ASTM_D_5465_-_93_2012.pdf

Designation:D546593(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 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 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 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 Filters4BCounting Colonies on Pour Plates5CCounting Colonies on Spread Plates62.2 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-quired in a microbiology laboratory while preparing,using,anddisposing of cultures,reagents,and materials.PRACTICEACOUNTING 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 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,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 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 of contact.4.3 Count the colonies with a stereoscopic(dissecting)microscope that provides a magnification of at least 10 to 15.4.4 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 thecount/reporting volume according to the following generalformula:CFU/mL5colonies countedvolume of sample filtered in mL31(1)CFU/100 mL 5colonies countedvolume of sample filtered in mL3100(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 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 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 general formula as follows:602031or3100!5 3or 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 then1These 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 1993.Last previous edition approved in 2004 as D6465 93(2004).DOI:10.1520/D5465-93R12.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 calculate 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:2613625 31(4)Report as 31 CFU/mL.26001360025 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 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,twov