ASTM-D4454 Standard Test Method for Simultaneous Enumeration of Total and Respiring Bacteria in Aquatic Systems by Microscopy (Withdrawn 2015)

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Standard Test Method for Simultaneous Enumeration of Total and Respiring Bacteria in Aquatic Systems by Microscopy (Withdrawn 2015)


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 μm.2

1.7 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.

1.8 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.

Significance and Use

Measurement of bacterial densities is generally the first step in establishing a relationship between bacteria and other biochemical processes. 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.


Water-formed deposits; Bacteria/bacterial control--water; Epifluorescence microscopy; Fluorescence--water analysis; Microscopic examination--water; Pathogens--water; ICS Number Code 07.100.20 (Microbiology of water)

To find similar documents by ASTM Volume:

11.02 (Water (II))

To find similar documents by classification:

07.100.20 (Microbiology of water Examination of biological properties of water, see 13.060.70)

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Document Number


Revision Level

1985 R09 EDITION



Modification Type


Publication Date

July 1, 2009

Document Type

Test Method

Page Count

3 pages

Committee Number