ASTM D5465-93(2012)
(Practice)Standard Practice for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods
Standard Practice for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods
SIGNIFICANCE AND USE
These practices provide a uniform set of counting, calculating, and reporting procedures for ASTM test methods in microbiology.
Section ACounting Colonies on Membrane Filters4 BCounting Colonies on Pour Plates5 CCounting Colonies on Spread Plates6
The counting rules provide a best attainable estimate of microorganisms in the sample, since the samples cannot be held and reanalyzed at a later date.
SCOPE
1.1 These practices cover recommended procedures for counting 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 the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
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Designation: D5465 − 93 (Reapproved 2012)
StandardPractice for
Determining Microbial Colony Counts from Waters Analyzed
by Plating Methods
This standard is issued under the fixed designation D5465; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 4.2 Thefluorescentlamptubeshouldbenearlyparallelwith
and directly over the membrane filter. Ideally, the lamp is
1.1 These practices cover recommended procedures for
attached to and surrounds the objective nosepiece of the
counting colonies and reporting colony-forming units (CFU)
stereoscopicmicroscope.Countthecoloniesindividually,even
on membrane filters (MF) and standard pour and spread plates.
iftheyareincontactwitheachother.Thetechnicianmustlearn
1.2 This standard does not purport to address all of the
to recognize the difference between two or more colonies that
safety concerns, if any, associated with its use. It is the
have grown into contact with each other and the single,
responsibility of the user of this standard to establish appro-
irregularly shaped colonies that sometimes develop on mem-
priate safety and health practices and determine the applica-
brane filters. The latter colonies are usually associated with a
bility of regulatory limitations prior to use.
fiber or particulate material and conform to the shape and size
of the fiber or particulates. Colonies that have grown together
2. Significance and Use
almost invariably show a very fine line of contact.
2.1 These practices provide a uniform set of counting,
4.3 Count the colonies with a stereoscopic (dissecting)
calculating, and reporting procedures for ASTM test methods
microscope that provides a magnification of at least 10 to 15×.
in microbiology.
Section 4.4 See Table 1 for guidance on acceptable counting limits.
A—Counting Colonies on Membrane Filters 4
4.5 Calculation of Results—Select the membrane with the
B—Counting Colonies on Pour Plates 5
C—Counting Colonies on Spread Plates 6
number of CFU in the acceptable range and calculate the
2.2 The counting rules provide a best attainable estimate of count/reporting volume according to the following general
formula:
microorganisms in the sample, since the samples cannot be
held and reanalyzed at a later date.
colonies counted
CFU/mL 5 31 (1)
volume of sample filtered in mL
3. Hazards
colonies counted
3.1 The analyst/technician must know and observe the
CFU/100 mL 5 3100 (2)
volume of sample filtered in mL
normal good laboratory practices and safety procedures re-
4.6 Counts Within the Acceptable Limits:
quiredinamicrobiologylaboratorywhilepreparing,using,and
disposing of cultures, reagents, and materials. 4.6.1 The acceptable range of counts on a membrane for
samples that are diluted is a function of the organism/test
PRACTICE A—COUNTING COLONIES ON
combination as given in Table 1.
MEMBRANE FILTERS
4.6.2 Assume that the filtration of volumes of 80, 20, 5, and
1 mL produced counts of 250, 60, 15, and 4, respectively. Do
4. Procedure
notcountthecoloniesonallfilters.SelecttheMF(s)withinthe
4.1 Thegridlineshelpincountingthecolonies.Countthem
acceptable counting range and then limit the actual counting to
fortheorganismofinterestfollowingapresetplansuchasthat
such membranes. After selecting the best MF for counting, in
shown in Fig. 1. Some colonies will be in contact with the grid
this case that with a 60-CFU count, the analyst counts CFU
lines. A suggested procedure for reducing error in counting is
according to the procedures shown in Fig. 1 and Fig. 2 and
shown in Fig. 2. Count the colonies in the squares indicated by
applies the general formula as follows:
the arrows.
31 ~or 3100! 5 3 ~or300! (3)
These practices are under the jurisdiction of ASTM Committee D19 on Water
and are the direct responsibility of Subcommittee D19.24 on Water Microbiology.
Report as 3 CFU/mL or 300 CFU/100 mL.
Current edition approved June 1, 2012. Published August 2012. Originally
4.6.3 Ifthereareacceptablecountsonreplicateplates,carry
approved in 1993. Last previous edition approved in 2004 as D6465–93 (2004).
DOI: 10.1520/D5465-93R12. the counts independently to final reporting units, and then
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D5465 − 93 (Reapproved 2012)
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 final
reported value. For example, assume that volumes of 0.3, 0.1,
0.03, and 0.01 mLproduced colony counts of too numerous to
count(TNTC),75,30,and8,respectively.Inthisexample,two
volumes, 0.1 and 0.03, produce colonies in an acceptable
counting range. Carry each MF count independently to a
count/mL or count/100 mL:
31 or 3100 5 750 CFU/mL or75000 CFU/100mL (6)
~ ! ~ !
0.1
31 ~ or 3100!
0.03
51000 CFU/mL or100000 CFU/100 mL
~ !
FIG. 1 Colony Counting Pathway (The Inner Circle Indicates the
Effective Filtering Area; the Dashed Line Indicates the Pathway)
Then calculate the arithmetic mean of these counts to obtain
the final reported value:
5 875 (7)
Report as 880 CFU/mL.
5 87500 (8)
Report as 88000 CFU/100 mL.
4.6.5 For finished drinking water samples only, countable
membranes may contain from one colony to the upper limit of
thetest(seeTable1).Countthetargetcolonies/volumefiltered.
Calculate and report the number of CFU/100 mL.
4.7 Counts Outside Acceptable Limits:
4.7.1 Zero counts recorded as < values/volume filtered are
acceptable for sample volumes of 100 mL or more.
FIG. 2 Enlarged Portion of Grid-Marked Square of Filter (Colo-
4.7.2 If full-volume samples are filtered, such as 25, 50, or
nies in Contact with Gridlines are Counted in Squares Indicated
100 mL, and the resulting count is 1 to 19 colonies, these
by the Arrow)
values are acceptable although <20. The count is adjusted to 1
or 100 mL for reporting. For example, a count of 1 colony/25
TABLE 1 Recommended Counting Range for High-Density
A
Samples mL is adjusted:
Microorganism Colony Count Remarks
31 or 3100 5,1 or4 (9)
Total coliform bacteria, MF, 47 mm 20 to 80 Upper limit, 200 ~ ! ~ !
colonies of all types
Fecal coliform bacteria, MF, 47 mm 20 to 60
Report as <1 CFU/mL (or 4 CFU/100 mL).
Fecal streptococci, MF, 47 mm 20 to 100
Heterotrophic spread plate count 20 to 200 4.7.3 If all MF counts are <20/volume filtered, select the
Heterotrophic pour plate count 30 to 300 Upper limit, 300
most nearly acceptable count (for non-drinking waters). For
A
Colony counts below or exceeding the limits cited above must be identified as
example, assume a count in which sample volumes of 1, 0.3,
outside of this range.
and 0.01 mL produced CFU counts of 14, 3, and 0, respec-
tively. No CFU count falls within recommended limits here.
Calculate on the basis of the most nearly acceptable plate
calculatethearithmeticmeanofthesecountstoobtainthefinal
count, 14, and report with a qualifying remark:
reportedvalue.Forexample,1mLvolumesproducedcountsof
26 and 36 CFU/mLor counts of 2600 and 3600 CFU/100 mL:
31 or 3100 5 14 or1400 (10)
~ ! ~ !
1.0
531 (4)
Report as estimated count: 14 CFU/mL (or 1400 CFU/100
mL).
Report as 31 CFU/mL.
4.7.4 Ifcountsfromallmembranesarezero,calculateusing
the count from largest filtration volume. For example, sample
5 3100 (5)
2 volumesof25,10,and2mLproducedCFUcountsof0,0,and
D5465 − 93 (Reapproved 2012)
0,respectively,andnoactualcalculationispossible,evenasan 4.9.3 Verification is also recommended for establishing
estimatedreport.Calculatethenum
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