© 2005 The Royal Microscopical Society Journal of Microscopy, Vol. 218, Pt 3 June 2005, pp. 240–246 Received 17 May 2004; accepted 15 March 2005 Blackwell Publishing, Ltd. A simple enzymatic method for examining calcite ossicles of Echinodermata C. G. TIAGO*, A. D. BRITES* , † & G. Y. KAWAUCHI* , † *Centro de Biologia Marinha, Universidade de São Paulo, São Sebastião, SP, 11600–000, Brazil †Instituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brazil Key words. Calcite ossicles, Echinodermata, enzymatic method, scanning electron microscopy. Summary The various shapes, size and fine details of ossicles are of para- mount importance as systematic characters in the Echinoder- mata. 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). Obser- vations of material prepared using the hypochlorite and the enzyme methods were made using scanning electron micro- scopy (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. Received 17 May 2004; accepted 15 March 2005 Introduction Echinoderm identification is based mainly on the morphology of the calcite skeleton. This skeleton is composed of numerous ossicles, united by muscles and other tissues (Ophiuroidea, Asteroidea, Echinoidea and Crinoidea) or scattered in the der- mal tissues of the animal (Holothuroidea). The classification of the echinoderms is based on the type, shape and fine details of the calcareous skeleton. The identification of these animals depends on the use of suitable techniques to clean the skele- ton, or to isolate the ossicles. The idea of developing a new method to isolate echinoderm ossicles arose because we had started to work with holothu- rians with minute ossicles, that usually are damaged with the traditional method which uses hypochlorite solution (house- hold bleach) to dissolve the tissues (Deichmann, 1954; Pawson, 1986; Ditadi, 1987; Hendler et al ., 1995; Cutress, 1996). However, this hypochlorite solution, when not properly buffered, can corrode the calcareous ossicles, resulting in modifi- cation of their shape, dimensions and fine details sometimes are completely lost (A. S. F. Ditadi, personal communication). Alterations in the characteristics of the ossicles can even com- promise the species identification, especially when identifica- tion is based on slight differences in dimensions and surface details of ossicles or when the ossicles in young individuals are very different from those in adults (Cutress, 1996). Proteolytic enzymes are used in several cleaning processes, such as in domestic protein-cleaning process of contact lenses, in clarification of halacarid mites (Newell, 1947) or in the pro- cedure of clearing and counterstaining cartilages and bones of fishes (Taylor & Van Dyke, 1985). The new method, developed to isolate the ossicles of echino- derm tissues, is based on the step of clearing the muscle tissue with proteolytic enzyme in a borax buffer in the Taylor & Van Dyke (1985) procedure. Materials and method The following materials were used for the method (see Fig. 1): pieces of body tissues of echinoderms (fresh or preserved); distilled water; sodium tetraborate (borax: Na 2 B 4 O 7 ·10H 2 O); proteolytic enzyme; glycerine jelly (Green & Macquitty, 1987), Permount® or Entellan®. The enzymatic method for isolating ossicles was tested with pieces of body tissues of three species of three different orders of holothurians: Holothuria grisea Selenka, 1867 (Aspido- chirotida), Duasmodactyla seguroensis (Deichmann, 1930) (Dendrochirotida) and Synaptula secreta Ancona Lopez (1957) (Apodida). It is possible to use either fresh or preserved pieces of body tissues, unless the animal or the tissues were preserved in non-buffered formalin, which corrodes the ossicles and precludes the process of identification in echinoderms. Correspondence: Dr Claudio G. Tiago. Tel.: +55 12 3862 7149; fax: +55 12 3862 6646; e-mail: clgtiago@usp.br