A simple cleaning and fluorescent staining protocol for recent and fossil diatom frustules

The analysis of diatom frustules by fluorescence microscopy and, in particular, confocal laser scanning microscopy requires adequate cleaning and staining methods prior to imaging. Here, a set of tools for an imaging procedure for fossil and living diatoms is presented. A simple and inexpensive cleaning protocol and four easily synthesised fluorescent dyes will give diatomists the possibility to analyse mostly organic-free diatom frustules at different wavelengths. The cleaning of cultured diatoms can easily be conducted at ambient temperature with commercially available bleaches based on hypochlorite solutions. This method quickly leads to clean and stainable frustules. Besides the known fluorescent dye, fluorescein isothiocyanate-(3-aminopropyl)trimethoxysilane (FITC-APS), three related new dyes, rhodamine B isothiocyanate-(3-aminopropyl)trimethoxysilane (RBITC-APS), eosin 5-isothiocyanate-(3-aminopropyl)trimethoxysilane (EITC-APS) and tetramethylrhodamine B isothiocyanate-(3-aminopropyl)trimethoxysilane (TRITC-APS) are introduced. This protocol is suitable for confocal laser scanning microscopy and the creation of three-dimensional models of diatom frustules for palaeontologists and biologists likewise allowing detailed morphological studies.