Isolation of living Algae growing in the shells of Molluscs and Barnacles with EDTA (ethylenediaminetetraacetic acid)

Several decalcifying mixtures or aqueous solutions of inorganic or organic acids are generally used for releasing algae growing in the shells of molluscs and barnacles, for instance dilute hydrochloric, nitric, citric, or acetic acid (4), a mixture of nitric acid, chromic acid and alcolhol (1), nitric acid and alcohol (9), chlorine dioxide and acetic acid (diaphanol) (3, 6, 11, 12), and formic acid and formiate (8). For a review see (7). Such mixtures or solutions generate carbondioxide bubbles that more or less disorganise the histological structure of the thallus. They also hydrolyse cell-contents and cell-wall material. Diaphanol and formiate appeared to be relatively useful for conserving the histological structure of the thalli. Recently (5, 10, 13) chelating agents, e.g. EDTA (ethylenediaminetetraacetic acid) were introduced as histological decalcifiers. The fact that EDTA is widely used as a chelating ingredient of nutrient media for algal cultures suggested its possible use for releasing living algae growing in animal shells. However, the concentration necessary for decalcification is a multiple of that used in culture media (1 % to 5 % and 0.0005 %. respectively). It is possible to vary the pH from 5—10, in accordance with the pH of the natural habitat of the alga.