 15 mm. Required to plate
6.2 Anaerobic Jar or Incubator—Any incubator or appara-
samples and control.
tus in an incubator that creates an environment having a level
NOTE 3—Pre-sterilized/disposable plastic petri dishes are available
ofoxygenthatdoesnotsupportthegrowthofoxygen-requiring
from most local laboratory supply houses.
microorganisms. Required only for organisms that need an
anaerobic environment to grow.
7.7 Physiological Saline—Sterile. Used to prepare inocu-
lum.
6.3 Balance—Any suitable laboratory balance with a mini-
mum readability of 0.01 g.
7.8 Pipet Tips—Sterile. 0.1mL and 1.0 mL capacity.
6.4 Beakers and Magnetic Stir Bars—For 100 mL sample
7.9 Positive Displacement Pipet Tips—Sterile. 1.0 mL ca-
size. A 250 mL beaker containing a 51mm by 8mm 62mm
pacity.
magnetic stir bar. The beaker and stir bar should be sterile.
7.10 Solid Growth and Plating Medium—Sterile soybean-
6.5 Colony Counter—Any of several types may be used.
casein digest agar (tryptic soy agar) or other solid media
6.6 Incubator—Any incubator capable of maintaining a
appropriate to support growth of the test organism(s) with
suitable temperature 62°C may be used.
appropriately validated neutralizers. Perform Test Method
E1054 to determine what neutralizers are required. Should be
6.7 Laboratory Centrifuge—Any centrifuge capable of pro-
tempered to between 40°C to 50°C if using a pour plate
ducing 3200 r/min (1520 RCF).
method.
6.8 Magnetic Stirring Plate—Any rotor powered magnetic
7.11 Water—Sterile deionized or distilled water.
stirrer.
6.9 Positive Displacement Pipet—1.0 mL capacity. Re-
8. Hazards
quired for viscous test materials.
8.1 Alltestorganismslistedforuseinthismethodfallunder
6.10 Sterilizer—Any suitable steam sterilizer capable of
the Biosafety level 1 or 2 categories and should be handled in
producing the conditions of sterility.
accordance with CDC and NIH guidelines.
6.11 Sterile Container—Any container of sufficient size to
dilute test material into.
6.12 Test Tubes with Cap—Sterile. Alternate sample con-
Horowitz, W., (Ed.), Offıcial Methods of Analysis of the AOAC, 17th Ed., Sec.
tainer to (7.5) for 10 mL sample size. Size of test tube should
6.3.03 A.(f), Chapter 6, 2000, p.10. Official Methods of Analysis of AOAC
allow the thorough mixing of samples. International, Gaithersburg, MD.
Richmond, J.Y. and McKinney, R. W. (eds.), 1999, Biosafety in Microbiologi-
6.13 Timer (Stop Clock)—One that displays minutes and
cal and Biomedical Laboratories, 4th ed., Washington DC, U.S. Government
seconds. Printing Office.
E2783 − 22
9. Calibration and Standardization 11.7.2 Inoculate a sufficient number of solid growth media
slants or plates (7.10).
9.1 Ensure that all equipment used in this test method has
11.7.3 Incubateatappropriatetemperatureandenvironment
been calibrated and standardized as required for that piece of
for the organism.
equipment.
11.7.4 Wash each slant by adding 5 to 10 mLof physiologi-
10. Test Organisms
cal saline (7.7) to each slant.
10.1 The following list of organisms may be used in this 11.7.5 Usingbacteriologicalloop(7.1),gentlyscrapeorrub
procedure. This list is not all inclusive and other organisms surface of agar to remove growth.
may be used. Organisms selected may be representative of the
11.7.6 Usingasterilepipet,collectthewashingsina50mL
microbialfloraencounteredundertheconditionsofuse,ormay
centrifuge tube (7.5). Repeat for all slants, adding washing to
representstandardizedstrains.Theorganismshouldbecapable
the centrifuge tube.
of providing reproducible results under the test conditions.
11.7.7 Centrifuge at conditions appropriate to sediment the
culture completely.
10.2 The organisms listed shall be maintained as specified
by ATCC or other validated methods.
11.7.8 Decantorpipetsupernatantandre-suspendorganism
10.2.1 Acinetobacter species.
pellet in 20 mL physiological saline (7.7).
10.2.2 Candida albicans ATCC 10231.
11.7.9 Centrifuge at conditions appropriate to sediment the
10.2.3 Enterobacter species.
culture completely.
10.2.4 Enterococcus faecalis ATCC 29212.
11.7.10 Decant or pipet supernatant and re-suspend organ-
10.2.5 Enterococcus faecium.
ism in an amount of physiological saline (7.7) sufficient to
10.2.6 Escherichia coli ATCC 8739, 11229, or 25922.
achieveaminimumfinalsuspensionof1.0×10 CFU/mL.Use
10.2.7 Klebsiella pneumoniae subsp. pneumoniae ATCC
McFarland Barium Sulfate Standard #2, turbidimetry, optical
10031 or 51504.
density, or other technique that correlates to an aerobic plate
10.2.8 Micrococcus yunnanensis Zhao et al. ATCC 7468.
count.
10.2.9 Pseudomonas aeruginosa ATCC 9027, 15442, or
11.8 Inoculum Prepared Directly from Broth:
27853.
11.8.1 The stock culture, frozen, or lyophilized should be at
10.2.10 Proteus mirabilis ATCC 4675 or 7002.
10.2.11 Salmonella enterica subsp. enterica serovar Chol- least one 24 h liquid growth media (7.3) transfer from the
original source.
eraesuis ATCC 10708.
10.2.12 Serratia marcescens ATCC 14756.
11.8.2 Incubateatappropriatetemperatureandenvironment
10.2.13 Shigella species.
for the organism.
10.2.14 Staphylococcus aureus ATCC 6538, 29213, 33591,
11.8.3 Centrifuge at conditions appropriate to sediment the
or 33592.
culture completely.
10.2.15 Staphylococcus epidermidis ATCC 12228.
11.8.4 Decantorpipetsupernatantandre-suspendorganism
10.2.16 Staphylococcus haemolyticus.
pellet in 20 mL physiological saline (7.7).
10.2.17 Staphylococcus hominis.
11.8.5 Centrifuge at conditions appropriate to sediment the
10.2.18 Staphylococcus saprophyticus.
culture completely.
10.2.19 Streptococcus pyogenes.
11.8.6 Decantorpipetsupernatantandre-suspendorganism
10.2.20 Streptococcus pneumoniae.
in an amount of physiological saline (7.7) sufficient to achieve
a minimum final suspension of 1.0 × 10 CFU/mL. Use
11. Preparation of Organism
McFarland Barium Sulfate Standard #2, turbidimetry, optical
11.1 Allorganismsusedinthetestmethodshallbeprepared
density, or other technique that correlates to an aerobic plate
using a validated method. The same method shall be used to
count.
prepare all organisms for the test.
NOTE 4—Antimicrobials sensitive to organic material (for example
11.2 Aminimum starting inoculum level of 1.0 CFU/mL ×
8 alcohol and iodine) may have reduced activity by even the slightest
10 CFU/mL should be used for the test.
organic load and therefore only use thoroughly washed inoculum
11.3 Other starting inoculum levels may be used depending suspensions, whether initially grown in broth or from solid media.
on the organisms growth potential.
12. Test Conditions
11.4 All organisms shall be no more than five passages
removed from the original source.
12.1 Test should be performed at room temperature.
11.5 The inoculum shall be used within4hof preparation.
12.2 Testmaterialsanddiluentsshouldbeatroomtempera-
ture.
11.6 If a validated method is not available, the following
instructions shall be used to prepare the inoculum.
12.3 Test materials may require a lower or higher tempera-
11.7 Inoculum Preparation Directly from Agar: ture than room temperature (for example, solids that require
11.7.1 The stock culture, frozen or lyophilized should be at warming to and be held at 45°C, or test material may only be
least one 24 h broth growth m
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
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: E2783 − 11 (Reapproved 2016) E2783 − 22
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
1.1 This test method measures the changes of a population of aerobic and anaerobic microorganisms within a specific sampling
time when tested against antimicrobial test materials in vitro. The organisms used are standardized as to growth requirements and
inoculum preparation and must grow under the conditions of the test. The primary purpose of this test method is to provide a set
of standardized conditions and test organisms to facilitate comparative assessments of antimicrobial materials miscible in aqueous
systems.
1.2 This test method allows the option of using a test sample size of 10 mL or 100 mL.
1.3 Knowledge of microbiological techniques is required for this procedure.
1.4 Aseptic technique should be practiced at all times.
1.5 In this test method, SI units are used for all applications, except for distance in which case inches are used and SI units follow
in parentheses.
1.6 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 safety, health, and healthenvironmental practices and determine the
applicability of regulatory limitations prior to use.
1.7 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:
E691 Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
E1054 Practices for Evaluation of Inactivators of Antimicrobial Agents
3. Terminology
3.1 Definitions:
This test method 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 April 1, 2016April 1, 2022. Published May 2016June 2022. Originally approved in 2010. Last previous edition approved in 20112016 as
E2783 – 11.E2783 – 11(2016). DOI: 10.1520/E2783–11R16.10.1520/E2783–22.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2783 − 22
3.1.1 antimicrobial, n—adj—describes an agent that kills or inactivates microorganisms or suppresses their growth or
reproduction.
3.1.2 drug, n—articles intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease in man or other
animals. Drugs are intended to affect the structure or any function of the body of man or other animals.
3.1.3 initial microbial population, n—bacterial count (CFU/mL) in the final volume of test material. Also known as initial bacterial
population, numbers control or control.
3.1.4 inoculum, n—the viable microorganisms used to contaminate a sample, device or surface, often expressed as to number and
type.in microbiology, a specimen compromised of living spores, bacteria, yeast or the multicellular filamentous fungi, or
combination of two or more types of microorganisms, that are introduced into a test medium or onto a specimen to be tested in
order to investigate the ability of the medium or specimen to support microbial growth or to investigate its antimicrobial properties.
3.1.5 microbiocide, n—a physical or chemical agent that kills microorganisms.
3.1.6 neutralization, n—the process for inactivating or quenching the activity of a microbiocide. Oftenmicrobiocide, often
achieved through chemical or physical means (for example, filtration or dilution).dilution) or chemical means.
3.1.7 room temperature, n—temperature in the range of 2020 °C to 30°C (6830 °C (68 °F to 85°F).85 °F).
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 the volume of inoculum suspension.
4. Summary of Test Method
4.1 A dilution/aliquot of the test material is brought into contact with a known population of test organisms for specified periods
of time, at a specified temperature. The activity of the test material is quenched at specified sampling intervals (example 15, 30,
and 60 s, or any range covering several minutes or hours) with an appropriate neutralizing technique. The test material is
neutralized at the sampling time and the surviving microorganisms enumerated. The percent and log reduction, from an initial
microbial population is calculated.
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 Adjustable or Fixed Volume Pipet—Capable of dispensing 0.1 mL and 1.0 mL.
6.2 Anaerobic Jar or Incubator—Any incubator or apparatus in an incubator that creates an environment having a level of oxygen
that does not support the growth of oxygen-requiring microorganisms. Required only for organisms that need an anaerobic
environment to grow.
6.3 Balance—Any suitable laboratory balance with a minimum readability of 0.01 g.
6.4 Beakers and Magnetic Stir Bars—For 100 mL sample size. A 250 mL beaker containing a 5151mm by 88 mm 6 2 mm
magnetic stir bar. The beaker and stir bar should be sterile.
6.5 Colony Counter—Any of several types may be used.
6.6 Incubator—Any incubator capable of maintaining a suitable temperature 62ºC62 °C may be used.
E2783 − 22
6.7 Laboratory Centrifuge—Any centrifuge capable of producing 3200 r/min (1520 RCF).
6.8 Magnetic Stirring Plate—Any rotor powered magnetic stirrer.
6.9 Positive Displacement Pipet—1.0 mL capacity. Required for viscous test materials.
6.10 Sterilizer—Any suitable steam sterilizer capable of producing the conditions of sterility.
6.11 Sterile Container—Any container of sufficient size to dilute test material into.
6.12 Test Tubes with Cap—Sterile. Alternate sample container to (7.5) for 10 mL sample size. Size of test tube should allow the
thorough mixing of samples.
6.13 Timer (Stop Clock)—One that displays minutes and seconds.
6.14 Vortex Mixer—Any suitable vortex mixer capable of mixing test material and diluents.
6.15 Waterbath—Any waterbath capable of maintaining suitable temperature.
7. Reagents and Materials
7.1 Bacteriological Loops—Any type of disposable sterile or sterilizable bacteriological loop is suitable.
7.2 Bacteriological Pipets—Sterile. 1.1, 2.0, 5.0, 1.1 mL, 2.0 mL, 5.0 mL, 10.0 mL capacity.
NOTE 1—Pre-sterilized/disposable bacteriological pipets are available from most local laboratory supply houses.
7.3 Broth Growth Medium—Sterile soybean-casein digest broth (tryptic soy broth) or other broth media appropriate to support
growth of the test organisms.
7.4 Dilution Fluid or Diluent—Sterile Butterfield’s buffered phosphate diluent or other suitable diluent with appropriately
validated neutralizers. Perform Test Method E1054 to determine what diluent or neutralizers are required. Volume is 9.0 mL after
sterilization.
7.5 Flip Top Centrifuge Tubes—Sterile. For 10 mL sample size. Minimum of 50 mL capacity.
NOTE 2—Pre-sterilized/ disposable flip-top centrifuge tubes are available from most laboratory supply houses.
7.6 Petri Dishes—100100 mm by 15 mm. Required to plate samples and control.
NOTE 3—Pre-sterilized/disposable plastic petri dishes are available from most local laboratory supply houses.
7.7 Physiological Saline—Sterile. Used to prepare inoculum.
7.8 Pipet Tips—Sterile. 0.10.1 mL and 1.0 mL capacity.
7.9 Positive Displacement Pipet Tips—Sterile. 1.0 mL capacity.
7.10 Solid Growth and Plating Medium—Sterile soybean-casein digest agar (tryptic soy agar) or other solid media appropriate to
Horowitz, W., (Ed.), Offıcial Methods of Analysis of the AOAC, 17th Ed., Sec. 6.3.03 A.(f), Chapter 6, 2000, p.10. Official Methods of Analysis of AOAC International,
Gaithersburg, MD.
E2783 − 22
support growth of the test organism(s) with appropriately validated neutralizers. Perform Test Method E1054 to determine what
neutralizers are required. Should be tempered to between 4040 °C to 50°C50 °C if using a pour plate method.
7.11 Water—Sterile deionized or distilled water.
8. Hazards
8.1 All test organisms listed for use in this method fall under the Biosafety level 1 or 2 categories and should be handled in
accordance with CDC and NIH guidelines.
9. Calibration and Standardization
9.1 Ensure that all equipment used in this test method has been calibrated and standardized as required for that piece of equipment.
10. Test Organisms
10.1 The following list of organisms may be used in this procedure. This list is not all inclusive and other organisms may be used.
Organisms selected may be representative of the microbial flora encountered under the conditions of use, or may represent
standardized strains. The organism should be capable of providing reproducible results under the test conditions.
10.2 The organisms listed shall be maintained as specified by ATCC or other validated methods.
10.2.1 Acinetobacter species.
10.2.2 Candida albicans ATCC 10231.
10.2.3 Enterobacter species.
10.2.4 Enterococcus faecalis ATCC 29212.
10.2.5 Enterococcus faecium.
10.2.6 Escherichia coli ATCC 8739, 11229, or 25922.
10.2.7 Klebsiella pneumoniae subsp. pneumoniae ATCC 10031 or 51504.
10.2.8 Micrococcus luteus yunnanensis Zhao et al. ATCC 7468.
10.2.9 Pseudomonas aeruginosa ATCC 9027, 15442, or 27853.
10.2.10 Proteus mirabilis ATCC 4675 or 7002.
10.2.11 Salmonella enterica subsp. enterica serovar Choleraesuis ATCC 10708.
10.2.12 Serratia marcescens ATCC 14756.
10.2.13 Shigella species.
10.2.14 Staphylococcus aureus ATCC 6538, 29213, 33591, or 33592.
10.2.15 Staphylococcus epidermidis ATCC 12228.
10.2.16 Staphylococcus haemolyticus.
10.2.17 Staphylococcus hominis.
Richmond, J. Y. and McKinney, R. W. (eds.), 1999, Biosafety in Microbiological and Biomedical Laboratories, 4th ed., Washington DC, U.S. Government Printing Office.
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10.2.18 Staphylococcus saprophyticus.
10.2.19 Streptococcus pyogenes.
10.2.20 Streptococcus pneumoniae.
11. Preparation of Organism
11.1 All organisms used in the test method shall be prepared using a validated method. The same method shall be used to prepare
all organisms for the test.
11.2 A minimum starting inoculum level of 1.0 CFU/mL × 10 CFU/mL should be used for the test.
11.3 Other starting inoculum levels may be used depending on the organisms growth potential.
11.4 All organisms shall be no more than five passages removed from the original source.
11.5 The inoculum shall be used within 4 h of preparation.
11.6 If a validated method is not available, the following instructions shall be used to prepare the inoculum.
11.7 Inoculum Preparation Directly from Agar:
11.7.1 The stock culture, frozen or lypholizedlyophilized should be at least one 24 h broth growth media (7.3) transfer from the
original source.
11.7.2 Inoculate a sufficient number of solid growth media slants or plates (7.10).
11.7.3 Incubate at appropriate temperature and environment for the organism.
11.7.4 Wash each slant by adding 5 to 10 mL of physiological saline (7.7) to each slant.
11.7.5 Using bacteriological loop (7.1), gently scrape or rub surface of agar to remove growth.
11.7.6 Using a sterile pipet, collect the washings in a 50 mL centrifuge tube (7.5). Repeat for all slants, adding washing to the
centrifuge tube.
11.7.7 Centrifuge at conditions appropriate to sediment the culture completely.
11.7.8 Decant or pipet supernatant and re-suspend organism pellet in 20 mL physiological saline (7.7).
11.7.9 Centrifuge at conditions appropriate to sediment the culture completely.
11.7.10 Decant or pipet supernatant and re-suspend organism in an amount of physiological saline (7.7) sufficient to achieve a
minimum final suspension of 1.0 × 10 CFU/mL. Use McFarland Barium Sulfate Standard #2, turbidimetry, optical density, or
other technique that correlates to an aerobic plate count.
11.8 Inoculum Prepared Directly from Broth:
11.8.1 The stock culture, frozen, or lypholizedlyophilized should be at least one 24 h liquid growth media (7.3) transfer from the
original source.
11.8.2 Incubate at appropriate temperature and environment for the organism.
11.8.3 Centrifuge at conditions appropriate to sediment the culture completely.
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11.8.4 Decant or pipet supernatant and re-suspend organism pellet in 20 mL physiological saline (7.7).
11.8.5 Centrifuge at conditions appropriate to sediment the culture completely.
11.8.6 Decant or pipet supernatant and re-suspend organism in an amount of physiological saline (7.7) sufficient to achieve a
minimum final suspension of 1.0 × 10 CFU/mL. Use McFarland Barium Sulfate Standard #2, turbidimetry, optical density, or
other technique that correlates to an aerobic plate count.
NOTE 4—Antimicrobials sensitive to organic material (for example alcohol and iodine) may have reduced activity by even the slightest organic load and
therefore only use thoroughly washed inoculum suspensions, whether initially grown in broth or from so
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