1、Designation: E1326 08E1326 13Standard Guide forEvaluating Nonconventional 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,
2、 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.1. Scope1.1 The purpose of this guide is to assist users and producers of nonconventional tests in determining the ap
3、plicability of thetest for processing different types of samples and evaluating the accuracy of the results. Conventional procedures such as theHeterotrophic (Standard) Plate Count, the Most Probable Number (MPN) method and the Spread Plate Count are widely cited andaccepted for the enumeration of m
4、icroorganisms. However, these methods have their limitations, such as performance time anddegree of accuracy. It is these limitations that have recently led to the marketing of a variety of non-conventional procedures, testkits and instruments.1.2 Aconventional test is one that is widely accepted an
5、d published as a standard microbiological method or related procedure.A new, nonconventional test method will attempt to provide the same information through the measurement of a differentparameter. This guide is designed to assist investigators in assessing the accuracy and precision of nonconventi
6、onal methodsintended for the determination of microbial population densities or activities.1.3 It is recognized that the Heterotrophic Plate Count does not recover all microorganisms present in a product or a system (1,2).2 When this problem occurs during the characterization of a microbiological po
7、pulation, alternative standard enumerationprocedures may be necessary, as in the case of sulfate-reducing bacteria. At other times, chemical methods that measure the ratesof appearance of metabolic derivatives or the utilization of contaminated product components might be indicated. In evaluatingnon
8、conventional tests, the use of these alternative standard procedures may be the only means available for establishingcorrelation. In such cases, this guide can serve as a reference for those considerations.1.4 Since there are so many types of tests that could be considered nonconventional, it is imp
9、ossible 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 a
10、re to be regarded as standard. No other units of measurement are included in this standard.2. Referenced Documents2.1 ASTM Standards:3D1129 Terminology Relating to WaterD3870 Practice for Establishing Performance Characteristics for Colony Counting Methods in Microbiology (Withdrawn 2000)4D4012 Test
11、 Method for Adenosine Triphosphate (ATP) Content of Microorganisms in WaterD5245 Practice for Cleaning Laboratory Glassware, Plasticware, and Equipment Used in Microbiological AnalysesD5465 Practice for Determining Microbial Colony Counts from Waters Analyzed by Plating MethodsD7687 Test Method for
12、Measurement of Cellular Adenosine Triphosphate in Fuel, Fuel/Water Mixtures, and Fuel-AssociatedWater with Sample Concentration by FiltrationD7847 Guide for Interlaboratory Studies for Microbiological Test MethodsE691 Practice for Conducting an Interlaboratory Study to Determine the Precision of a T
13、est MethodE2694 Test Method for Measurement of Adenosine Triphosphate in Water-Miscible Metalworking Fluids1 This guide is under the jurisdiction of ASTM Committee E35 on Pesticides, Antimicrobials, and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicrobia
14、l Agents.Current edition approved Oct. 1, 2008Oct. 1, 2013. Published October 2008October 2013. Originally approved in 1990. Last previous edition approved in 20062008 asE1326 06.E1326 08. DOI: 10.1520/E1326-08.10.1520/E1326-13.2 The boldface numbers in parentheses refer to the list of references at
15、 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 standards Document Summary page on the ASTM website.4 The last approved version of this hi
16、storical standard is referenced on www.astm.org.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 accurate
17、ly, 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 States1
18、E2756 Terminology Relating to Antimicrobial and Antiviral Agents3. Terminology3.1 For definitions of terms used in this guide refer to Terminologies D1129 and E2756.4. Summary of Guide4.1 ASTM standard practices are referenced for use by producers and users to determine the potential utility of then
19、onconventional test. Users of tests who are unequipped for performing standard microbiological tests are given recommendationsfor seeking out microbiological laboratories that could perform collaborative studies to evaluate and verify the informationgenerated with the nonconventional tests.5. Signif
20、icance and Use5.1 This guide should be used by producers and potential producers of nonconventional tests to determine the accuracy,selectivity, specificity, and reproducibility of the tests, as defined in Practices E691 and D3870. Results of such studies shouldidentify the limitations and indicate
21、the utility or applicability of the nonconventional test, or both, for use on different types ofsamples.5.2 Nonconventional test users and potential users should employ this guide to evaluate results of the nonconventional test ascompared to their present methods. Practices D5245 and D5465 should be
22、 reviewed in regards to the conventional microbiologicalmethods employed. If conventional methods have not been used for monitoring the systems, then guidelines are included forobtaining microbiological expertise.5.3 Utilization of a nonconventional test may reduce the time required to determine the
23、 microbiological status of the system andenable an improvement in the overall operating efficiency. In many cases, the findings of a significantly high level of bacteriaindicates the need for an addition of an antimicrobial agent. By accurately determining this in a shorter time period than byconven
24、tional methods, treatment with antimicrobial agents may circumvent more serious problems than if the treatment werepostponed until conventional results were available. If the antimicrobial treatment program relies on an inaccurate nonconventionaltest, then unnecessary loss of product and problems as
25、sociated with inappropriate selection or improper dosing with antimicrobialagents would exist.5.4 Since many methods based on entirely different chemical and microbiological principles are considered, it is not possibleto establish a unique design and recommend a specific method of statistical analy
26、ses 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.5.5 There are various ways for categorizing microbiological test methods. One valid approach is to dif
27、ferentiate betweenmethods intended to quantify a particular microbe from those intended to quantify overall bioburden.5.5.1 Methods used to quantify a single microbe typically can be evaluated for precision (Practice E691). Even though it isunlikely that reference standards exist, often these method
28、s can also be evaluated for bias relative to other methods used to detectthe same microbe.5.5.2 Methods used to quantify total populations are more problematic in terms of precision and bias testing. Guide D7847addresses many of the factors that confound efforts to determine the precision of microbi
29、ological test methods used to quantifymicrobial contamination in fuels and fuel systems. Many of these issues are broadly relevant to the challenge of developingrelevant precision terms for microbiological test methods used to quantify total bioburdens in industrial systems.6. Procedures6.1 In order
30、 to determine the utility of the nonconventional test, evaluate and compare the results to those obtained with apreviously accepted standard method.The Heterotrophic Plate Count (Practice D5465) may be entirely satisfactory for this purpose(3); however, understand its limitations before it is used a
31、s the basis for evaluating methods that measure other parametersindicative of microbial life (metabolic activity, concentration of cell constituents, or whole cell numbers). Several methods usedfor the Heterotrophic Plate Count are listed in Table 1. When the Heterotrophic Plate Count is not a suita
32、ble refereed method,Adenosine Triphosphate Concentration (Test MethodMethods D4012, E2694, and D7687) or the Most Probable Number (MPN)technique (4) may be more appropriate. Alternative standard enumeration methods or methods for measuring the rate of theappearance of derivatives or the rate of disa
33、ppearance of components of the product in which the microbial contamination is beingmeasuredwhere such phenomena are known to be correlated to microbial contamination levelsmay also be used as refereemethods for assessing the accuracy and precision of a novel nonconventional method. No single method
34、 is universally applicable;consequently, it is imperative to determine the rationale for employing any given measurement procedure and to select a standardthat will permit the determination of whether or not the nonconventional method achieves the objectives defined in the scope ofthe procedure.6.2
35、Aknowledge of standard microbiological technique is required for this procedure. If that expertise is not currently availablein-house, consult an outside testing laboratory. Many industrial microbiology laboratories are certified for the analysis of drinkingE1326 132water by the EPA or the state gov
36、ernment (a listing of these laboratories can be obtained from the regional EPA office or the stategovernment). There are also other microbiology laboratories that specialize in processing samples from different industries; theseare often listed as “LaboratoriesTesting” in the telephone book. It is i
37、mportant that this document be referenced whenundertaking an evaluation with an outside laboratory.6.3 For each method, first make an enumeration of all major sources of variability. For example, if a nonconventional testmethod is involved and if more than a single analysis can be conducted with a s
38、ingle test, consider the variability within andbetween tests. For plates, it is important to consider the variability between plates obtained from aliquots of the same sample. Itis also important to prepare samples covering the entire range of values (for example, counts per milliliter) of interest.
39、 Each suchvalue is referred to as a level. Thus, the levels must cover the range of interest.6.4 At each level, analyze replicate samples, both by the method under study, and by the standard method. The number ofreplicates depends on the number of sources of variability. Thus, in the previous-mentio
40、ned example of nonconventional test, itwould be advisable to analyze at each level at least two replicates of each (preferably more) in at least two nonconventional tests(preferably more). At the same time, analyze replicates by the Heterotrophic Plate Count, resulting in several replicate plates. T
41、hescheme shown in Table 2 illustrates such a procedure; in this case, three replicates are analyzed at any given level using threenonconventional tests, while five replicate plates are counted by the Heterotrophic Plate Count. (These numbers will varyaccording to the method.)6.5 Using the example of
42、 Table 2, the data of the new method would be analyzed and compared with the Heterotrophic PlateCount method for determining precision, as well as (1) within-test variability; (2) between-test variability; and (3) between-platevariability.6.6 Again, using the example of Table 2, the nine values by t
43、he new method and the five values by the Heterotrophic Plate Countare averaged for all levels and then plotted. A curve, using appropriate statistical procedures, must then be fitted to these points.This curve is the calibration line of the new method versus the Heterotrophic Plate Count, and it can
44、 be used to convert valuesobtained by the new method into equivalent units of the Heterotrophic Plate Count.7. Report7.1 The standard deviations obtained by the new method can be converted, by appropriate statistical procedures, into equivalentunits of the standard method by using the calibration li
45、ne for conversion.Acomparison with the standard method can then be madeto determine the precision of the new method.TABLE 1 Comparison of Selected Heterotrophic Plate Count Procedures for Samples from Various SourcesWater (5) Dairy (6) Environment (7) Food (4) Cosmetic (4) Paper (8) Pharmaceutical (
46、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 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 Pla
47、te) 1012 10+ 1215 Spread Plates 1520 152015 (Spread Plates)5 (Membrane Filter)TGE = Tryptone Glucose Extract AgarSM = Standard Methods Agar (Tryptone Glucose Yeast Agar)ML = Modified Letheen AgarMLB = Modified Letheen BrothSCD = Soybean Casein Digest AgarR2A = Low-Nutrient Media (which may not be av
48、ailable in dehydrated form)m-HPC = Formerly called m-SPC Agar (used for membrane filtration)TABLE 2 Scheme for Analysis at a Given LevelTestNew Method Heterotrophic Plate CountReplicateWithin Test Determination Plate Determination1 1 1 1 12 2 2 23 3 3 34 45 52 1 42 53 63 1 72 83 9E1326 1337.2 In vie
49、w of the complexity of the problem and variety of situations that can arise, it is not possible to recommend furtherprocedures and statistical methods, or both. A more detailed discussion of statistical methods may be found in the StatisticalManual of the Association of Offcial Analytical Chemists(10) and in Chapter 14, “The Comparison of Method of Measurements,”of The Statistical Analysis of Experimental Data(11).8. Precision and Bias8.1 A precision and bias statement cannot be made for this guide.REFERENCES(1) Roszak, D. B., and Colwell, R. R.,
