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    ASTM D4454-85(2009)

    Standard Test Method for Simultaneous Enumeration of Total and Respiring Bacteria in Aquatic Systems by Microscopy (Withdrawn 2015)

    Withdrawn Standard: ASTM D4454-85(2009) | Developed by Subcommittee: D19.24


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    Withdrawn Rationale:

    This test method covers the detection and enumeration of aquatic bacteria by the use of an acridine-orange epifluorescence direct-microscopic counting procedure.

    Formerly under the jurisdiction of Committee D19 on Water, this test method was withdrawn in December 2015 in accordance with section 10.6.3 of the Regulations Governing ASTM Technical Committees, which requires that standards shall be updated by the end of the eighth year since the last approval date.

    1. 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 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.



    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


    Referencing This Standard
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    DOI: 10.1520/D4454-85R09

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    Citation Format

    ASTM D4454-85(2009), Standard Test Method for Simultaneous Enumeration of Total and Respiring Bacteria in Aquatic Systems by Microscopy (Withdrawn 2015), ASTM International, West Conshohocken, PA, 2009, www.astm.org

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