Use of Membrane Filters for Microscopic Preparations of Sponge Spicules

Abstract

A new method for preparation of sponge spicules for microscopy utilizes membrane filters for separation of spicules from cleaning fluids. The membranes with adherent spicules are cleared during mounting on microscope slides for direct analysis by light microscopy. The basic filtration procedure is also used to produce preparations for scanning electron microscopy. The main advantages of this procedure over a variety of traditional methods include preclusion of spicule loss during cleaning and significant reduction of procedure-introduced variation. The technique described offers promise for quantitative analysis of spicule complements. Incentive to develop a standardized procedure for spicule preparation stems from several problems noted in publications of both veteran and neophyte sponge taxonomists. Most prevalent is a general absence of detailed reporting of methods employed in spicule examinations. The result is a disturbing lack of certainty in taxonomic identifications, deriving partly from real differences in the specimens themselves, but also is an artifact of the methods of spicule preparation and analysis. The most commonly reported problems are the rare occurrence or absence of a spicule type and differences in size of spicules from specimens that otherwise appear to belong to the same species. We surveyed 123 primary taxonomic papers on Porifera published during the last 40 years3. Although the sample is biased by both ease of access and language of publication (untranslated Eastern languages were excluded), it does include all of the important contemporary treatments: 75% (n = 92) of these papers include no mention of the method of skeletal preparation; 19% (n = 24) include a short mention of, or reference to, a technique; and only 6% (n = 7) provide a description of methods in sufficient detail to enable duplication. Potential investigators looking to these publications of methods to use in identification, and, more importantly, seeking an authoritative format for preparation of taxonomic publications, are provided with little or no guidance. A detailed set of standard procedures for (1) making spicule preparations, (2) examining them microscopically, and (3) carrying out and reporting quantitative statistical analyses on the data generated has not been formally proposed or tested. Our purpose is to put forward a basic procedure for producing permanent spicule preparations. This procedure is new, requires minimal speSupported by a Natural Sciences and Engineering Research Council of Canada Operating Grant