ASTM D5465-93(2004)
(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 A—Counting Colonies on Membrane Filters4 B—Counting Colonies on Pour Plates5 C—Counting 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 (Reapproved2004)
Standard Practice for
Determining Microbial Colony Counts from Waters Analyzed
1
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.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope lines. A suggested procedure for reducing error in counting is
shown in Fig. 2. Count the colonies in the squares indicated by
1.1 These practices cover recommended procedures for
the arrows.
counting colonies and reporting colony-forming units (CFU)
on membrane filters (MF) and standard pour and spread plates.
4.2 Thefluorescentlamptubeshouldbenearlyparallelwith
1.2 This standard does not purport to address all of the and directly over the membrane filter. Ideally, the lamp is
safety concerns, if any, associated with its use. It is the attached to and surrounds the objective nosepiece of the
responsibility of the user of this standard to establish appro- stereoscopicmicroscope.Countthecoloniesindividually,even
priate safety and health practices and determine the applica-
iftheyareincontactwitheachother.Thetechnicianmustlearn
bility of regulatory limitations prior to use. to recognize the difference between two or more colonies that
have grown into contact with each other and the single,
2. Significance and Use
irregularly shaped colonies that sometimes develop on mem-
brane filters. The latter colonies are usually associated with a
2.1 These practices provide a uniform set of counting,
fiber or particulate material and conform to the shape and size
calculating, and reporting procedures for ASTM test methods
of the fiber or particulates. Colonies that have grown together
in microbiology.
almost invariably show a very fine line of contact.
Section
A—Counting Colonies on Membrane Filters 4
4.3 Count the colonies with a stereoscopic (dissecting)
B—Counting Colonies on Pour Plates 5
C—Counting Colonies on Spread Plates 6 microscope that provides a magnification of at least 10 to 15×.
2.2 The counting rules provide a best attainable estimate of
4.4 See Table 1 for guidance on acceptable counting limits.
microorganisms in the sample, since the samples cannot be
4.5 Calculation of Results—Select the membrane with the
held and reanalyzed at a later date.
number of CFU in the acceptable range and calculate the
3. Hazards count/reporting volume according to the following general
formulae:
3.1 The analyst/technician must know and observe the
normal good laboratory practices and safety procedures re- colonies counted
CFU/mL 5 31 (1)
quiredinamicrobiologylaboratorywhilepreparing,using,and volume of sample filtered in mL
disposing of cultures, reagents, and materials.
colonies counted
CFU/100 mL 5 3100 (2)
volume of sample filtered in mL
PRACTICE A—COUNTING COLONIES ON
MEMBRANE FILTERS 4.6 Counts Within the Acceptable Limits:
4.6.1 The acceptable range of counts on a membrane for
4. Procedure
samples that are diluted is a function of the organism/test
4.1 Thegridlineshelpincountingthecolonies.Countthem combination as given in Table 1.
fortheorganismofinterestfollowingapresetplansuchasthat
4.6.2 Assume that the filtration of volumes of 80, 20, 5, and
shown in Fig. 1. Some colonies will be in contact with the grid
1 mL produced counts of 250, 60, 15, and 4, respectively. Do
notcountthecoloniesonallfilters.SelecttheMF(s)withinthe
acceptable counting range and then limit the actual counting to
1
These practices are under the jurisdiction of ASTM Committee D19 on Water
such membranes. After selecting the best MF for counting, in
and are the direct responsibility of Subcommittee D19.24 on Water Microbiology.
this case that with a 60-CFU count, the analyst counts CFU
Current edition approved June 1, 2004. Published June 2004. Originally
according to the procedures shown in Fig. 1 and Fig. 2 and
approved in 1993. Last previous edition approved in 1993 as D6465–93 (1998).
DOI: 10.1520/D5465-93R04. applies the general formula as follows:
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
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D5465 − 93 (2004)
26136
531 (4)
2
Report as 31 CFU/mL.
260013600
5 3100 (5)
2
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
...
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