Analysing bacteria and fungi colonising Cultural Heritage by Fluorescence In Situ Hybridisation

Abstract

The FISH protocol developed by us for analysing filamentous fungi in suspension were used as a first approach to analyse the cells harvested from pure liquid cultures of yeast (S. cerevisiae and Rhodotorula sp) and bacteria (Bacillus sp and E. coli). The physiological state of the cells, their RNA content and enzymatic activity, as expected, show strong influence on the results. However, good FISH signals have been obtained, independently of the variation of these cell properties, by fixing the cells in ethanol/PBS solutions (50, 80 and 100% v/v) for 3, 15 and 60 min. The concentration of the cells and of the FISH probes, EUK516-Cy3 and EUB338-Cy3, reveal to be crucial factors for yeast and bacteria detection. Thus, they need to be controlled for ensuring the detection of these microorganisms. Since: I) yeast and bacteria have been successfully detected by applying the FISH protocol independently of the fixation conditions (ethanol/PBS solutions 50, 80 or 100% v/v for 3, 15 and 60 min); and II) these conditions have also been previously applied by us for analysing filamentous fungi isolated from the Portuguese CH obtaining good results, this work have result in the development of a rapid FISH protocol for analysing fungi as well as bacteria in suspension, reducing the fixation time to 3 min. This will be the basis for using RNA FISH for simultaneous analysis of these microorganisms in CH materials.