Use of historesin for viewing Batrachochytrium dendrobatidis in the mouth of Lithobates catesbeianus tadpoles (Rana catesbeiana Shaw, 1802), bullfrog

In 1998 an emerging disease, chytridiomycosis, was diagnosed, caused by the fungus, Batrachochytrium dendrobatidis, capable of infecting amphibians (Urodela and Anura) and it was considered to be the main cause of the global decline of these animals. Continuing the research work on the presence of this fungus in commercial flocks, tadpoles of bullfrogs were studied, where its presence was possibly found in the mouth of these animals using special histological techniques like historesin. Starting from the visual observation of the mouth of the tadpoles, it was observed that there was a discoloration of the partial structures of the denticles, which were typically dark in color and appeared whitish instead. The mouth of tadpoles with denticles was collected, and fixed in 10% buffered formalin. The inclusion of the biological material in glycol methacrylate (GMA) has been widely used due to a number of advantages over the usual histological methods, such as faster processing, water soluble resins eliminate the use of solvents, easy handling, obtaining semifine sections from 0.5 to 5 μm, highest resolution light microscope, and material is processed at room temperature thereby reducing distortions and artifacts. We used the commercial kit historesin FONTOVIT 7100 (Kulzer)®. After fixation in cacodylate buffer, dehydration increased in ethanol alcohol, pre-infiltrated with historesin diluted in alcohol, infiltrated with historesin pure and polymerized. Following mounting in acrylic and cut with automatic microtome. The staining can be performed with techniques used for inclusion in paraffin and is not necessary to hydrate or diaphanized cuts and color dyes suitable for fungi such as cotton blue and Congo red and hematoxylin-eosin. Slides were covered with Entellan® and coverslip. Characteristics of fungal structures, such as zoosporangia, single or grouped globular structures with discharge pipes for the removal of spores, were observed.