ASTM D4455-85(2002)
(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.4
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.5
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 1014 cells/mL are required for detection by this test method.
1.6 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.
General Information
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Standards Content (Sample)
NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation: D 4455 – 85 (Reapproved 2002)
Standard Test Method for
Enumeration of Aquatic Bacteria by Epifluorescence
Microscopy Counting Procedure
This standard is issued under the fixed designation D 4455; 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 (e) indicates an editorial change since the last revision or reapproval.
1. Scope 4.2 The membrane filter is stained with acridine orange
solution.
1.1 This test method describes a procedure for detection and
4.3 The stained filter is examined for fluorescing bacteria
enumeration of aquatic bacteria by the use of an acridine-
cells using a fluorescent microscope.
orange epifluorescence direct-microscopic counting procedure.
4.4 Thefluorescentbacteriaarecounted.Dilutionsaretaken
It is applicable to environmental waters.
into consideration and bacterial concentrations established.
1.2 Certain types of debris and other microorganisms may
fluoresce in acridine orange-stained smears.
5. Significance and Use
1.3 The test method requires a trained microbiologist or
5.1 Bacterial populations, as part of the microbial commu-
technician who is capable of distinguishing bacteria from other
nity in aquatic systems are actively involved in nutrient
fluorescing bodies on the basis of morphology when viewed at
2 cycling. The significance of these populations is often difficult
higher magnifications.
to ascertain because of the presence of many physiological
1.4 Use of bright light permits differentiation of single
types. However, measurement of bacterial densities is usually
bacteriawherereducedformazanisdepositedatthepolarends.
the first step in trying to establish any relationship that might
1.5 Approximately 10 cells/mL are required for detection
2 exist between bacteria and other biochemical processes.
by this test method.
5.2 Acridine-orange epifluorescence direct-counting proce-
1.6 This standard does not purport to address the safety
dure cannot differentiate between viable and nonviable cells.
concerns, if any, associated with its use. It is the responsibility
5.3 This procedure cannot be used to convert directly the
of the user of this standard to establish appropriate safety and
numbers to total carbon biomass because of the natural
health practices and determine the applicability of regulatory
variations in bacterial cell size.
limitations prior to use.
5.4 The acridine-orange epifluorescence direct-microscopic
2. Referenced Documents count is both quantitative and precise.
5.5 Thisprocedureisidealforenumeratingbothpelagicand
2.1 ASTM Standards:
3 epibenthic bacteria in all fresh water and marine environ-
D 1129 Terminology Relating to Water
ments.
D 1193 Specification for Reagent Water
5.6 The process can be employed in survey activities to
D 3370 Practices for Sampling Water from Closed Con-
characterize the bacteriological densities of environmental
duits
waters.
3. Terminology
5.7 The procedure can also be used to estimate bacterial
densities in cooling tower waters, process waters, and waters
3.1 Definitions—For definitions of terms used in this test
associated with oil drilling wells.
method, refer to Terminology D 1129.
6. Apparatus
4. Summary of Test Method
6.1 Fluorescence Microscope, with oil-immersion objective
4.1 Enumeration of aquatic bacteria is obtained by passing a
lens (1003).
water sample through a 0.2-µm polycarbonate membrane filter.
6.2 Eye pieces, 12.53, equipped with a net micrometer (10
by 10 mm) (25 by 2-mm squares).
This test method is under the jurisdiction of ASTM Committee D19 on Water
and is the direct responsibility of Subcommittee D19.24 on Water Microbiology. Cherry, et al, “Temperature Influence on Bacterial Populations in Aquatic
Current edition approved Jan. 25, 1985. Published March 1985. Systems,” Water Research, Vol 8, 1974, pp. 149–155.
2 5
DIFCOTechnicalInformation—BactoAcridineOrangeStain,isavailablefrom Daley, R. J., “Direct Epifluorescence Enumeration of NativeAquatic Bacteria,”
Difco Laboratories, P.O. Box 1058, Detroit, MI 48201. Native Aquatic Bacteria: Enumeration, Activity, and Ecology, ASTM STP 695,
Annual Book of ASTM Standards, Vol 11.01. ASTM, 1979, pp. 29–45.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
D 4455 – 85 (2002)
6.3 Condenser, 1.253, suitable for the microscope. 8.7 Place membrane on a clean microscope slide on which
6.4 High-Pressure Mercury Lamp, 200 W, on a UV light has been added 2 drops of fluorescence-free immersion oil.
source giving vertical illumination and a filter unit H2 (Leitz)
8.8 Placeanotherdropofimmersionoilontopofmembrane
with BG12 and BG38 transmission filters or equivalent.
and apply cover slip.
6.5 Stage Micrometer, 2 by 200 parts.
8.9 Count cells using incident fluorescent illumination in
6.6 Membrane Filter Support (25 mm), sterile, particle-
violet light wavelength range (410 nm).
free, fritted-glass.
8.10 Count 20 fields at random within the stained portion of
6.7 Funnel, 15-mL capacity or equivalent.
the membrane.
6.8 Membrane Filter, sterile plain regular polycarbonate-25
8.11 Count only that portion of the field which lies within
mm, (0.2-µm pore size).
the micrometer area.
6.9 Filter Apparatus, containing vacuum source, filtering
8.12 Calculate the average number of bacteria per microme-
flask, and a filtering flask as a water trap.
ter area.
6.10 Forceps (flat tip), Alcohol, Bunsen Burner, Clean
8.13 Use the procedure outlined in Section 9 to determine
Glass Slides, and Cover Slips.
bacterial density per millilitre of water sample.
8.14 Type IA water is used as a negative control and
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