ASTM D4454-85(2002)
(Test Method)Standard Test Method for Simultaneous Enumeration of Total and Respiring Bacteria in Aquatic Systems by Microscopy
Standard Test Method for Simultaneous Enumeration of Total and Respiring Bacteria in Aquatic Systems by Microscopy
SIGNIFICANCE AND USE
Measurement of bacterial densities is generally the first step in establishing a relationship between bacteria and other biochemical processes.5 It is known that the classical plate count procedure underestimates bacterial densities while the epifluorescence direct microscopic procedure more accurately depicts the total numbers of nonviable or dormant and viable cells in a water sample. The acridine-orange INT-formazan reduction technique provides information on the total concentrations of bacteria as well as that proportion which are actively respiring and thus involved in degradative processes.
The acridine-orange INT-formazan reduction technique 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 studies to characterize the bacteriological densities and activities of environmental waters.
SCOPE
1.1 This test method covers the detection and enumeration of aquatic bacteria by the use of an acridine-orange epifluorescence direct-microscopic counting procedure. This test method is applicable to environmental waters and potable waters.
1.2 Certain types of debris and other microorganisms may fluoresce in acridine-orange stained smears.
1.3 The procedure described 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 Minimal cell size which allows the detection of formazan deposits is represented by bacteria of 0.4 m.2
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.
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Designation:D4454–85(Reapproved2002)
Standard Test Method for
Simultaneous Enumeration of Total and Respiring Bacteria
in Aquatic Systems by Microscopy
This standard is issued under the fixed designation D 4454; 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. Summary of Test Method
1.1 This test method covers the detection and enumeration 4.1 A water sample is treated with an aqueous solution of
of aquatic bacteria by the use of an acridine-orange epifluo- INT-dye(2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazo-
rescence direct-microscopic counting procedure. This test lium chloride) for 20 min. The reaction then is stopped by
method is applicable to environmental waters and potable adding a 37 % solution of formaldehyde. Sample is filtered
waters. through a 0.1-µm pore size polycarbonate membrane filter
1.2 Certain types of debris and other microorganisms may (presoaked in sudan black solution or equivalent), and stained
fluoresce in acridine-orange stained smears. with acridine orange for 3 min.
1.3 The procedure described requires a trained microbiolo- 4.2 The filter is then air-dried and examined under oil
gist or technician who is capable of distinguishing bacteria immersion for total bacteria under epifluorescence illumination
fromotherfluorescingbodiesonthebasisofmorphologywhen and for respiring bacteria under transmitted bright light illu-
viewed at higher magnifications. mination.
1.4 Use of bright light permits differentiation of single
5. Significance and Use
bacteriawherereducedformazanisdepositedatthepolarends.
5.1 Measurement of bacterial densities is generally the first
1.5 Approximately 10 cells/mL are required for detection
by this test method. step in establishing a relationship between bacteria and other
biochemical processes. It is known that the classical plate
1.6 Minimal cell size which allows the detection of forma-
zan deposits is represented by bacteria of 0.4 µm. count procedure underestimates bacterial densities while the
epifluorescence direct microscopic procedure more accurately
1.7 This standard does not purport to address the safety
depicts the total numbers of nonviable or dormant and viable
concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety and cells in a water sample. The acridine-orange INT-formazan
reduction technique provides information on the total concen-
health practices and determine the applicability of regulatory
limitations prior to use. trationsofbacteriaaswellasthatproportionwhichareactively
respiring and thus involved in degradative processes.
2. Referenced Documents
5.2 The acridine-orange INT-formazan reduction technique
2.1 ASTM Standards: is both quantitative and precise.
D 1129 Terminology Relating to Water 5.3 Thisprocedureisidealforenumeratingbothpelagicand
D 1193 Specification for Reagent Water epibenthic bacteria in all fresh water and marine environments.
D 3370 Practices for Sampling Water from Closed Con- 5.4 The process can be employed in survey studies to
duits characterize the bacteriological densities and activities of
environmental waters.
3. Terminology
6. Apparatus
3.1 Definitions—For definitions of terms used in this test
method, refer to Terminology D 1129. 6.1 Fluorescence Microscope, with an oil immersion objec-
tive lens (1003).
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. Zimmerman, et al, “Simultaneous Determination of Total Number of Aquatic
Current edition approved Jan. 25, 1985. Published March 1985. Bacteria and the Number Thereof Involved in Respiration,” Applied and Environ-
DIFCOTechnicalInformation—BactoAcridineOrangeStain,isavailablefrom mental Microbiology, Vol 36, 1978, pp. 926–935
Difco Laboratories, P.O. Box 1058, Detroit, MI 48201. Cherry, et al, “Temperature Influence on Bacterial Populations in Aquatic
Annual Book of ASTM Standards, Vol 11.01. Systems,” Water Res., Vol 8, 1974, pp. 149–155.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
D4454–85 (2002)
6.2 Eye Pieces, 12.53, equipped with a net micrometer (10 8.1.3 Stop the reaction by adding 0.1 mL of 37 % formal-
by 10 mm) (25 3 2-mm squares). dehyde that also acts as preservative (at this stage the sample
6.3 Condenser, 1.253, suitable for the microscope. can be stored at 4°C up to one month).
6.4 High-Pressure Mercury Lamp, 200-W, on a UV light
8.2 Membrane Filtration and Microscopic Examination:
sourcegivingverticalillumination,andafilterunitH2(Leitz)
8.2.1 Filter 1 mL of the (INT) treated/preserved sample
with BG12 and BG38 transmission filters or equivalents.
through 0.1-µm polycarbonate membrane which has been
6.5 Stage Micrometer, 2 by 200 parts.
presoaked for 24 h in a solution of sudan black B (BDH) in
6.6 Membrane Filter Support, sterile, particle-free, fritted-
50 % ethanol.
glass, 25 mm.
8.2.2 Stain the filter with 3 mLof acridine orange for 3 min.
6.7 Funnel, 15-mL capacity or equivalent.
8.2.3 Filter the acridine orange.
6.8 Membrane Filter, sterile plain regular polycarbonate,
8.2.4 Remove the filter, and air-dry for 15 s.
25-mm (0.1-µm pore size).
8.2.5 Place the membrane on a clean slide on which has
6.9 Filter Apparatus, that should contain vacuum source,
been added.1 to 2 drops of very low fluorescing immersion
filtering flask, and a filtering flask as a water trap.
oil.
6.10 Forceps (flat tip), Alcohol, Bunsen Burner, Clean
Glass Slides, and Cover Slips. 8.2.6 Place another drop of the immersion oil on top of the
membrane and apply the cover slip.
7. Reagents and Materials
8.2.7 Countcellsusingincidentfluorescentilluminationina
7.1 Purity of Reagents—Reagent grade chemicals shall be
violet light wavelength range (410 nm) for total bacteria.
used in all tests. Unless otherwise indicated, it is intended that
8.2.8 Switch to bright field illumination and count cells
all reagents conform to the specifications of the Co
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