1、Designation: E 1891 97 (Reapproved 2002)Standard 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 E 1891; the number immediately following t
2、he designation indicates 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 (e) indicates an editorial change since the last revision or reapproval.INTRODUCTIONA variety of testing p
3、rocedures have been devised almost from the beginning of disinfection andantisepsis as disciplines. From the first, there was a recognition of the importance of time and ratesof kill. After many decades and numerous test procedures involving carriers, the approach ofestablishing a death rate curve (
4、often described as a survivor curve) is reclaiming its importance inestablishing 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, processingcalculations and rates of kill ar
5、e discussed in many texts. 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 d
6、etermining D-values and 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 appl
7、ication 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 killcurve. The user should establish a protocol for the process that best fits their needs.An experimental kill curve provides data f
8、or a calculated D-value 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 tes
9、ts, while Europeans have emphasized suspension tests combined with practicalapplied 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 theactivity of antimicrobials. The earl
10、y history of microbiology 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
11、.Kronig and Paul (1897) 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 ofs
12、urvivors after disinfectant exposure, when plotted against time of treatment, produced a straight linethat showed similarity to chemical, eqstetimolecular reactions. Distortions in the expected straight-linereactions were noted by Chick as well as in subsequent investigations. Over the years, the mo
13、stcommon type of deviation 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 tha
14、t can be used to calculateD-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, veget
15、ative bacterial cells, or mycobacteria in devising thermal processing toeliminate Clostidium botulinium in the canning industry. They also devised end-point analysis forinterpretation of the results of heat exposure and for processing calculations. Their procedure involvedsampling multiple tubes or
16、other containers of product and analysis of the number remaining positiveto determine the number of survivors by Most Probable Number (MPN) analysis using the pattern ofpositive and negative tubes. (1)2This analysis is done after an exposure period when there are fewerbacterial cells or spores in th
17、e container and positive and negative tubes 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
18、is exposed to a disinfectantsterilant and 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
19、often used (1, 2, 3). A common method derived 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 construc
20、t a survivor curve and plotted to determineD-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
21、 h, denotes the effect of dilution on theactivity 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
22、determining the number ofsurvivors after 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 defin
23、e a survivor curve and tosubsequently calculate 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
24、from exposure at differentdilutions of antimicrobial can be used to show the effect ofdilution by calculation of the concentration exponent, h (2).1.1.2 As an example of potential use of kill curve data, thepublished FDA, OTC Tentative Final Monograph for Health-Care Antiseptic Drug Products, Propos
25、ed 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 reaction mixture is stirre
26、d 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 antimicrobial ingredients in
27、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 as thestandard.1.4 This standard does not purport to address all of thesafety concerns, if any, associate
28、d 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 Definitions:2.1.1 D-value or decimal reduction time(often referred toas log death time)
29、 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 Fn = Fraction negative (FN) data(quantal data) areexperimental results in the for
30、m of a dichotomous response: theunit tested is either positive (showing growth) or negative(showing no growth).2.1.3 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 a1This gui
31、de is under the jurisdiction of ASTM Committee E35 on Pesticides and Alternative Control Agents and is the direct responsibility of Subcommittee E35.15 onAntimicrobial Agents.Current edition approved Oct. 10, 2002. Published March 2003. Originally approved in 1997. Last previous edition approved in
32、1997 as E 1891 97.2The boldface numbers given in parentheses refer to a list of references at the end of the text.E 1891 97 (2002)2comparable degree of death in a bacterial suspension at leasttwo different concentrations is measured (D-value) (6).2.1.4 Most Probable Number (MPN)data in which afracti
33、on 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 Method3.1 This test method is conducted on selected microbialspecies cultured to produce high-count susp
34、ensions 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,neutralized and cultured to determine survivors, using standardprocedures. A D-value is calculated fro
35、m 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 107to 108cfu/mL, to achieve a 106cfu/mL when addedto the reaction chamber and exposed to disinfectant
36、 and tosporicidal chemicals.3.3 A growth medium for the inoculum must produce a highnumbers of vegetative cells or spores within a reasonable timeperiod with consistent resistance to chemical disinfectants.3.4 Where possible agitation of the reaction chamber isrecommended.3.5 Currently a test temper
37、ature 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 study of manyantimicrobials is increased with increasing temperature. Analternative temperature may be selected
38、 for testing, but must becontrolled and constant.3.6 An alternative testing technique to single sequentialtimed samples may be included in execution of this methodbecause a major problem has occurred with many reportedstudies. Many kill or survival curves have shown a rapid kill ofseveral logs after
39、 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 termed, tailing. A changefrom single sampling to replicate - unit sampling is recom-mended as a means t
40、o 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. Significance and Use4.1 The different procedures and methods are designed to beused to produce survival data
41、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, often applied testmethods.4.2 The data from these test procedures may be used in theselection and design
42、 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 as the initial information onwhich a testing program can be built.5. Materials and Reagents5.1 Some ba
43、sic 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 types may be used.5.1.2 Membrane Filter Holders and Microbially RetentiveMembranes, (0.22 m) with vacuum equipmen
44、t 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.5.1.4 A Glass Reaction Vessel, of appropriate size anddesign to permit required sampling.5.1.5 A Realistic Means
45、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 TimersAn interval timer, such as a stop watch fordetermining elapsed time to remove test samples from thereaction ch
46、amber.6. 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 required for performingstandard plate count3.6.1.2 Bacteriologic Pipets, 10.0 and 2.2 or 1.1. mL capacity.Micropipet types may
47、also be used4.6.1.3 Liquid Media, appropriate for the test microorganism.A soybean case in digest agar5or 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 samp
48、les.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 Flasks and Tubes, required for culturingof the test
49、microorganisms.6.1.8 Diluent Tubes, for dilution of the test and controlsamples. Diluent may have phosphate-buffered normal salineor other appropriate diluent for specific microorganisms andneutralizers specific for the test disinfectant should be added tothe diluent.6.1.9 An Automatic Mixer, such as a Vortex mixer.3Presterilizeddisposable plastic dishes are available from most local laboratorysupply houses.4Presterilizeddisposable bacteriological pipets are available from most locallaboratory supply houses.5United States Pharmacoepia XX; United States Pharmacopeial Conve
