ASTM E1326-2015a Standard Guide for Evaluating Non-culture Microbiological Tests《评估非培养微生物试验的标准指南》.pdf

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1、Designation: E1326 15E1326 15aStandard Guide forEvaluating Non-culture Microbiological Tests Used forEnumerating Bacteria1This standard is issued under the fixed designation E1326; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, th

2、e 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.1. Scope1.1 The purpose of this guide is to assist users and producers of non-culture microbiological tests in determini

3、ng theapplicability of the test for processing different types of samples and evaluating the accuracy of the results. Culture test proceduressuch as the Heterotrophic (Standard) Plate Count, the Most Probable Number (MPN) method and the Spread Plate Count arewidely cited and accepted for the enumera

4、tion of microorganisms. However, these methods have their limitations, such asperformance time and degree of accuracy. Moreover time. Moreover, any given culture test method typically recovers only afractionportion of the total viable microbes present in a sample. It is these limitations that have r

5、ecently led to the marketing ofa variety of non-culture procedures, test kits and instruments.1.2 Culture test methods estimate microbial population densities based on the ability of mircoorganisms in a sample toproliferate in or on a specified growth medium, under specified growth conditions. Non-c

6、ulture test methods attempt to providethe same or complimentary information through the measurement of a different parameter. This guide is designed to assistinvestigators in assessing the accuracy and precision of non-culture methods intended for the determination of microbial populationdensities o

7、r activities.1.3 It is recognized that the Heterotrophic Plate Count (HPC) does not recover all microorganisms present in a product or asystem (1, 2).2 When this problem occurs during the characterization of a microbiological population, alternative standardenumeration procedures may be necessary, a

8、s in the case of sulfate-reducing bacteria. At other times, chemical methods thatmeasure the rates of appearance of metabolic derivatives, the utilization of contaminated product components or genetic profileof the microbial population might be indicated. In evaluating non-culture tests, it is possi

9、ble that the use of these alternativestandard procedures might be the only means available for establishing correlation. In such cases, this guide can serve as areference for those considerations.1.4 Because there are so many types of tests that could be considered non-culture based, it is impossibl

10、e to recommend a specifictest protocol with statistical analyses for evaluating the tests. Instead, this guide should assist in determining what types of testsshould be considered to verify the utility and identify the limitations of the nonconventional test.1.5 The values stated in SI units are to

11、be regarded as standard. No other units of measurement are included in this standard.2. Referenced Documents2.1 ASTM Standards:3D1129 Terminology Relating to WaterD4012 Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in WaterD5245 Practice for Cleaning Laboratory Glassware, Pl

12、asticware, and Equipment Used in Microbiological AnalysesD5465 Practice for Determining Microbial Colony Counts from Waters Analyzed by Plating MethodsE177 Practice for Use of the Terms Precision and Bias in ASTM Test MethodsE691 Practice for Conducting an Interlaboratory Study to Determine the Prec

13、ision of a Test MethodE1601 Practice for Conducting an Interlaboratory Study to Evaluate the Performance of an Analytical MethodE2756 Terminology Relating to Antimicrobial and Antiviral Agents1 This guide is under the jurisdiction of ASTM Committee E35 on Pesticides, Antimicrobials, and Alternative

14、Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved May 1, 2015Oct. 1, 2015. Published June 2015November 2015. Originally approved in 1990. Last previous edition approved in 20132015 asE1326 13.E1326 15. DOI: 10.1520/E1326-15.10.1520

15、/E1326-15A.2 The boldface numbers in parentheses refer to the list of references at the end of this guide.3 For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to the standar

16、ds Document Summary page on the ASTM website.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. Becauseit may not be technically possible to adequately depict all changes accurately,

17、 ASTM recommends that users consult prior editions as appropriate. In all cases only the current versionof the standard as published by ASTM is to be considered the official document.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States13.

18、Terminology3.1 Defintions:3.1.1 For definitions of terms used in this guide refer to Terminologies D1129, E2756, and E177.3.2 Abbreviations:3.2.1 HPCHeterotrophic Plate Count4. Summary of Guide4.1 ASTM standard methods and practices are referenced for use by producers and users in order to determine

19、 the potentialutility of a non-standard, non-culture test.4.2 Recognizing that potential users of non-culture test methods might not have the resources with which or capabilities forevaluating the utility of non-standard, non-culture test methods, recommendations are provided to assist those users i

20、n identifyingthe capabilities that qualify microbiological laboratories to perform collaborative studies to evaluate those methods.5. Significance and Use5.1 This guide should be used by producers and potential producers of non-culture tests to determine the accuracy, selectivity,specificity, and re

21、producibilityprecision of the tests, as defined in Practice E691. Results of such studies should identify thelimitations and indicate the utility or applicability of the non-culture test, or both, for use on different types of samples.5.2 Non-culture test users and potential users should employ this

22、 guide to evaluate results of the non-culture test as comparedto their present methods. Practices D5245 and D5465 should be reviewed in regards to the microbiological methods employed.If culture methods have not been used for monitoring the systems, then guidelines are included for obtaining microbi

23、ologicalexpertise.5.3 Utilization of a non-culture test can reduce the time required to determine the microbiological status of the system and detectmicrobe that are not detected by culture testing. Consequently, non-culture tests can contribute to the improvement in the overalloperating efficiency

24、of microbial contamination condition monitoring and diagnostic efforts, and microbicide performanceevaluations.5.4 Detecting microbial contamination levels that exceed predetermined upper control limits indicates the need for an additionof an antimicrobial agent or other corrective maintenance actio

25、n. By accurately determining this in a shorter time period than ispossible than by culture methods, treatment with antimicrobial agents may circumvent more serious problems than if the treatmentwere postponed until culture results were available. If the antimicrobial treatment program relied on an i

26、naccurate non-culture test,then unnecessary loss of product and problems associated with inappropriate selection or improper dosing with antimicrobialagents would exist.5.5 Since many methods based on entirely different chemical and microbiological principles are considered, it is not possibleto est

27、ablish a unique design and recommend a specific method of statistical analyses for the comparisons to be made. It is onlypossible to present guides that should be followed while performing the experiments. It is also recommended that a statistician beinvolved in the study.6. Procedures6.1 Practice E

28、1601 provides guidance on the evaluation of analytical method performance. The guidance provided belowamplifies the processes described in Practice E1601 as they apply to microbiological test methods.6.2 Although the heterotrophic plate count (HPC) has been used historically to determine the utility

29、 of newly developednon-culture methods, and can be an appropriate reference method in many cases (3), there are cases for which HPC is not anappropriate referreference method6.2.1 The choice of referee method to use for validating a new or proposed non-culture method should be determined based onthe

30、 parameter the new method purports to be measuring.6.2.2 Several methods used for the HPC are listed in Table 1.6.2.3 When none of the Table 1 variations of the HPC (Heterotrophic Plate Count) are suitable refereedreference methods,Adenosine Triphosphate Concentration (Test Method D4012) or the Most

31、 Probable Number (MPN) technique (7) may be moreappropriate.6.2.4 Alternative standard enumeration methods or methods for measuring the rate of the appearance of derivatives or the rateof disappearance of components of the product in which the microbial contamination is being measuredwhere such phen

32、omenaare known to be correlated to microbial contamination levelsmay also be used as referee methods for assessing the accuracy andprecision of a novel non-culture method.6.2.5 No single method is universally applicable; consequently, it is imperative to determine the rationale for employing anygive

33、n measurement procedure and to select a standard that will permit the determination of whether or not the nonconventionalmethod achieves the objectives defined in the scope of the procedure.E1326 15a26.3 Aknowledge of standard microbiological technique is required in order to conduct microbiological

34、 test method evaluations.If that expertise is not currently available in-house, consult an outside testing laboratory.6.3.1 Many industrial microbiology laboratories are certified for the analysis of drinking water by the EPA or the stategovernment, or both (a listing of these laboratories can be ob

35、tained from the regional EPA office or the state government).6.3.2 These and other independent microbiology laboratories often specialize in processing samples from different industries6.3.3 Suitable microbiology laboratories are typically often listed as “LaboratoriesTesting” in the telephone book

36、or indirectories such as the ASTM International Directory of Testing Laboratories3. It is important that this document be referencedwhen undertaking an evaluation with an outside laboratory.6.4 For each method, first list of all known major sources of variability.6.4.1 For example, major sources of

37、variability can include:6.4.1.1 Sample heterogeneitynon-uniform distribution of physical (for example: temperature and viscosity), chemical (forexample: layering caused by eutrophication) and microbiological (for example: population density, taxonomic diversity andphysiological state of microbes).6.

38、4.1.2 Sample perishabilitychanges in taxonomic profile (diversity and relative abundance of individual taxa contained insample).6.4.1.3 Storage and handling conditions.6.4.2 Measures must be taken to minimize the individual and net contributions of these factors when evaluating test methodprecision.

39、6.4.3 When designing a non-culture test method evaluation, ensure that the microbial bioburdens in the samples cover the newmethods expected quantification range. Minimally the test plan shall include three samples (test levels) of each test matrix forwhich the candidate method is expected to be app

40、ropriate:Low bioburden microbial contamination just above the methods expected lowlower limit of quantificationMedium bioburden microbial contamination in the mid-range of the methods detection rangeHigh bioburden microbial contamination near the upper limits of the methods detection range6.4.3.1 Fo

41、r the purposes of this practice, each bioburden range is a test level. Thus the levels must cover the range ofinterest.interest for each intended application.6.4.3.2 A test matrix is the type material in which the microbes are found (for example: water, industrial fluids, soils, coatings,etc.)6.5 At

42、 each test level, analyze replicate samples, by both the method being evaluated, and by the standard or reference method.The number of replicates depends on the number of sources of variability. Thus, in the previous-mentioned example of non-culturetest (6.4.2), it is necessary to analyze at least t

43、wo replicate samples at each level (preferably more) by both the reference andcandidate method.6.5.1 The standard or reference method used will often be one of the methods listed in Table 1, however, in matrices from whichculture test results are likely to be inaccurate or suspected of being inaccur

44、ate, data from the candidate method can be comparedwith data form non-microbiological parameters known to covary with bioburden.6.6 A suitable test plan is shown in Table 2.6.6.1 In this example, at each level, three replicates are analyzed by the non-culture, candidate method and by the HPC method.

45、These numbers of replicates will vary according to the method.6.6.2 Although Practice E1601 prescribes a minimum of duplicate tests per analyst/laboratory, a minimum of three replicatessubstantially improves the robustness of the method validation effort.TABLE 1 Comparison of Selected Heterotrophic

46、Plate Count Procedures for Samples from Various SourcesWater (4) Dairy (5) Environment (6) Food (7) Cosmetic (7) Paper (8) Pharmaceutical (9)Media TGE, SM, R2A or m-HPC SM SM or TGE SM ML TGE SCDDilution, H2O KH2PO4 + MgCl2 KH2PO4 KH2PO4 KH2PO4 MLB H2O KH2PO4Incubation, C 35 0.5 20 or 28 (R2A) 32 1

47、35 0.5 35 30 2 36 0.5 3035Incubation, h 48 3 72 4 48 3 48 48 2 48 48 4872(bottled water)72168 (R2A medium)Amount of Agar, mL 1012 (Pour Plate) 1012 10+ 1215 Spread Plates 1520 152015 (Spread Plates)5 (Membrane Filter)TGE = Tryptone Glucose Extract AgarSM = Standard Methods Agar (Tryptone Glucose Yea

48、st Agar)ML = Modified Letheen AgarMLB = Modified Letheen BrothSCD = Soybean Casein Digest AgarR2A = Low-Nutrient Media (which may not be available in dehydrated form)m-HPC = Formerly called m-SPC Agar (used for membrane filtration)E1326 15a36.6.3 A full interlaboratory study requires at least 30 deg

49、rees of freedom, including participation of no fewer than sixlaboratories and a sufficient range of samples to address the issues outlined in 6.4. See Table 2 and Practice E691.6.6.4 For initial test method robustness evaluations it is sufficient to have two participants (either individual analysts or differentlaboratories) so that preliminary repeatability and reproducibility estimates can be computed.6.6.5 Although the correlation between the candidate test parameter and bioburden can be determined from data produced byreplicate