A cold bath for a formalin-free laboratory: alternative fixative methods in early developmental stages of the sea urchin Paracentrotus lividus (Lamarck, 1816)

Fixatives are widespread in biological and medical research because they allow preserving specimens for a long time. Historically, formaldehyde has been the most used fixative so far, but new solutions are needed because of its carcinogenicity. In this study, we tested alternative fixative methods to find a harmless, economic, and simple-to-use methodology to fix samples for larval morphological analysis in Paracentrotus lividus. In two separate experiments, P. lividus embryos were fixed after 48 h post-fertilization by adding Formalin Free Tissue AccustainTM, NaOH-buffered Formalin Free Tissue AccustainTM, glacial ethanol and denatured ethanol at different concentrations (from 10 % to 70 %) and by submerging the vials containing the larvae in seawater at 0 °C and maintained at 4 °C for 144 h. Our results suggested that all the alternative fixatives tested do not guarantee a good quality of larvae for morphological purposes, while larvae that faced the thermal shock and were kept at 4 °C did not show any evidence of damage throughout time. The results of this study candidate this method as a good and safe substitute of formalin in studies that require morphological and taxonomic recognition and shed light on its use in other kinds of studies as well.