Biological test method for determining toxicity of sediment using luminescent bacteria: chapter 4

Section 2: Test Organisms

• 2.1 Species
• 2.2 Source and Holding

2.1 Species

Organisms used in this test come from a standardized culture (strain NNRL B-11177; see Table 10, Appendix E), and belong to a particular species of luminescent marine bacteria (i.e., Vibrio fischeri; formerly classified as  Photobacterium phosphoreum). This is a bacterium which normally lives in the ocean, and produces a continuous blue-green light by enzymatic reactions if sufficient oxygen is available (EC, 1992).

2.2 Source and Holding

Standard cultures of V. fischeri can be purchased from Strategic Diagnostics Inc.^Footnote 1 Bacteria are marketed as a uniform strain of lyophilized (i.e., freeze-dried under vacuum) bacteria (“Bacterial Reagent”), harvested during the exponential phase of growth. Production lots are sold in packages which each contain ≥ 10 sealed vials. Each vial contains about 100 million (1 × 10⁶) lyophilized organisms.

Containers of lyophilized Bacterial Reagent are reportedly stable for up to one year when kept in a freezer at -20°C (EC, 1992).^Footnote 2 Storage temperature should be constant and range within -20 to -25°C; “frost-free” freezers which warm up during the defrosting phase should not be used. The viability of new lots of bacteria, or of individual lots used over an extended period, should be determined by a solid-phase reference toxicity test performed with one or more chemicals using the procedures and conditions described in Section 5. Each lot is suitable for at least two hours (EC, 1992) and up to three hours (Gaudet, 1998) of testing, after they have been reconstituted to an active state.

The number and expiry date of the lot of bacteria used in each toxicity test must be recorded; this information must be included in the test-specific report together with the species and strain of the test organism (see Section 7.1.2). Other data specific to the test organisms, including their source, date of receipt, and temperature during storage or holding, must either be included in the test-specific report or the general report, or held on file for a minimum of five years (see Section 7.2.2).