1、Designation: E 1326 08Standard Guide forEvaluating Nonconventional Microbiological Tests Used forEnumerating Bacteria1This standard is issued under the fixed designation E 1326; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the y
2、ear 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 producersof nonconventional tests in determining the applicabi
3、lity of thetest for processing different types of samples and evaluating theaccuracy of the results. Conventional procedures such as theHeterotrophic (Standard) Plate Count, the Most ProbableNumber (MPN) method and the Spread Plate Count are widelycited and accepted for the enumeration of microorgan
4、isms.However, these methods have their limitations, such as perfor-mance time and degree of accuracy. It is these limitations thathave recently led to the marketing of a variety of non-conventional procedures, test kits and instruments.1.2 A conventional test is one that is widely accepted andpublis
5、hed as a standard microbiological method or relatedprocedure. A new, nonconventional test method will attempt toprovide the same information through the measurement of adifferent parameter. This guide is designed to assist investiga-tors in assessing the accuracy and precision of nonconventionalmeth
6、ods intended for the determination of microbial popula-tion densities or activities.1.3 It is recognized that the Heterotrophic Plate Count doesnot recover all microorganisms present in a product or a system(1, 2).2When this problem occurs during the characterizationof a microbiological population,
7、alternative standard enumera-tion procedures may be necessary, as in the case of sulfate-reducing bacteria. At other times, chemical methods thatmeasure the rates of appearance of metabolic derivatives or theutilization of contaminated product components might beindicated. In evaluating nonconventio
8、nal tests, the use of thesealternative standard procedures may be the only means avail-able for establishing correlation. In such cases, this guide canserve as a reference for those considerations.1.4 Since there are so many types of tests that could beconsidered nonconventional, it is impossible to
9、 recommend aspecific test protocol with statistical analyses for evaluating thetests. Instead, this guide should assist in determining whattypes of tests should be considered to verify the utility andidentify the limitations of the nonconventional test.2. Referenced Documents2.1 ASTM Standards:3D 38
10、70 Practice for Establishing Performance Characteris-tics for Colony Counting Methods in Microbiology4D 4012 Test Method for Adenosine Triphosphate (ATP)Content of Microorganisms in WaterD 5245 Practice for Cleaning Laboratory Glassware, Plas-ticware, and Equipment Used in Microbiological AnalysesD
11、5465 Practice for Determining Microbial Colony Countsfrom Waters Analyzed by Plating MethodsE 691 Practice for Conducting an Interlaboratory Study toDetermine the Precision of a Test Method3. Summary of Guide3.1 ASTM standard practices are referenced for use byproducers and users to determine the po
12、tential utility of thenonconventional test. Users of tests who are unequipped forperforming standard microbiological tests are given recom-mendations for seeking out microbiological laboratories thatcould perform collaborative studies to evaluate and verify theinformation generated with the nonconve
13、ntional tests.4. Significance and Use4.1 This guide should be used by producers and potentialproducers of nonconventional tests to determine the accuracy,selectivity, specificity, and reproducibility of the tests, asdefined in Practices E 691 and D 3870. Results of such studiesshould identify the li
14、mitations and indicate the utility orapplicability of the nonconventional test, or both, for use ondifferent types of samples.4.2 Nonconventional test users and potential users shouldemploy this guide to evaluate results of the nonconventionaltest as compared to their present methods. Practices D 52
15、45and D 5465 should be reviewed in regards to the conventionalmicrobiological methods employed. If conventional methods1This guide is under the jurisdiction ofASTM Committee E35 on Pesticides andAlternative Control Agents and is the direct responsibility of Subcommittee E35.15on Antimicrobial Agents
16、.Current edition approved Oct. 1, 2008. Published October 2008. Originallyapproved in 1990. Last previous edition approved in 2006 as E 1326 06.2The boldface numbers in parentheses refer to the list of references at the end ofthis guide.3For referenced ASTM standards, visit the ASTM website, www.ast
17、m.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.4Withdrawn.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United State
18、s.have not been used for monitoring the systems, then guidelinesare included for obtaining microbiological expertise.4.3 Utilization of a nonconventional test may reduce thetime required to determine the microbiological status of thesystem and enable an improvement in the overall operatingefficiency
19、. In many cases, the findings of a significantly highlevel of bacteria indicates the need for an addition of anantimicrobial agent. By accurately determining this in a shortertime period than by conventional methods, treatment withantimicrobial agents may circumvent more serious problemsthan if the
20、treatment were postponed until conventional resultswere available. If the antimicrobial treatment program relies onan inaccurate nonconventional test, then unnecessary loss ofproduct and problems associated with inappropriate selectionor improper dosing with antimicrobial agents would exist.4.4 Sinc
21、e many methods based on entirely different chemi-cal and microbiological principles are considered, it is notpossible to establish a unique design and recommend a specificmethod of statistical analyses for the comparisons to be made.It is only possible to present guides that should be followedwhile
22、performing the experiments. It is also recommended thata statistician be involved in the study.5. Procedures5.1 In order to determine the utility of the nonconventionaltest, evaluate and compare the results to those obtained with apreviously accepted standard method. The Heterotrophic PlateCount (Pr
23、actice D 5465) may be entirely satisfactory for thispurpose (3); however, understand its limitations before it isused as the basis for evaluating methods that measure otherparameters indicative of microbial life (metabolic activity,concentration of cell constituents, or whole cell numbers).Several m
24、ethods used for the Heterotrophic Plate Count arelisted in Table 1. When the Heterotrophic Plate Count is not asuitable refereed method, Adenosine Triphosphate Concentra-tion (Test Method D 4012) or the Most Probable Number(MPN) technique (9) may be more appropriate. Alternativestandard enumeration
25、methods or methods for measuring therate of the appearance of derivatives or the rate of disappear-ance of components of the product in which the microbialcontamination is being measuredwhere such phenomena areknown to be correlated to microbial contamination levelsmay also be used as referee method
26、s for assessing the accuracyand precision of a novel nonconventional method. No singlemethod is universally applicable; consequently, it is imperativeto determine the rationale for employing any given measure-ment procedure and to select a standard that will permit thedetermination of whether or not
27、 the nonconventional methodachieves the objectives defined in the scope of the procedure.5.2 A knowledge of standard microbiological technique isrequired for this procedure. If that expertise is not currentlyavailable in-house, consult an outside testing laboratory. Manyindustrial microbiology labor
28、atories are certified for the analy-sis of drinking water by the EPA or the state government (alisting of these laboratories can be obtained from the regionalEPA office or the state government). There are also othermicrobiology laboratories that specialize in processing samplesfrom different industr
29、ies; these are often listed as“LaboratoriesTesting” in the telephone book. It is importantthat this document be referenced when undertaking an evalu-ation with an outside laboratory.5.3 For each method, first make an enumeration of all majorsources of variability. For example, if a nonconventional t
30、estmethod is involved and if more than a single analysis can beconducted with a single test, consider the variability within andbetween tests. For plates, it is important to consider thevariability between plates obtained from aliquots of the samesample. It is also important to prepare samples cover
31、ing theentire range of values (for example, counts per milliliter) ofinterest. Each such value is referred to as a level. Thus, thelevels must cover the range of interest.5.4 At each level, analyze replicate samples, both by themethod under study, and by the standard method. The numberof replicates
32、depends on the number of sources of variability.Thus, in the previous-mentioned example of nonconventionaltest, it would be advisable to analyze at each level at least tworeplicates of each (preferably more) in at least two nonconven-tional tests (preferably more). At the same time, analyzereplicate
33、s by the Heterotrophic Plate Count, resulting in severalreplicate plates. The scheme shown in Table 2 illustrates sucha procedure; in this case, three replicates are analyzed at anygiven level using three nonconventional tests, while fivereplicate plates are counted by the Heterotrophic Plate Count.
34、(These numbers will vary according to the method.)TABLE 1 Comparison of Selected Heterotrophic Plate Count Procedures for Samples from Various SourcesWater (6) Dairy (7) Environment (8) Food (9) Cosmetic (9) Paper (10) Pharmaceutical (11)Media TGE, SM, R2A or m-HPC SM SM or TGE SM ML TGE SCDDilution
35、, H2OKH2PO4+ MgCl2KH2PO4KH2PO4KH2PO4MLB H2OKH2PO4Incubation, C 35 6 0.5 20 or 28 (R2A) 32 6 1356 0.5 35 30 6 2366 0.5 3035Incubation, h 48 6 3726 4486 348 486 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 (M
36、embrane 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 available in dehydrated form)m-HPC = Formerly called m-SPC Agar (
37、used for membrane filtration)E 1326 0825.5 Using the example of Table 2, the data of the newmethod would be analyzed and compared with the Het-erotrophic Plate Count method for determining precision, aswell as (1) within-test variability; (2) between-test variability;and (3) between-plate variabilit
38、y.5.6 Again, using the example of Table 2, the nine values bythe new method and the five values by the Heterotrophic PlateCount are averaged for all levels and then plotted. A curve,using appropriate statistical procedures, must then be fitted tothese points. This curve is the calibration line of th
39、e newmethod versus the Heterotrophic Plate Count, and it can beused to convert values obtained by the new method intoequivalent units of the Heterotrophic Plate Count.6. Report6.1 The standard deviations obtained by the new methodcan be converted, by appropriate statistical procedures, intoequivalen
40、t units of the standard method by using the calibrationline for conversion. A comparison with the standard methodcan then be made to determine the precision of the newmethod.6.2 In view of the complexity of the problem and variety ofsituations that can arise, it is not possible to recommend furtherp
41、rocedures and statistical methods, or both. A more detaileddiscussion of statistical methods may be found in the StatisticalManual of the Association of Offcial Analytical Chemists (4)and in Chapter 14, “The Comparison of Method of Measure-ments,” of The Statistical Analysis of Experimental Data (5)
42、.7. Precision and Bias7.1 A precision and bias statement cannot be made for thisguide.REFERENCES(1) Roszak, D. B., and Colwell, R. R., “Survival Strategies of Bacteria inthe Natural Environment,” Microbiological Reviews, Vol 51, No. 3,Sept. 1987, pp. 365379.(2) Oliver, J. D., “The Viable but Noncult
43、urable State in Bacteria,”Journal of Microbiology, Vol 43, No. S (Special Issue), Feb. 2005, pp.93100.(3) Buck, J. D., “The Plate Count in Aquatic Microbiology,” Symposiumon Native Aquatic Bacteria: Enumeration, Activity, and Ecology,edited by J. W. Costerton and R. R. Colwell, ASTM STP 695, ASTM,19
44、79, pp. 1928.(4) Youden, W. J., and Steiner, E. H., Statistical Manual of the Associationof Offcial Analytical Chemists, Second Printing, Association ofOfficial Analytical Chemists, Arlington, VA 22209, 1979.(5) Mandel, J., The Statistical Analysis of Experimental Data, Dover,1984.(6) “Standard Meth
45、ods for the Examination of Water and Wastewater,”American Public Health Association, New York, NY, 19th ed., 1995 ormost current.(7) “Standard Methods for the Examination of Dairy Products,” AmericanPublic Health Association, New York, NY, 16th ed., 1993 or mostcurrent.(8) “Microbiological Methods f
46、or Monitoring the Environment,” Environ-mental Monitoring and Support Laboratory, Office of Research andDevelopment, U.S. Environmental Protection Agency, Cincinnati,Ohio, EPA 600/8-78-017, December 1978.(9) FDA Bacteriological Analytical Manual, Food and Drug Administra-tion Staff, 1995, AOAC Inter
47、national, Arlington, VA, 8th ed., or mostcurrent.(10) “Microbiological Examination of Process Water and Slush Pulp,”(proposed review of Official Method T631 om-79), Technical Asso-ciation of the Pulp and Paper Industry, Technology Park,Atlanta, GA,April 5, 1984, or most current.(11) “Microbial Limit
48、s-Total Aerobic Microbial Count,” U.S. Pharmaco-poeia XXIII-National Formulary, U.S. Pharmacopoeia Convention,Inc., Rockville, MD, 1995 or most current.ASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentionedin this standard. Us
49、ers of this standard are expressly advised that determination of the validity of any such patent rights, and the riskof infringement of such rights, are entirely their own responsibility.This standard is subject to revision at any time by the responsible technical committee and must be reviewed every five years andif not revised, either reapproved or withdrawn. Your comments are invited either for revision of this standard or for additional standardsand should be addressed to ASTM International Headquarters. Your comments will receive careful consideration at a meet
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