1、Designation: E1891 10aStandard Guide forDetermination of a Survival Curve for Antimicrobial AgentsAgainst Selected Microorganisms and Calculation of aD-Value and Concentration Coefficient1This standard is issued under the fixed designation E1891; the number immediately following the designation indi
2、cates the year oforiginal adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.INTRODUCTIONA variety of testing procedures have been
3、devised almost from the beginning of disinfection andantisepsis as disciplines. From the first, there was recognition of the importance of time and rates ofkill.After many decades and numerous test procedures involving carriers, the approach of establishinga death rate curve (often described as a su
4、rvivor curve) is reclaiming its importance in establishing thebasic kinetics of the killing process after exposure to antimicrobial chemicals.D-values (historically, log death time or decimal reduction time), kill or survivor curves, processingcalculations and rates of kill are discussed in many tex
5、ts. There is extensive theoretical discussion butlittle applied material on how to perform testing to establish kill curves and D-values and associatedcalculations.The guideline form has been selected to permit the inclusion of background information and amodel procedure for determining D-values and
6、 their calculation. A related function, the concentrationcoefficient (h) can be calculated from a series of D-values calculated for different concentrations ofthe test antimicrobial and defines the loss of activity as the material is diluted. This information hasvalue for application in disinfectant
7、s because many are sold to be diluted in use.Specific procedural details are presented in descriptions of methods routinely used to establish a killcurve. 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
8、 derived from test data used toconstruct the kill curve.BACKGROUNDScientists concerned about antimicrobial testing have debated the value of suspension tests incontrast to tests using simulant carriers with dried microorganisms. U.S. regulation has beencommitted to carrier tests, while Europeans hav
9、e emphasized suspension tests combined with practicalapplied 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 theactivity of antimicrobials. The early history of microbiol
10、ogy reveals a strong momentum directedtoward clarification of these reactions. From the earliest years of microbiology, the ideas of rate-of-killand killing reactions as first order reactions (from chemical kinetics) have been involved in theestimation of antimicrobial activity.Kronig and Paul (1897
11、) were the early pioneers who developed the concept of bacterial destructionas a process. They used anthrax spores dried on garnet crystals and assessed the survivors by platingwashings from the garments after treatment with disinfectants. Chick (1908) found that the number ofsurvivors after disinfe
12、ctant exposure, when plotted against time of treatment, produced a straight linethat showed similarity to chemical, equimolecular reactions. Distortions in the expected straight-linereactions were noted by Chick as well as in subsequent investigations. Over the years, the mostcommon type of deviatio
13、n from the expected, straight-line survivor curve is a sigmodial onedisplaying a shoulder, a lag or delay in logarithmic kill, and ending in distinct tailing, sometimesindicating a resistant population.There has been a variety of procedures advanced for accumulating data that can be used to calculat
14、eD-values and construct survivor curves.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.Esty and Meyer (1922) introduced the terminology we currently use in relation to bacterial killwhether for spores or vegetative bacterial cells i
15、n devising thermal processing to eliminate Clostidiumbotulinium in the canning industry. They also devised end-point analysis for interpretation of theresults of heat exposure and for processing calculations. Their procedure involved sampling multipletubes or other containers of product and analysis
16、 of the number remaining positive to determine thenumber of survivors by Most Probable Number (MPN) analysis using the pattern of positive andnegative tubes. (1)2This analysis is done after an exposure period when there are fewer bacterial cellsor spores in the container and positive and negative tu
17、bes can be expected on recovery.Single-sample subculturing of aliquot samples from a reaction vessel containing the test organismand the test antimicrobial has been the basic means for establishing survival curves. Usually asuspension of target microorganisms is exposed to a disinfectantsterilant an
18、d aliquots are withdrawnat specific time intervals and assessed for survivors, usually with plate counts. Because of tailingproblems and difficulty in enumerating small numbers, when only a few survivors are left, MPNmethods of enumeration are recommended and often used (1, 2, 3). A common method de
19、rived fromthermal processing in the canning industry is the end-point method, described above, in which thenumber of positive and negative tubes from replicate sampling (such as tubes or cans) is used aloneor in the combination with single sampling to construct a survivor curve and plotted to determ
20、ineD-values. (4)Many antimicrobial formulations available for test are diluted in use. When D-values aredetermined and calculated at more than one concentration (dilution) of an antimicrobial, theconcentration coefficient, designated as the Greek letter eta or h, denotes the effect of dilution on th
21、eactivity of a chemical or formulation.1. Scope1.1 This guide covers the methods for determining the deathrate kinetics expressed as D-values. These values can bederived from the construction of a kill curve (or survivor curve)or by using other procedures for determining the number ofsurvivors after
22、 exposure to antimicrobial chemicals or formu-lations. Options for calculations will be presented as well as themethod for calculation of a concentration coefficient.1.1.1 The test methods are designed to evaluate antimicro-bial agents in formulations to define a survivor curve and tosubsequently ca
23、lculate a D-value. The tests are designed toproduce data and calculate values that provide basic informa-tion of the rate-of-kill of antimicrobial formulations testedagainst single, selected microorganisms. In addition, calculatedD-values from survivor curves from exposure at differentdilutions of a
24、ntimicrobial can be used to show the effect ofdilution by calculation of the concentration exponent, h (2).D-value determination assumes the ideal of first-order killingreactions that are reflected in a straight-line reduction in countwhere a count-versus-time plot is done. The goal here is not tode
25、termine the time at which no survivors are found, but todetermine a standard value that can be used in processing andexposure determinations or used to estimate dilutions.1.1.2 As an example of potential use of kill curve data, thepublished FDA, OTC Tentative Final Monograph for Health-Care Antisept
26、ic Drug Products, Proposed Rule, June 17, 1994has suggested the testing of topically applied antimicrobialproducts using survival curve (or kill curve) calculations. Themethods described in this guide are applicable to these prod-ucts, but adjustments such as the use of antifoaming agentswhen the re
27、action mixture is stirred may be necessary tocounteract the presence of detergents in many formulations.Frequently the sampling for these tests is done after very shortintervals of exposure to the formulation, such as 30 and 60 s.This methodology also has been applied to preservative testingof antim
28、icrobial ingredients in more complex cosmetic formu-lations (5).1.2 The test methods discussed should be performed only bythose trained in microbiological techniques.1.3 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.4 This s
29、tandard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Terminology2.1 Definit
30、ions:2.1.1 D-value or decimal reduction time(often referred toas log death time) relates reaction kinetics and inactivationrate. It is defined as the time (usually in minutes) to reduce themicrobiologic population one log10or to reduce it to 90 % orreduce it to 10 % of the initial population.2.1.2 F
31、n = Fraction negative (FN) data(quantal data) areexperimental results in the form of a dichotomous response: theunit tested is either positive (showing growth) or negative(showing no growth).1This guide is under the jurisdiction of ASTM Committee E35 on Pesticides and Alternative Control Agents and
32、is the direct responsibility of Subcommittee E35.15 onAntimicrobial Agents.Current edition approved Oct. 1, 2010. Published November 2010. Originally approved in 1997. Last previous edition approved in 2010 as E1891 10. DOI:10.1520/E1891-10A.2The boldface numbers given in parentheses refer to a list
33、 of references at the end of the text.E1891 10a22.1.3 Concentration exponent, h: (dilution coeffcient)measures the effect of changes in concentration (or dilution) oncell death rate. To measure h, the time necessary to produce acomparable degree of death in a bacterial suspension for atleast two dif
34、ferent concentrations is measured (D-value) (6).2.1.4 Most Probable Number (MPN)data in which afraction of the replicate units are negative and can be analyzedstatistically using the MPN technique to yield the probablenumber of survivors at the respective exposure time.3. Summary of a Basic Test Met
35、hod3.1 This test method is conducted on selected microbialspecies cultured to produce high-count suspensions that areexposed to the test antimicrobial agent or formulation(s) understandardized conditions of temperature and agitation. Samplesfrom this reaction mixture are withdrawn at pre-set times,n
36、eutralized and cultured to determine survivors, using standardprocedures. A D-value is calculated from the post exposuresurvivor data utilizing published and accepted methods.3.2 This test method involves testing a high count suspen-sion of a microorganism as the initial challenge inoculum; atleast
37、107to 108cfu/mL, to achieve a 106cfu/mL when addedto the reaction chamber and exposed to disinfectant and tosporicidal chemicals.3.3 Agrowth medium for the inoculum must produce a highnumbers of vegetative cells or spores within a reasonable timeperiod with consistent resistance to chemical disinfec
38、tants.3.4 Where possible agitation of the reaction chamber isrecommended.3.5 Currently a test temperature of 20 6 1C is recom-mended. This temperature is lower than most environmentaltemperatures in practice (room temperature). A more typicaltemperature range is suggested at 22 6 1C. The activity of
39、many antimicrobials is increased with increasing temperature.An alternative temperature may be selected for testing, butmust be controlled and constant.3.6 An alternative testing technique to single sequentialtimed samples may be included in execution of this methodbecause a major problem has occurr
40、ed with many reportedstudies. Many kill or survival curves have shown a rapid kill ofseveral logs after an exposure period expected to eliminatesurvivors, yet leaving a few survivors, usually ten or fewerranging to 1000. This number fluctuates for an extended timewith repeated sampling and has been
41、termed, tailing. A changefrom single sampling to replicate - unit sampling is recom-mended as a means to alleviate this problem.3.7 Repetition of the estimation of a survival curve isrecommended. Recommendations for three to five replicationswith sampling at five time points have been made.4. Signif
42、icance and Use4.1 The different procedures and methods are designed to beused to produce survival data after microorganisms are ex-posed to antimicrobial agents in order to calculate values thatcan be used to analyze and rationalize the effectiveness ofantimicrobial agents when tested using other, o
43、ften applied testmethods.4.2 The data from these test procedures may be used in theselection and design of other tests of effectiveness of antimi-crobial agents, some of which may be required by regulatoryagencies to establish specific claims. Basic kinetic informationabout killing rate often serves
44、 as the initial information onwhich a testing program can be built.5. Basic Materials and Reagents5.1 Some basic materials will be required regardless of thespecific method selected. This list may need to be supple-mented depending on the techniques selected.5.1.1 Colony Counter, any of several type
45、s may be used.5.1.2 Membrane Filter Holders and Microbially RetentiveMembranes, (0.22 m) with vacuum equipment for filtration.5.1.3 IncubatorAny incubator capable of maintaining atemperature within a 6 2C of the recommended optimaltemperature for the growth of a specific microorganism undertest.5.1.
46、4 A Glass Reaction Vessel, of appropriate size anddesign to permit required sampling.5.1.5 A Realistic Means of Agitation, such as a hot-platewith a magnetic stirring feature.5.1.6 Temperature Controlled Water Bath, with agitation,when available.5.1.7 Sterilizer.5.1.8 Spectrophotometer.5.1.9 TimersA
47、n interval timer, such as a stop watch fordetermining elapsed time to remove test samples from thereaction chamber.6. Additional Materials and Reagents6.1 Depending on the specific method used, additions mayhave to be made to the materials and reagents tested.6.1.1 Petri Dishes, 100 by 15 mm require
48、d for performingstandard plate count.NOTE 1Presterilizeddisposable plastic dishes are available from mostlocal laboratory supply houses.6.1.2 Bacteriologic Pipets, 10.0 and 2.2 or 1.1. mL capacity.Micropipet types may also be used.NOTE 2Presterilizeddisposable bacteriological pipets are availablefro
49、m most local laboratory supply houses.6.1.3 Liquid Media, appropriate for the test microorganism.A soybean casein digest agar3or equivalent may be used forculturing the test microorganism.6.1.4 Agar Plates, (spread- or pour-plates) of appropriate,optimal media for the test microorganism for culture of test andcontrol samples.6.1.5 Neutralizer Solution, specific for each antimicrobialtested incorporated into diluent and optionally into recoverymedium.6.1.6 Test Tubes, with closures of appropriate size forsamples and ten-fold dilution of samples.6.1.7 A Selection of Flas
