ASTM D4455-85(2009)
(Test Method)Standard Test Method for Enumeration of Aquatic Bacteria by Epifluorescence Microscopy Counting Procedure
Standard Test Method for Enumeration of Aquatic Bacteria by Epifluorescence Microscopy Counting Procedure
SIGNIFICANCE AND USE
Bacterial populations, as part of the microbial community in aquatic systems are actively involved in nutrient cycling. The significance of these populations is often difficult to ascertain because of the presence of many physiological types. However, measurement of bacterial densities is usually the first step in trying to establish any relationship that might exist between bacteria and other biochemical processes.
Acridine-orange epifluorescence direct-counting procedure cannot differentiate between viable and nonviable cells.
This procedure cannot be used to convert directly the numbers to total carbon biomass because of the natural variations in bacterial cell size.
The acridine-orange epifluorescence direct-microscopic count is both quantitative and precise.
This procedure is ideal for enumerating both pelagic and epibenthic bacteria in all fresh water and marine environments.
The process can be employed in survey activities to characterize the bacteriological densities of environmental waters.
The procedure can also be used to estimate bacterial densities in cooling tower waters, process waters, and waters associated with oil drilling wells.
SCOPE
1.1 This test method describes a procedure for detection and enumeration of aquatic bacteria by the use of an acridine-orange epifluorescence direct-microscopic counting procedure. It is applicable to environmental waters.
1.2 Certain types of debris and other microorganisms may fluoresce in acridine orange-stained smears.
1.3 The test method requires a trained microbiologist or technician who is capable of distinguishing bacteria from other fluorescing bodies on the basis of morphology when viewed at higher magnifications.
1.4 Use of bright light permits differentiation of single bacteria where reduced formazan is deposited at the polar ends.
1.5 Approximately 104 cells/mL are required for detection by this test method.
1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.7 This standard does not purport to address 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.
General Information
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Standards Content (Sample)
NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation:D4455 −85(Reapproved2009)
StandardTest Method for
Enumeration of Aquatic Bacteria by Epifluorescence
Microscopy Counting Procedure
This standard is issued under the fixed designation D4455; 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 3. Terminology
1.1 Thistestmethoddescribesaprocedurefordetectionand
3.1 Definitions—For definitions of terms used in this test
enumeration of aquatic bacteria by the use of an acridine-
method, refer to Terminology D1129.
orangeepifluorescencedirect-microscopiccountingprocedure.
It is applicable to environmental waters.
4. Summary of Test Method
1.2 Certain types of debris and other microorganisms may
4.1 Enumerationofaquaticbacteriaisobtainedbypassinga
fluoresce in acridine orange-stained smears.
watersamplethrougha0.2-µmpolycarbonatemembranefilter.
1.3 The test method requires a trained microbiologist or
4.2 The membrane filter is stained with acridine orange
technicianwhoiscapableofdistinguishingbacteriafromother
solution.
fluorescing bodies on the basis of morphology when viewed at
4.3 The stained filter is examined for fluorescing bacteria
higher magnifications.
cells using a fluorescent microscope.
1.4 Use of bright light permits differentiation of single
4.4 Thefluorescentbacteriaarecounted.Dilutionsaretaken
bacteriawherereducedformazanisdepositedatthepolarends.
into consideration and bacterial concentrations established.
1.5 Approximately 10 cells/mL are required for detection
by this test method.
5. Significance and Use
1.6 The values stated in SI units are to be regarded as
5.1 Bacterial populations, as part of the microbial commu-
standard. No other units of measurement are included in this
nity in aquatic systems are actively involved in nutrient
standard.
cycling. The significance of these populations is often difficult
1.7 This standard does not purport to address the safety
to ascertain because of the presence of many physiological
concerns, if any, associated with its use. It is the responsibility
types. However, measurement of bacterial densities is usually
of the user of this standard to establish appropriate safety and
the first step in trying to establish any relationship that might
health practices and determine the applicability of regulatory
exist between bacteria and other biochemical processes.
limitations prior to use.
5.2 Acridine-orange epifluorescence direct-counting proce-
2. Referenced Documents
dure cannot differentiate between viable and nonviable cells.
2.1 ASTM Standards:
5.3 This procedure cannot be used to convert directly the
D1129Terminology Relating to Water
numbers to total carbon biomass because of the natural
D1193Specification for Reagent Water
variations in bacterial cell size.
5.4 The acridine-orange epifluorescence direct-microscopic
This test method is under the jurisdiction ofASTM Committee D19 on Water
count is both quantitative and precise.
and is the direct responsibility of Subcommittee D19.24 on Water Microbiology.
Current edition approved May 1, 2009. Published June 2009. Originally
5.5 Thisprocedureisidealforenumeratingbothpelagicand
approved in 1985. Last previous edition approved in 2002 as D4455–85 (2002).
epibenthic bacteria in all fresh water and marine environ-
DOI: 10.1520/D4455-85R09.
ments.
The sole source of supply of the apparatus, Bacto Acridine Orange Stain,
known to the committee at this time is Difco Laboratories, P.O. Box 1058, Detroit,
MI 48201. If you are aware of alternative suppliers, please provide this information
to ASTM International Headquarters. Your comments will receive careful consid-
erationatameetingoftheresponsibletechnicalcommittee, whichyoumayattend. Cherry, et al, “ Temperature Influence on Bacterial Populations in Aquatic
For referenced ASTM standards, visit the ASTM website, www.astm.org, or Systems,” Water Research, Vol 8, 1974, pp. 149–155.
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Daley, R. J., “ Direct Epifluorescence Enumeration of Native Aquatic
Standards volume information, refer to the standard’s Document Summary page on Bacteria,” Native Aquatic Bacteria: Enumeration, Activity, and Ecology, ASTM
the ASTM website. STP 695, ASTM, 1979, pp. 29–45.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D4455−85(Reapproved2009)
5.6 The process can be employed in survey activities to 7.6 Xylene.
characterize the bacteriological densities of environmental
7.7 Immersion Oil, very low fluorescing (equivalent to
waters.
Cargille Type A).
5.7 The procedure can also be used to estimate bacterial
densities in cooling tower waters, process waters, and waters
8. Procedure
associated with oil drilling wells.
8.1 Place a 0.2-µm membrane filter on the filter base and
attach the funnel. Add 10 mL of buffered water to the funnel
6. Apparatus
then add 1 mL of the water sample or dilution (use 9-mL
6.1 Fluorescence Microscope, with oil-immersion objective
dilution blanks). Turn on the vacuum.
lens (100×).
8.2 Rinse the membrane with 5 mLof sterile reagent water.
6.2 Eye pieces, 12.5×, equipped with a net micrometer (10
8.3 Turn off the vacuum and flood the membrane with the
by 10 mm) (25 by 2-mm squares).
acridine orange solution. Allow to stand for 3 to 4 min, then
6.3 Condenser, 1.25×, suitable for the microscope.
turn on the vacuum and filter through.
6.4 High-Pressure Mercury Lamp , 200 W, on a UV light
8.4 Rinsethemembranewith0.5mLofisopropanol.Donot
source giving vertical illumination and a filter unit H2 (Leitz)
exceed 10-s contact time.
with BG12 and BG38 transmission filters or equivalent.
8.5 Rinse the membrane with 0.4 mL of xylene.
6.5 Stage Micrometer, 2 by 200 parts.
8.6 Remove the membrane and air dry for 15 s.
6.6 Membrane Filter Support(25mm),sterile,particle-free,
8.7 Place membrane on a clean microscope slide on which
fritted-glass.
has been add
...
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