1、Designation: D5465 93 (Reapproved 2012)D5465 16Standard PracticePractices forDetermining Microbial Colony Counts from Waters Analyzedby Plating Methods1This standard is issued under the fixed designation D5465; the number immediately following the designation indicates the year oforiginal adoption o
2、r, 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.1. Scope Scope*1.1 These practices cover recommended procedures for counting colonies and
3、reporting colony-forming units (CFU) onmembrane filters (MF) and standard pour and spread plates.1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.3 This standard does not purport to address all of the safety concerns, if
4、any, associated with its use. It is the responsibilityof the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatorylimitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D1129 Terminology Relating to WaterD5259 Test Metho
5、d for Isolation and Enumeration of Enterococci from Water by the Membrane Filter ProcedureD5392 Test Method for Isolation and Enumeration of Escherichia Coli in Water by the Two-Step Membrane Filter ProcedureD6161 Terminology Used for Microfiltration, Ultrafiltration, Nanofiltration and Reverse Osmo
6、sis Membrane ProcessesD6974 Practice for Enumeration of Viable Bacteria and Fungi in Liquid FuelsFiltration and Culture ProceduresE2563 Practice for Enumeration of Non-Tuberculosis Mycobacteria in Aqueous Metalworking Fluids by Plate Count Method2.2 Other Standards:9215 Heterotrophic Plate Count33.
7、Terminology3.1 Definitions:3.1.1 For definitions of terms use in this standard, see Terminologies D1129 and D6161.3.1.2 colony forming unit (CFU), nin microbiology, a visible mass of cells (algae, bacteria or fungi) originating from eitheran individual cell or cluster of cells that have been placed
8、onto or dispersed into a solid or semi-solid nutrient medium andsubsequently incubated under prescribed conditions.3.1.2.1 DiscussionPrescribed growth conditions can include, but are not limited to: growth medium pH and nutrient composition, incubationtemperature, incubation environment (for example
9、: gas mixture, pressure and relative humidity), and incubation interval. Anygiven set of growth conditions will select for the culture recovery of a fraction of a samples microbiome and against the culturerecovery of the balance of that microbiome.3.1.2.2 Discussion1 These practices are under the ju
10、risdiction of ASTM Committee D19 on Water and are the direct responsibility of Subcommittee D19.24 on Water Microbiology.Current edition approved June 1, 2012June 1, 2016. Published August 2012June 2016. Originally approved in 1993. Last previous edition approved in 20042012 asD6465 93 (2004).(2012)
11、. DOI: 10.1520/D5465-93R12.10.1520/D5465-16.2 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.3 Available onlin
12、e from http:/standardmethods.org/store/ProductView.cfm?ProductID=102, or from American Public Health Association (APHA), Standard Methods forthe Examination of Water and Wastewater, 800 I Street, NW Washington, DC 20001, http:/www.apha.org.This document is not an ASTM standard and is intended only t
13、o 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, ASTM recommends that users consult prior editions as appropriate. In all cases only the current versiono
14、f the standard as published by ASTM is to be considered the official document.*A Summary of Changes section appears at the end of this standardCopyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1Recognizing that all culture test methods a
15、re selective, CFU data invariably underestimate the population densities of viablemicrobes in samples tested by those methods. Moreover, incomplete disaggregation of masses of microbial cells during samplepreparation contributes to decreasing the ratio of CFU to total viable microbes in the sample.3
16、.1.2.3 DiscussionColonies normally become visible to the naked eye only after approximately 1 billion cells have amassed.Assuming that the colonyderived from a single cell, it requires approximately 30 generations for a single cell to proliferate to a mass of 1 billion cells.Consequently, the time l
17、apse between inoculation and detection of a CFU is directly dependent on the generation time(s) of taxapresent in sample. Moreover, because colony diameter increases as the cells within the colony continue proliferate, in samplescontaining microbes with different generation times, colonies of microb
18、es with longer generation times are likely to be eclipsed(and therefore undetected) by colonies of microbes with shorter generation times. This phenomenon further contributes to CFUdata underestimating total viable cell numbers.4. Significance and Use4.1 Numerous ASTM test methods and practices (for
19、 example: Test Methods D5259 and D5392, and Practices D6974 andE2563) report colony counts as their measured parameter.4.2 These practices provide a uniform set of counting, calculating, and reporting procedures for ASTM test methods inmicrobiology.SectionACounting Colonies on Membrane Filters 4ACou
20、nting Colonies on Membrane Filters 6BCounting Colonies on Pour Plates 5BCounting Colonies on Pour Plates 7CCounting Colonies on Spread Plates 6CCounting Colonies on Spread Plates 84.3 The counting rules provide a best attainable estimate of microorganisms in the sample, since the samples cannot be h
21、eld andreanalyzed at a later date.5. Hazards5.1 The analyst/technician must know and observe the normal good laboratory practices and safety procedures required in amicrobiology laboratory while preparing, using, and disposing of cultures, reagents, and materials.PRACTICE ACOUNTINGACOUNTING COLONIES
22、 ON MEMBRANE FILTERS6. Procedure6.1 The grid lines help in counting the colonies. Count them for the organism of interest following a preset plan such as thatshown in Fig. 1. Some colonies will be in contact with the grid lines.Asuggested procedure for reducing error in counting is shownin Fig. 2. C
23、ount the colonies in the squares indicated by the arrows.FIG. 1 Colony Counting Pathway (The Inner Circle Indicates the Effective Filtering Area; the Dashed Line Indicates the Pathway)D5465 1626.2 The fluorescent lamp tube should be nearly parallel with and directly over the membrane filter. Ideally
24、, the lamp is attachedto and surrounds the objective nosepiece of the stereoscopic microscope. Count the colonies individually, even if they are in contactwith each other. The technician must learn to recognize the difference between two or more colonies that have grown into contactwith each other a
25、nd the single, irregularly shaped colonies that sometimes develop on membrane filters. The latter colonies areusually associated with a fiber or particulate material and conform to the shape and size of the fiber or particulates. Colonies thathave grown together almost invariably show a very fine li
26、ne of contact.6.3 Count the colonies with a stereoscopic (dissecting) microscope that provides a magnification of at least 10 to 15.6.4 See Table 1 for guidance on acceptable counting limits.6.5 Calculation of ResultsSelect the membrane with the number of CFU in the acceptable range and calculate th
27、ecount/reporting volume according to the following general formula:CFU/mL5 colonies countedvolume of sample filtered in mL31 (1)CFU/100mL5 colonies countedvolume of sample filtered in mL3100 (2)FIG. 2 Enlarged Portion of Grid-Marked Square of Filter (Colonies in Contact with Gridlines are Counted in
28、 Squares Indicated by theArrow)TABLE 1 Recommended Counting Range for High-DensitySamplesAMicroorganism Colony Count RemarksTotal coliform bacteria, MF, 47 mm 20 to 80 Upper limit, 200colonies of all typesTotal coliform bacteria, MF, 47 mm 20 to 60 Upper limit, 200colonies of all typesFecal coliform
29、 bacteria, MF, 47 mm 20 to 60Fecal streptococci, MF, 47 mm 20 to 100Heterotrophic spread plate count 20 to 200Heterotrophic pour plate count 30 to 300 Upper limit, 300A Colony counts below or exceeding the limits cited above must be identified asoutside of this range.D5465 1636.6 Counts Within the A
30、cceptable Limits:6.6.1 The acceptable range of counts on a membrane for samples that are diluted is a function of the organism/test combinationas given in Table 1.6.6.2 Assume that the filtration of volumes of 80, 20, 5, and 1 mL produced counts of 250, 60, 15, and 4, respectively. Do notcount the c
31、olonies on all filters. Select the MF(s) within the acceptable counting range and then limit the actual counting to suchmembranes. After selecting the best MF for counting, in this case that with a 60-CFU count, the analyst counts CFU accordingto the procedures shown in Fig. 1 and Fig. 2 and applies
32、 the general formula as follows:602031or3100!53or 300! (3)Report as 3 CFU/mL or 300 CFU/100 mL.6.6.3 If there are acceptable counts on replicate plates, carry the counts independently to final reporting units, and then calculatethe arithmetic mean of these counts to obtain the final reported value.
33、For example, 1 mL volumes produced counts of 26 and 36CFU/mL or counts of 2600 and 3600 CFU/100 mL:261362 531 (4)Report as 31 CFU/mL.2600136002 53100 (5)Report as 3100 CFU/100 mL.6.6.4 If more than one dilution produced acceptable counts, count the colonies for each dilution, carry the counts indepe
34、ndentlyto final reporting units, and then average for the final reported value. For example, assume that volumes of 0.3, 0.1, 0.03, and 0.01mL produced colony counts of too numerous to count (TNTC), 75, 30, and 8, respectively. In this example, two volumes, 0.1 and0.03, produce colonies in an accept
35、able counting range. Carry each MF count independently to a count/mL or count/100 mL:750.131or3100!5750 CFU/mL or 75000 CFU/100 mL! (6)300.0331or3100!51000 CFU/mL or100000 CFU/100 mL!Then calculate the arithmetic mean of these counts to obtain the final reported value:750110002 5875 (7)Report as 880
36、 CFU/mL.7500011000002 587500 (8)Report as 88 000 CFU/100 mL.6.6.5 For finished drinking water samples only, countable membranes may contain from one colony to the upper limit of thetest (see Table 1). Count the target colonies/volume filtered. Calculate and report the number of CFU/100 mL.6.7 Counts
37、 Outside Acceptable Limits:6.7.1 Zero counts recorded as 800 000 CFU/100 mL.6.7.6.1 Alternatively, small sample volumes or sample dilutions can be used to minimize the TNTC problem. Replicates ofsmaller sample volumes or sample dilutions may be filtered and the results combined. If the MF technique
38、is not applicable, themost probable number (MPN) is useful.6.7.7 If there is no result because of confluency, laboratory accident, etc., report as no result and specify the reason.6.8 Reporting ResultsReport bacterial densities as CFU/mL or CFU/100 mL of sample, as the method requires.6.9 Verificati
39、on A verified MF test establishes the validity of organism identification on a differential or selective medium.Obviously, verification is not applied to nonselective media.6.9.1 A percent verification can be determined for any colony validation test:colonies meeting verification testcolonies subjec
40、ted to verification31005percent verification (15)6.9.2 Adjust the original count according to the percent of CFU verified. The verification of all colonies up to at least 10 isrecommended. This verification number is required for all positive MF results from potable waters.6.9.3 Verification is also
41、 recommended for establishing quality control in research with new test waters, procedures, ortechnicians; for identifying atypical colonies; and as support for data used in legal actions. The worker is cautioned not to applythe percent of verification determined for one sample to other samples. The
42、 careful worker may also pick non-typical colonies andfollow the verification procedure to determine whether false negative responses do occur.PRACTICE BCOUNTING COLONIES ON POUR PLATES7. Procedure7.1 Manual CountingCount the colonies with the aid of magnification under uniform light, using a tally.
43、 Alternately, use aQuebec-type colony counter equipped with a guide plate ruled in square centimeters. Do not mistake particles of undissolvedmedium, sample, or precipitated matter in plates for pinpoint colonies.D5465 1657.2 Automatic CountersDo not use plates having scratched surfaces, stippled ag
44、ar surfaces, or particles or air bubbles in theagar or plates with fingerprints or films on the bottoms of the plates. The colony counters should yield counts within 6 10 %610 % of those obtained manually, 90 % of the time.7.3 Count plates containing between 30 and 300 colonies. Count the number of
45、colonies below 30 if the sample is anexceptionally clean water, such as a drinking water. Count all colonies, including those of pinpoint size, and record the samplevolume and dilution used.7.4 Report the pour plate count as CFU per mL.PRACTICE CCOUNTINGCCOUNTING COLONIES ON SPREAD PLATES8. Procedur
46、e8.1 Count plates containing between 20 and 200 colonies. The maximum number of colonies per spread plate is fewer than thatfor other plate techniques because surface colonies are larger than subsurface colonies and crowding can result at lower countlevels. If the water sample is exceptionally clean
47、, count the actual number of colonies below 20. Report as CFU/mL or CFU/100mL, depending on the use of the data.9. Significant Figures9.1 To prevent false precision in the reporting of counts, the counts must be limited to the digit(s) known definitely plus onedigit that is in doubt. These combined
48、digits are termed the significant figures (SFs).9.1.1 For example, if an analyst reports a plate count of 124 to three SFs, he is indicating that he is certain of the first two digits,1 and 2, but is uncertain whether the last digit is 3, 4, or 5. If the analyst were reporting that same number to tw
49、o SFs, he wouldreport the first figure, 1, as certain, the second figure, 2, as uncertain, and the third figure, 4, as unknown. Large counts of 1200,12 000, and 12 000 000 contain only two significant figures. Of course, zeros can be significant in actual counts of 10, 60, 105,and so forth.9.2 In plate count and MF methods, the number of significant digits that can be reported is dictated by the test method itself,as follows: within the acceptable counting range of the test method itself, that is, 20 to 60, 20 to 80, 20 to 100, or 30 to 300, thea
