Standard Historical Last Updated: Mar 12, 2014
ASTM D4455-85(2009)

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.


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.

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Book of Standards Volume: 11.02
Developed by Subcommittee: D19.24
Pages: 3
DOI: 10.1520/D4455-85R09
ICS Code: 07.100.20