ASTM D4455 - 85(2014)

    Standard Test Method for Enumeration of Aquatic Bacteria by Epifluorescence Microscopy Counting Procedure

    Active Standard ASTM D4455 | Developed by Subcommittee: D19.24

    Book of Standards Volume: 11.02


      Format Pages Price  
    PDF 3 $38.00   ADD TO CART
    Hardcopy (shipping and handling) 3 $38.00   ADD TO CART


    Significance and Use

    5.1 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

    5.2 Acridine-orange epifluorescence direct-counting procedure cannot differentiate between viable and nonviable cells.

    5.3 This procedure cannot be used to convert directly the numbers to total carbon biomass because of the natural variations in bacterial cell size.

    5.4 The acridine-orange epifluorescence direct-microscopic count is both quantitative and precise.

    5.5 This procedure is ideal for enumerating both pelagic and epibenthic bacteria in all fresh water and marine environments.5

    5.6 The process can be employed in survey activities to characterize the bacteriological densities of environmental waters.

    5.7 The procedure can also be used to estimate bacterial densities in cooling tower waters, process waters, and waters associated with oil drilling wells.

    1. 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.2

    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.2

    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.


    2. Referenced Documents (purchase separately) The documents listed below are referenced within the subject standard but are not provided as part of the standard.

    ASTM Standards

    D1129 Terminology Relating to Water

    D1193 Specification for Reagent Water


    ICS Code

    ICS Number Code 07.100.20 (Microbiology of water)

    UNSPSC Code

    UNSPSC Code 50121700(Aquatic invertebrates); 41111722(Fluorescent microscopes)


    Referencing This Standard

    DOI: 10.1520/D4455

    ASTM International is a member of CrossRef.

    Citation Format

    ASTM D4455-85(2014), Standard Test Method for Enumeration of Aquatic Bacteria by Epifluorescence Microscopy Counting Procedure, ASTM International, West Conshohocken, PA, 2014, www.astm.org

    Back to Top