ASTM E1891-97(2002)
(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
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.
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.
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Designation:E1891–97 (Reapproved 2002)
Standard Guide for
Determination of a Survival Curve for Antimicrobial Agents
Against Selected Microorganisms and Calculation of a
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 a 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.Arelated function, the concentration
coefficient (h) 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
committedtocarriertests,whileEuropeanshaveemphasizedsuspensiontestscombinedwithpractical
applied test 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
thatshowedsimilaritytochemical,eqstetimolecularreactions.Distortionsintheexpectedstraight-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.
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E1891–97 (2002)
Esty and Meyer (1922) introduced the terminology we currently use in relation to bacterial kill
whether for spores, vegetative bacterial cells, or mycobacteria 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 of
positive and negative tubes. (1) This analysis is done after an exposure period when there are fewer
bacterial cells or spores in the container and positive and negative tubes can be expected on recovery.
Single-sample subculturing of aliquot samples from a reaction vessel containing the test organism
and the test antimicrobial has been the basic means for establishing survival curves. Usually a
suspension of target microorganisms is exposed to a disinfectant\sterilant and aliquots are withdrawn
at specific time intervals and assessed for survivors, usually with plate counts. Because of tailing
problems and difficulty in enumerating small numbers, when only a few survivors are left, MPN
methods of enumeration are recommended and often used (1, 2, 3). A common method derived from
thermal processing in the canning industry is the end-point method, described above, in which the
number of positive and negative tubes from replicate sampling (such as tubes or cans) is used alone
or in the combination with single sampling to construct a survivor curve and plotted to determine
D-values. (4)
Many antimicrobial formulations available for test are diluted in use. When D-values are
determined and calculated at more than one concentration (dilution) of an antimicrobial, the
concentration coefficient, designated as the Greek letter eta or h, denotes the effect of dilution on the
activity of a chemical or formulation.
This guide is under the jurisdiction of ASTM Committee E35 on Pesticides and Alternative Control Agents and is the direct responsibility of Subcommittee E35.15 on
Antimicrobial Agents.
Current edition approved Oct. 10, 2002. Published March 2003. Originally approved in 1997. Last previous edition approved in 1997 as E1891 – 97. DOI:
10.1520/E1891-97R02.
The boldface numbers given in parentheses refer to a list of references at the end of the text.
1. Scope This methodology also has been applied to preservative testing
of antimicrobial ingredients in more complex cosmetic formu-
1.1 This guide covers the methods for determining the death
lations (5).
rate kinetics expressed as D-values. These values can be
1.2 Thetestmethodsdiscussedshouldbeperformedonlyby
derivedfromtheconstructionofakillcurve(orsurvivorcurve)
those trained in microbiological techniques.
or by using other procedures for determining the number of
1.3 The values stated in SI units are to be regarded as the
survivors after exposure to antimicrobial chemicals or formu-
standard.
lations.Optionsforcalculationswillbepresentedaswellasthe
1.4 This standard does not purport to address all of the
method for calculation of a concentration coefficient.
safety concerns, if any, associated with its use. It is the
1.1.1 The test methods are designed to evaluate antimicro-
responsibility of the user of this standard to establish appro-
bial agents in formulations to define a survivor curve and to
priate safety and health practices and determine the applica-
subsequently calculate a D-value. The tests are designed to
bility of regulatory limitations prior to use.
produce data and calculate values that provide basic informa-
tion of the rate-of-kill of antimicrobial formulations tested
2. Terminology
against single, selected microorganisms. In addition, calculated
2.1 Definitions:
D-values from survivor curves from exposure at different
2.1.1 D-value or decimal reduction time—(often referred to
dilutions of antimicrobial can be used to show the effect of
as log death time) relates reaction kinetics and inactivation
dilution by calculation of the concentration exponent, h (2).
rate. It is defined as the time (usually in minutes) to reduce the
1.1.2 As an example of potential use of kill curve data, the
microbiologic population one log or to reduce it to 90 % or
published FDA, OTC Tentative Final Monograph for Health-
reduce it to 10 % of the initial population.
Care Antiseptic Drug Products, Proposed Rule, June 17, 1994
2.1.2 Fn = Fraction negative (FN) data—(quantal data) are
has suggested the testing of topically applied antimicrobial
experimentalresultsintheformofadichotomousresponse:the
products using survival curve (or kill curve) calculations. The
unit tested is either positive (showing growth) or negative
methods described in this guide are applicable to these prod-
(showing no growth).
ucts, but adjustments such as the use of antifoaming agents
2.1.3 Concentration exponent, h: (dilution coeffıcient)—
when the reaction mixture is stirred may be necessary to
measures the effect of changes in concentration (or dilution) on
counteract the presence of detergents in many formulations.
cell death rate. To measure h, the time necessary to produce a
Frequently the sampling for these tests is done after very short
comparable degree of death in a bacterial suspension at least
intervals of exposure to the formulation, such as 30 and 60 s. two different concentrations is measured (D-value) (6).
E1891–97 (2002)
2.1.4 Most Probable Number (MPN)—data in which a 5. Materials and Reagents
fraction of the replicate units are negative and can be analyzed
5.1 Some basic materials will be required regardless of the
statistically using the MPN technique to yield the probable
specific method selected. This list may need to be supple-
number of survivors at the respective exposure time.
mented depending on the techniques selected.
5.1.1 Colony Counter, any of several types may be used.
3. Summary of a Basic Test Method
5.1.2 Membrane Filter Holders and Microbially Retentive
3.1 This test method is conducted on selected microbial
Membranes, (0.22 µm) with vacuum equipment for filtration.
species cultured to produce high-count suspensions that are
5.1.3 Incubator—Any incubator capable of maintaining a
exposed to the test antimicrobial agent or formulation(s) under
temperature within a 6 2°C of the recommended optimal
standardized conditions of temperature and agitation. Samples
temperature for the growth of a specific microorganism under
from this reaction mixture are withdrawn at pre-set times,
test.
neutralized and cultured to determine survivors, using standard
5.1.4 A Glass Reaction Vessel, of appropriate size and
procedures. A D-value is calculated from the post exposure
design to permit required sampling.
survivor data utilizing published and accepted methods.
5.1.5 A Realistic Means of Agitation, such as a hot-plate
3.2 This test method involves testing a high count suspen-
with a magnetic stirring feature.
sion of a microorganism as the initial challenge inoculum; at
5.1.6 Temperature Controlled Water Bath, with agitation,
7 8 6
least 10 to 10 cfu/mL, to achieve a 10 cfu/mL when added
when available.
to the reaction chamber and exposed to disinfectant and to
5.1.7 Sterilizer.
sporicidal chemicals.
5.1.8 Spectrophotometer.
3.3 Agrowth medium for the inoculum must produce a high
5.1.9 Timers—An interval timer, such as a stop watch for
numbers of vegetative cells or spores within a reasonable time
determining elapsed time to remove test samples from the
period with consistent resistance to chemical disinfectants.
reaction chamber.
3.4 Where possible agitation of the reaction chamber is
recommended.
6. Materials and Reagents
3.5 Currently a test temperature of 20 6 1°C is recom-
mended. This temperature is lower than most environmental
6.1 Depending on the specific method used, additions may
temperatures in practice (room temperature). A more typical
have to be made to the materials and reagents tested.
temperaturerangeissuggestedat22 61°C.Thestudyofmany
6.1.1 Petri Dishes, 100 by 15 mm required for performing
antimicrobials is increased with increasing temperature. An
standard plate count .
alternativetemperaturemaybeselectedfortesting,butmustbe
6.1.2 Bacteriologic Pipets, 10.0 and 2.2 or 1.1. mLcapacity.
controlled and constant.
Micropipet types may also be used .
3.6 An alternative testing technique to single sequential
6.1.3 Liquid Media, appropriate for the test microorganism.
timed samples may be included in execution of this method 5
A soybean case in digest agar or equivalent may be used for
because a major problem has occurred with many reported
culturing the test microorganism.
studies. Many kill or survival curves have shown a rapid kill of
6.1.4 Agar Plates, (spread- or pour-plates) of appropriate,
several logs after an exposure period expected to eliminate
optimalmediaforthetestmicroorganismforcultureoftestand
survivors, yet leaving a few survivors, usually ten or fewer
control samples.
ranging to 1000. This number fluctuates for an extended time
6.1.5 Neutralizer Solution, specific for each antimicrobial
with repeated sampling and has been termed, tailing.Achange
tested incorporated into diluent and optionally into recovery
from single sampling to replicate - unit sampling is recom-
medium.
mended as a means to alleviate this problem.
6.1.6 Test Tubes, with closures of appropriate size for
3.7 Repetition of the estimation of a survival curve is
samples and ten-fold dilution of samples.
recommended. Recommendations for three to five replications
6.1.7 ASelection of Flasks and Tubes, required for culturing
with sampling at five time points have been made.
of the test microorganisms.
6.1.8 Diluent Tubes, for dilution of the test and control
4. Significance and Use
samples. Diluent may have phosphate-buffered normal saline
4.1 Thedifferentproceduresandmethodsaredesignedtobe
or other appropriate diluent for specific microorganisms and
used to produce survival data after microorganisms are ex-
neutralizers specific for the test disinfectant should be added to
posed to antimicrobial agents in order to calculate values that
the diluent.
can be used to analyze and rationalize the effectiveness of
6.1.9 An Automatic Mixer, such as a Vortex mixer.
antimicrobial agents when tested using other, often applied test
methods.
4.2 The data from these test procedures may be used in the
selection and design of other tests of effectiveness of antimi-
Presterilized\disposable plastic dishes are available from
...
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