J Microsc 2005 Jun 01;218Pt 3:240-6. doi: 10.1111/j.1365-2818.2005.01486.x.

A simple enzymatic method for examining calcite ossicles of Echinodermata.

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The various shapes, size and fine details of ossicles are of paramount importance as systematic characters in the Echinodermata. The ossicles in this group are endodermal and a method to free them from the dermis is essential for their study. To date, a small quantity of hypochlorite solution (usually household liquid bleach) has been used to dissolve a small piece of tissue and, consequently, free these ossicles. A new method, using a proteolytic enzyme instead of hypochlorite solution to dissolve holothurian tissues, is proposed. This method was tested on fragments of body tissues from three species in three different orders of holothurians: Holothuria grisea Selenka, 1867 (Aspidochirotida), Duasmodactyla seguroensis (Deichmann, 1930) (Dendrochirotida) and Synaptula secreta Ancona Lopez (1957) (Apodida). Observations of material prepared using the hypochlorite and the enzyme methods were made using scanning electron microscopy (SEM). The SEM images show the striking differences in the surface of the ossicles obtained by each method, and reveal the advantages of the enzyme method.

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