ASTM D4454-85(1997)

Standard Test Method for Simultaneous Enumeration of Total and Respiring Bacteria in Aquatic Systems by Microscopy

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.

General Information

• Status: Historical
• Publication Date: 09-Dec-1997
• Technical Committee: D19 - Water
• Drafting Committee: D19.24 - Water Microbiology

Relations

• Replaced By: ASTM D4454-85(2002) - Standard Test Method for Simultaneous Enumeration of Total and Respiring Bacteria in Aquatic Systems by Microscopy
• Effective Date: 25-Jan-1985

Standards Content (Sample)

Designation: D 4454 – 85 (Reapproved 1997)
AMERICAN SOCIETY FOR TESTING AND MATERIALS
100 Barr Harbor Dr., West Conshohocken, PA 19428
Reprinted from the Annual Book of ASTM Standards. Copyright ASTM
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-phenyl tetrazo-
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
from other fluorescing bodies on the basis of morphology when 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
bacteria where reduced formazan is deposited at the polar ends.
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
concerns, if any, associated with its use. It is the responsibility depicts the total numbers of nonviable or dormant and viable
cells in a water sample. The acridine-orange INT-formazan
of the user of this standard to establish appropriate safety and
health practices and determine the applicability of regulatory reduction technique provides information on the total concen-
trations of bacteria as well as that proportion which are actively
limitations prior to use.
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 This procedure is ideal for enumerating both pelagic and
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).
6.2 Eye Pieces, 12.53, equipped with a net micrometer (10
by 10 mm) (25 3 2-mm squares).
This test method is under the jurisdiction of ASTM Committee D-19 on Water
6.3 Condenser, 1.253, suitable for the microscope.
and is the direct responsibility of Subcommittee D19.24 on Water Microbiology.
6.4 High-Pressure Mercury Lamp, 200-W, on a UV light
Current edition approved Jan. 25, 1985. Published March 1985.
DIFCO Technical Information—Bacto Acridine Orange Stain, is available from
Difco Laboratories, P.O. Box 1058, Detroit, MI 48201.
Zimmerman, et al, “Simultaneous Determination of Total Number of Aquatic
Annual Book of ASTM Standards, Vol 11.01.
Bacteria and the Number Thereof Involved in Respiration,” Applied and Environ-
mental Microbiology, Vol 36, 1978, pp. 926–935
Cherry, et al, “Temperature Influence on Bacterial Populations in Aquatic
Systems,” Water Res., Vol 8, 1974, pp. 149–155.
D 4454
source giving vertical illumination, and a filter unit H2 (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 mL of 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 8.2.6 Place another drop of the immersion oil on top of the
Glass Slides, and Cover Slips. membrane and apply the cover slip.
8.2.7 Count cells using incident fluorescent illumination in a
7. Reagents and Materials
violet light wavelength range (410 nm) for total bacteria.
8.2.8 Switch to bright field illumination and count cells
7.1 Purity of Reagents—Reagent grade chemicals shall be
used in all tests. Unless otherwise indicated, it is intended that showing only bright red spots (indication of respiring bacteria).
8.2.9 Count 20 fields at random within the s
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