How endo- is endo-?: surface sterilization of delicate samples: a Bryopsis (Bryopsidales, Chlorophyta) case study

摘要

In the search for endosymbiotic bacteria, elimination of ectosymbionts is a key point of attention. Commonly, the surface of the host itself or the symbiotic structures are sterilized with aggressive substances such as chlorine or mercury derivatives. Although these disinfectants are adequate to treat many species, they are not suitable for surface sterilization of delicate samples. In order to study the bacterial endosymbionts in the marine green alga Bryopsis, the host plant's cell wall was mechanically, chemically and enzymatically cleaned. Merely a chemical and enzymatical approach proved to be highly effective. Bryopsis thalli treated with cetyltrimethylammonium bromide (CTAB) lysis buffer, proteinase K and bactericidal cleanser Umonium Master showed no bacterial growth on agar plates or bacterial fluorescence when stained with a DNA fluorochrome. Moreover, the algal cells were intact after sterilization, suggesting endophytic DNA is still present within these algae. This new surface sterilization procedure opens the way to explore endosymbiotic microbial communities of other, even difficult to handle, samples.