ASTM E1891-21
(Guide)Standard Guide for Determination of a Survival Curve for Antimicrobial Agents Against Selected Microorganisms and Calculation of a D-Value and Concentration Coefficient
Standard Guide for Determination of a Survival Curve for Antimicrobial Agents Against Selected Microorganisms and Calculation of a D-Value and Concentration Coefficient
SIGNIFICANCE AND USE
5.1 The different procedures and methods are designed to be used to produce survival data after microorganisms are exposed to antimicrobial agents in order to calculate values that can be used to analyze and rationalize the effectiveness of antimicrobial agents when tested using other, often applied test methods.
5.2 The data from these test procedures may be used in the selection and design of other tests of effectiveness of antimicrobial agents, some of which may be required by regulatory agencies to establish specific claims. Basic kinetic information about killing rate often serves as the initial information on which a testing program can be built.
SCOPE
1.1 This guide covers the methods for determining the death rate kinetics expressed as D-values. These values can be derived from the construction of a kill curve (or survivor curve) or by using other procedures for determining the number of survivors after exposure to antimicrobial chemicals or formulations. Options for calculations will be presented as well as the method for calculation of a concentration coefficient.
1.1.1 The test methods are designed to evaluate antimicrobial agents in formulations to define a survivor curve and to subsequently calculate a D-value. The tests are designed to produce data and calculate values that provide basic information of the rate-of-kill of antimicrobial formulations tested against single, selected microorganisms. In addition, calculated D-values from survivor curves from exposure at different dilutions of antimicrobial can be used to show the effect of dilution by calculation of the concentration exponent, η (2). D-value determination assumes the ideal of first-order killing reactions that are reflected in a straight-line reduction in count where a count-versus-time plot is done. The goal here is not to determine the time at which no survivors are found, but to determine a standard value that can be used in processing and exposure determinations or used to estimate dilutions.
1.1.2 As an example of potential use of kill curve data, the published FDA, OTC Tentative Final Monograph for Health-Care Antiseptic Drug Products, Proposed Rule, June 17, 1994 has suggested the testing of topically applied antimicrobial products using survival curve (or kill curve) calculations. The methods described in this guide are applicable to these products, but adjustments such as the use of antifoaming agents when the reaction mixture is stirred may be necessary to counteract the presence of detergents in many formulations. Frequently the sampling for these tests is done after very short intervals of exposure to the formulation, such as 30 and 60 s. This methodology also has been applied to preservative testing of antimicrobial ingredients in more complex cosmetic formulations (5).
1.2 The test methods discussed should be performed only by those trained in microbiological techniques.
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 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.
1.5 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.
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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: E1891 − 21
Standard Guide for
Determination of a Survival Curve for Antimicrobial Agents
Against Selected Microorganisms and Calculation of a
1
D-Value and Concentration Coefficient
This standard is issued under the fixed designation E1891; 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.
INTRODUCTION
A variety of testing procedures have been devised almost from the beginning of disinfection and
antisepsis as disciplines. From the first, there was recognition of the importance of time and rates of
kill.Aftermanydecadesandnumeroustestproceduresinvolvingcarriers,theapproachofestablishing
a death rate curve (often described as a survivor curve) is reclaiming its importance in establishing the
basic kinetics of the killing process after exposure to antimicrobial chemicals.
D-values (historically, log death time or decimal reduction time), kill or survivor curves, processing
calculations and rates of kill are discussed in many texts. There is extensive theoretical discussion but
little applied material on how to perform testing to establish kill curves and D-values and associated
calculations.
The guideline form has been selected to permit the inclusion of background information and a
model procedure for determining D-values and their calculation.Arelated function, the concentration
coefficient(η)canbecalculatedfromaseriesofD-valuescalculatedfordifferentconcentrationsofthe
test antimicrobial and defines the loss of activity as the material is diluted. This information has value
for application in disinfectants because many are sold to be diluted in use.
Specificproceduraldetailsarepresentedindescriptionsofmethodsroutinelyusedtoestablishakill
curve. The user should establish a protocol for the process that best fits their needs.
An experimental kill curve provides data for a calculated D-value derived from test data used to
construct the kill curve.
BACKGROUND
Scientists concerned about antimicrobial testing have debated the value of suspension tests in
contrast to tests using simulant carriers with dried microorganisms. U.S. regulation has been
committedtocarriertests,whileEuropeanshaveemphasizedsuspensiontestscombinedwithpractical
applied tests using materials as carriers on which the disinfectant actually will be used.
The examination of the kinetics of kill for various disinfectants provides basic information on the
activity of antimicrobials. The early history of microbiology reveals a strong momentum directed
towardclarificationofthesereactions.Fromtheearliestyearsofmicrobiology,theideasofrate-of-kill
and killing reactions as first order reactions (from chemical kinetics) have been involved in the
estimation of antimicrobial activity.
Kronig and Paul (1897) were the early pioneers who developed the concept of bacterial destruction
as a process. They used anthrax spores dried on garnet crystals and assessed the survivors by plating
washings from the garments after treatment with disinfectants. Chick (1908) found that the number of
survivors after disinfectant exposure, when plotted against time of treatment, produced a straight line
that showed similarity to chemical, equimolecular reactions. Distortions in the expected straight-line
reactions were noted by Chick as well as in subsequent investigations. Over the years, the most
common type of deviation from the expected, straight-line survivor curve is a sigmodial one
displaying a shoulder, a lag or delay in logarithmic kill, and ending in distinct tailing, sometimes
indicating a resistant population.
There has been a variety of procedures advanced for accumulating data that can be used to calculate
D-values and construct survivor curves.
Esty and Meyer (1922) introduced the terminology we currently use in relation to bacterial kill
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
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E1891 − 21
whether for spores or vegetative bacterial cells in devising thermal processing to eliminate Clostidium
botulinium in the canning industry. They also devised end-point analysis for interpretation of the
results of heat exposure and for processing calculations. Their procedure involved sampling multiple
tubes or other containers of product and analysis of the number remaining positive to determine the
number of survivors by Most Probable Number (MPN) analysis using the pattern o
...
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: E1891 − 10a (Reapproved 2015) E1891 − 21
Standard Guide for
Determination of a Survival Curve for Antimicrobial Agents
Against Selected Microorganisms and Calculation of a
1
D-Value and Concentration Coefficient
This standard is issued under the fixed designation E1891; 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.
INTRODUCTION
A variety of testing procedures have been devised almost from the beginning of disinfection and
antisepsis as disciplines. From the first, there was recognition of the importance of time and rates of
kill. After many decades and numerous test procedures involving carriers, the approach of establishing
a death rate curve (often described as a survivor curve) is reclaiming its importance in establishing the
basic kinetics of the killing process after exposure to antimicrobial chemicals.
D-values (historically, log death time or decimal reduction time), kill or survivor curves, processing
calculations and rates of kill are discussed in many texts. There is extensive theoretical discussion but
little applied material on how to perform testing to establish kill curves and D-values and associated
calculations.
The guideline form has been selected to permit the inclusion of background information and a
model procedure for determining D-values and their calculation. A related function, the concentration
coefficient (η) can be calculated from a series of D-values calculated for different concentrations of the
test antimicrobial and defines the loss of activity as the material is diluted. This information has value
for application in disinfectants because many are sold to be diluted in use.
Specific procedural details are presented in descriptions of methods routinely used to establish a kill
curve. The user should establish a protocol for the process that best fits their needs.
An experimental kill curve provides data for a calculated D-value derived from test data used to
construct the kill curve.
BACKGROUND
Scientists concerned about antimicrobial testing have debated the value of suspension tests in
contrast to tests using simulant carriers with dried microorganisms. U.S. regulation has been
committed to carrier tests, while Europeans have emphasized suspension tests combined with practical
applied tests using materials as carriers on which the disinfectant actually will be used.
The examination of the kinetics of kill for various disinfectants provides basic information on the
activity of antimicrobials. The early history of microbiology reveals a strong momentum directed
toward clarification of these reactions. From the earliest years of microbiology, the ideas of rate-of-kill
and killing reactions as first order reactions (from chemical kinetics) have been involved in the
estimation of antimicrobial activity.
Kronig and Paul (1897) were the early pioneers who developed the concept of bacterial destruction
as a process. They used anthrax spores dried on garnet crystals and assessed the survivors by plating
washings from the garments after treatment with disinfectants. Chick (1908) found that the number of
survivors after disinfectant exposure, when plotted against time of treatment, produced a straight line
1
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 Oct. 1, 2015Oct. 1, 2021. Published November 2015October 2021. Originally approved in 1997. Last previous edition approved in 20102015
as E1891 – 10a.E1891 – 10a(2015). DOI: 10.1520/E1891-10AR15.10.1520/E1891-21.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1
---------------------- Page: 1 ----------------------
E1891 − 21
that showed similarity to chemical, equimolecular reactions. Distortions in the expected straight-line
reactions were noted by Chick as well as in subsequent investigations. Over the years, the most
common type of deviation from the expected, straight-line survivor curve is a sigmodial one
displaying a shoulder, a lag or delay in logarithmic kill, and ending in distinct tailing, sometimes
indicating a resistant population.
There has been a variety of procedures advanced for ac
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