1、Designation: D5465 93 (Reapproved 2012)Standard Practice 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 or, in the case of
2、 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. Scope1.1 These practices cover recommended procedures forcounting colonies and reporting colony-forming
3、units (CFU)on membrane filters (MF) and standard pour and spread plates.1.2 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine t
4、he applica-bility of regulatory limitations prior to use.2. Significance and Use2.1 These practices provide a uniform set of counting,calculating, and reporting procedures for ASTM test methodsin microbiology.SectionACounting Colonies on Membrane Filters 4BCounting Colonies on Pour Plates 5CCounting
5、 Colonies on Spread Plates 62.2 The counting rules provide a best attainable estimate ofmicroorganisms in the sample, since the samples cannot beheld and reanalyzed at a later date.3. Hazards3.1 The analyst/technician must know and observe thenormal good laboratory practices and safety procedures re
6、-quired in a microbiology laboratory while preparing, using, anddisposing of cultures, reagents, and materials.PRACTICE ACOUNTING COLONIES ONMEMBRANE FILTERS4. Procedure4.1 The grid lines help in counting the colonies. Count themfor the organism of interest following a preset plan such as thatshown
7、in Fig. 1. Some colonies will be in contact with the gridlines. A suggested procedure for reducing error in counting isshown in Fig. 2. Count the colonies in the squares indicated bythe arrows.4.2 The fluorescent lamp tube should be nearly parallel withand directly over the membrane filter. Ideally,
8、 the lamp isattached to and surrounds the objective nosepiece of thestereoscopic microscope. Count the colonies individually, evenif they are in contact with each other. The technician must learnto recognize the difference between two or more colonies thathave grown into contact with each other and
9、the single,irregularly shaped colonies that sometimes develop on mem-brane filters. The latter colonies are usually associated with afiber or particulate material and conform to the shape and sizeof the fiber or particulates. Colonies that have grown togetheralmost invariably show a very fine line o
10、f contact.4.3 Count the colonies with a stereoscopic (dissecting)microscope that provides a magnification of at least 10 to 153.4.4 See Table 1 for guidance on acceptable counting limits.4.5 Calculation of ResultsSelect the membrane with thenumber of CFU in the acceptable range and calculate thecoun
11、t/reporting volume according to the following generalformula:1These practices are under the jurisdiction of ASTM Committee D19 on Waterand are the direct responsibility of Subcommittee D19.24 on Water Microbiology.Current edition approved June 1, 2012. Published August 2012. Originallyapproved in 19
12、93. Last previous edition approved in 2004 as D6465 93 (2004).DOI: 10.1520/D5465-93R12.FIG. 1 Colony Counting Pathway (The Inner Circle Indicates theEffective Filtering Area; the Dashed Line Indicates the Pathway)1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, P
13、A 19428-2959, United States.CFU/mL 5colonies countedvolume of sample filtered in mL3 1 (1)CFU/100 mL 5colonies countedvolume of sample filtered in mL3 100 (2)4.6 Counts Within the Acceptable Limits:4.6.1 The acceptable range of counts on a membrane forsamples that are diluted is a function of the or
14、ganism/testcombination as given in Table 1.4.6.2 Assume that the filtration of volumes of 80, 20, 5, and1 mL produced counts of 250, 60, 15, and 4, respectively. Donot count the colonies on all filters. Select the MF(s) within theacceptable counting range and then limit the actual counting tosuch me
15、mbranes. After selecting the best MF for counting, inthis case that with a 60-CFU count, the analyst counts CFUaccording to the procedures shown in Fig. 1 and Fig. 2 andapplies the general formula as follows:60203 1 or 3 100! 5 3 or 300! (3)Report as 3 CFU/mL or 300 CFU/100 mL.4.6.3 If there are acc
16、eptable counts on replicate plates, carrythe counts independently to final reporting units, and thencalculate the arithmetic mean of these counts to obtain the finalreported value. For example, 1 mLvolumes produced counts of26 and 36 CFU/mL or counts of 2600 and 3600 CFU/100 mL:26 1 3625 31 (4)Repor
17、t as 31 CFU/mL.2600 1 360025 3100 (5)Report as 3100 CFU/100 mL.4.6.4 If more than one dilution produced acceptable counts,count the colonies for each dilution, carry the counts indepen-dently to final reporting units, and then average for the finalreported value. For example, assume that volumes of
18、0.3, 0.1,0.03, and 0.01 mL produced colony counts of too numerous tocount (TNTC), 75, 30, and 8, respectively. In this example, twovolumes, 0.1 and 0.03, produce colonies in an acceptablecounting range. Carry each MF count independently to acount/mL or count/100 mL:750.13 1 or 3 100! 5 750 CFU/mL or
19、 75 000 CFU/100 mL!(6)300.033 1 or 3 100!= 1 000 CFU/mL (or100 000 CFU/100 mL)Then calculate the arithmetic mean of these counts to obtainthe final reported value:750 1 1 00025 875 (7)Report as 880 CFU/mL.75 000 1 100 00025 87 500 (8)Report as 88 000 CFU/100 mL.4.6.5 For finished drinking water samp
20、les only, countablemembranes may contain from one colony to the upper limit ofthe test (see Table 1). Count the target colonies/volume filtered.Calculate and report the number of CFU/100 mL.4.7 Counts Outside Acceptable Limits:4.7.1 Zero counts recorded as 800 000 CFU/100 mL.4.7.6.1 Alternatively, s
21、mall sample volumes or sample di-lutions can be used to minimize the TNTC problem. Replicatesof smaller sample volumes or sample dilutions may be filteredand the results combined. If the MF technique is not applicable,the most probable number (MPN) is useful.4.7.7 If there is no result because of co
22、nfluency, laboratoryaccident, etc., report as no result and specify the reason.4.8 Reporting ResultsReport bacterial densities asCFU/mL or CFU/100 mL of sample, as the method requires.4.9 Verification A verified MF test establishes the validityof organism identification on a differential or selectiv
23、e me-dium. Obviously, verification is not applied to nonselectivemedia.4.9.1 A percent verification can be determined for anycolony validation test:colonies meeting verification testcolonies subjected to verification3 100 5 percent verification(15)4.9.2 Adjust the original count according to the per
24、cent ofCFU verified. The verification of all colonies up to at least 10is recommended. This verification number is required for allpositive MF results from potable waters.4.9.3 Verification is also recommended for establishingquality control in research with new test waters, procedures, ortechnician
25、s; for identifying atypical colonies; and as supportfor data used in legal actions. The worker is cautioned not toapply the percent of verification determined for one sample toother samples. The careful worker may also pick non-typicalcolonies and follow the verification procedure to determinewhethe
26、r false negative responses do occur.PRACTICE BCOUNTING COLONIES ON POURPLATES5. Procedure5.1 Manual Counting Count the colonies with the aid ofmagnification under uniform light, using a tally. Alternately,use a Quebec-type colony counter equipped with a guide plateruled in square centimeters. Do not
27、 mistake particles ofundissolved medium, sample, or precipitated matter in platesfor pinpoint colonies.5.2 Automatic CountersDo not use plates havingscratched surfaces, stippled agar surfaces, or particles or airbubbles in the agar or plates with fingerprints or films on thebottoms of the plates. Th
28、e colony counters should yield countswithin 6 10 % of those obtained manually, 90 % of the time.5.3 Count plates containing between 30 and 300 colonies.Count the number of colonies below 30 if the sample is anexceptionally clean water, such as a drinking water. Count allcolonies, including those of
29、pinpoint size, and record thesample volume and dilution used.5.4 Report the pour plate count as CFU per mL.PRACTICE CCOUNTING COLONIES ON SPREADPLATES6. Procedure6.1 Count plates containing between 20 and 200 colonies.The maximum number of colonies per spread plate is fewerthan that for other plate
30、techniques because surface coloniesare larger than subsurface colonies and crowding can result atlower count levels. If the water sample is exceptionally clean,count the actual number of colonies below 20. Report asCFU/mL or CFU/100 mL, depending on the use of the data.7. Significant Figures7.1 To p
31、revent false precision in the reporting of counts, thecounts must be limited to the digit(s) known definitely plus onedigit that is in doubt. These combined digits are termed thesignificant figures (SFs).7.1.1 For example, if an analyst reports a plate count of 124to three SFs, he is indicating that
32、 he is certain of the first twodigits, 1 and 2, but is uncertain whether the last digit is 3, 4, or5. If the analyst were reporting that same number to two SFs,he would report the first figure, 1, as certain, the second figure,2, as uncertain, and the third figure, 4, as unknown. Largecounts of 1200
33、, 12 000, and 12 000 000 contain only twosignificant figures. Of course, zeros can be significant in actualcounts of 10, 60, 105, and so forth.7.2 In plate count and MF methods, the number of signifi-cant digits that can be reported is dictated by the test methoditself, as follows: within the accept
34、able counting range of theD5465 93 (2012)3test method itself, that is, 20 to 60, 20 to 80, 20 to 100, or 30to 300, the actual number of colonies observed is the bestestimate of the true density. The number of SFs is equal to thenumber of colonies (see Table 2).7.3 Rounding Off CountsSince plate coun
35、ts must belimited to the number of SFs obtainable by test method, thenon-zero number that is not significant should be treated by thestandard scientific convention.7.3.1 If the insignificant digit is below five, replace it with azero, for example, 3530 becomes 3500.7.3.2 If the insignificant digit i
36、s five, round the precedingsignificant digit to the nearest even number, for example, withtwo SFs, 3450 becomes 3400, and 3550 becomes 3600.7.3.3 If the insignificant digit is greater than five, drop thedigit and increase the preceding significant number by one, forexample, 3480 becomes 3500.8. Keyw
37、ords8.1 calculating results; counting colonies; membrane filtermethods; pour plate methods; reporting results; rounding off;significant figures; spread plate methods; verification of countsASTM International takes no position respecting the validity of any patent rights asserted in connection with a
38、ny item mentionedin this standard. Users 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 technica
39、l 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
40、at a meeting of theresponsible technical committee, which you may attend. If you feel that your comments have not received a fair hearing you shouldmake your views known to the ASTM Committee on Standards, at the address shown below.This standard is copyrighted by ASTM International, 100 Barr Harbor
41、 Drive, PO Box C700, West Conshohocken, PA 19428-2959,United States. Individual reprints (single or multiple copies) of this standard may be obtained by contacting ASTM at the aboveaddress or at 610-832-9585 (phone), 610-832-9555 (fax), or serviceastm.org (e-mail); or through the ASTM website(www.as
42、tm.org). Permission rights to photocopy the standard may also be secured from the ASTM website (www.astm.org/COPYRIGHT/).TABLE 2 Number of Significant Figures ReportedActualColony CountPour Plate/Spread PlateMethodMembrane FiltrationMethod1to9 1SF 1SF10 to 99 2 SFs 2 SFs100 to 300 3 SFs .D5465 93 (2012)4
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