Abstract
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 enzymatic 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.
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Acknowledgments
This research was funded by ‘Fonds Wetenschappelijk Onderzoek’ FWO-Flanders project G.0045.08. We sincerely thank Valya Vassileva and Tom Beeckman for confocal microscopy assistance. We acknowledge John West for useful comments.
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Hollants, J., Leliaert, F., De Clerck, O. et al. How endo- is endo-? Surface sterilization of delicate samples: a Bryopsis (Bryopsidales, Chlorophyta) case study. Symbiosis 51, 131–138 (2010). https://doi.org/10.1007/s13199-010-0068-0
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DOI: https://doi.org/10.1007/s13199-010-0068-0