1、Designation: D 5465 93 (Reapproved 2004)Standard Practice forDetermining Microbial Colony Counts from Waters Analyzedby Plating Methods1This standard is issued under the fixed designation D 5465; the number immediately following the designation indicates the year oforiginal adoption or, in the case
2、of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 These practices cover recommended procedures forcounting colonies and reporting colony-formi
3、ng 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 determin
4、e the 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 5CCount
5、ing 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
6、 re-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 thatsho
7、wn 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. Ideal
8、ly, 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 that1These practices are under the jurisdicti
9、on of ASTM Committee D19 on Waterand are the direct responsibility of Subcommittee D19.24 on Water Microbiology.Current edition approved June 1, 2004. Published June 2004. Originallyapproved in 1993. Last previous edition approved in 1993 as D 6465 93 (1998).FIG. 1 Colony Counting Pathway (The Inner
10、 Circle Indicates theEffective Filtering Area; the Dashed Line Indicates the Pathway)FIG. 2 Enlarged Portion of Grid-Marked Square of Filter (Coloniesin Contact with Gridlines are Counted in Squares Indicated bythe Arrow)1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshoh
11、ocken, PA 19428-2959, United States.have grown into contact with each other and 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 particul
12、ates. Colonies that have grown togetheralmost invariably show a very fine line of 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
13、the membrane with thenumber of CFU in the acceptable range and calculate thecount/reporting volume according to the following generalformulae: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 Accept
14、able Limits:4.6.1 The acceptable range of counts on a membrane forsamples that are diluted is a function of the organism/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
15、 on all filters. Select the MF(s) within theacceptable counting range and then limit the actual counting tosuch membranes. 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 gener
16、al 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 acceptable 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 e
17、xample, 1 mLvolumes produced counts of26 and 36 CFU/mL or counts of 2600 and 3600 CFU/100 mL:26 1 3625 31 (4)Report 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-d
18、ently to final reporting units, and then average for the finalreported value. For example, assume that volumes of 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 acceptabl
19、ecounting range. Carry each MF count independently to acount/mL or count/100 mL:750.13 1 or 3 100! 5 750 CFU/mL or 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)Re
20、port 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 samples 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
21、 mL.4.7 Counts Outside Acceptable Limits:4.7.1 Zero counts recorded as 800 000 CFU/100 mL.4.7.6.1 Alternatively, small 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
22、 MF technique is not applicable,the most probable number (MPN) is useful.4.7.7 If there is no result because of confluency, 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.
23、9 Verification A verified MF test establishes the validityof organism identification on a differential or selective 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 testcolo
24、nies subjected to verification3 100 5 percent verification(15)4.9.2 Adjust the original count according to the percent 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 Verificat
25、ion is also recommended for establishingquality control in research with new test waters, procedures, ortechnicians; 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 samp
26、les. The careful worker may also pick non-typicalcolonies and follow the verification procedure to determinewhether 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
27、tally. Alternately,use a Quebec-type colony counter equipped with a guide plateruled in square centimeters. Do not mistake particles ofundissolved medium, sample, or precipitated matter in platesfor pinpoint colonies.5.2 Automatic CountersDo not use plates havingscratched surfaces, stippled agar sur
28、faces, or particles or airbubbles in the agar or plates with fingerprints or films on thebottoms of the plates. The 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
29、 30 if the sample is anexceptionally clean water, such as a drinking water. Count allcolonies, including those of 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 contain
30、ing between 20 and 200 colonies.The maximum number of colonies per spread plate is fewerthan that for other plate 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
31、 colonies below 20. Report asCFU/mL or CFU/100 mL, depending on the use of the data.7. Significant Figures7.1 To prevent 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 thesignific
32、ant figures (SFs).7.1.1 For example, if an analyst reports a plate count of 124to three SFs, he is indicating that 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
33、figure, 1, as certain, the second figure,2, as uncertain, and the third figure, 4, as unknown. Largecounts of 1200, 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 numb
34、er of signifi-cant digits that can be reported is dictated by the test methoditself, as follows: within the acceptable counting range of thetest 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 num
35、ber of SFs is equal to thenumber of colonies (see Table 2).D 5465 93 (2004)37.3 Rounding Off CountsSince plate counts 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 insignif
36、icant digit is below five, replace it with azero, for example, 3530 becomes 3500.7.3.2 If the insignificant digit is 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 t
37、han five, drop thedigit and increase the preceding significant number by one, forexample, 3480 becomes 3500.8. Keywords8.1 calculating results; counting colonies; membrane filtermethods; pour plate methods; reporting results; rounding off;significant figures; spread plate methods; verification of co
38、untsASTM International takes no position respecting the validity of any patent rights asserted in connection with any 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,
39、 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 sta
40、ndardsand should be addressed to ASTM International Headquarters. Your comments will receive careful consideration 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 Com
41、mittee on Standards, at the address shown below.This standard is copyrighted by ASTM International, 100 Barr Harbor 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 aboveaddr
42、ess or at 610-832-9585 (phone), 610-832-9555 (fax), or serviceastm.org (e-mail); or through the ASTM website(www.astm.org).TABLE 2 Number of Significant Figures ReportedActualColony CountPour Plate/Spread PlateMethodMembrane FiltrationMethod1to9 1SF 1SF10 to 99 2 SFs 2 SFs100 to 300 3 SFs .D 5465 93 (2004)4
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