Clearing and staining small vertebrates for demonstrating ossification of the skeletal system using the KOH and glycerine clearing method

Abstract

Preparing articulated skeletons of small vertebrates is extremely difficult due to the presence of vast amounts of cartilage and/or the extremely small size of skeletal components. Clearing the tissues surrounding small skeletons and staining the bones in toto reveals the skeletal system without the risk of displacing the bony components. The specimens are first fixed and bleached which renders the tissues preserved and transparent. These specimens may then be exposed to alizarin red S which stains bone a pink to red colour making them visible through the transparent soft tissues. Toluidine blue may also be used to stain cartilage a blue colour. The Spalteholz staining method is cited, though the focus will be on the KOH and glycerine clearing method for macroscopic observation.