Enhancing the contrast and visibility of daily growth increments in fish otoliths etched by proteinase K buffer

The otoliths of the amphidromous gobies Stiphodon elegans and Sicyopterus japonicus , a marine eel Strophidon sathete and a freshwater fish Varicorhinus barbatulus were digested by proteinase K buffer (PKb), which removed the proteins and retained the major calcified structure to reveal conspicuous daily increments. Compared with etching by ethylenediaminetetraacetate (EDTA), etching with PKb revealed more visible daily increments and enhanced the contrast. Moreover, by using PKb, both the daily increments and annuli could be observed simultaneously. The better performance of PKb as an etching agent can be attributed to digesting of predominantly only the organic matrix and leaving the calcified structure almost intact. PKb provides another effective means to reveal otolith microstructure so as to examine and count daily growth increments.