Colonisers of Cultural Heritage: The influence of the physiological stage on Fluorescence In Situ Hybridisation analysis

Two yeast and two bacteria (one Gram-negative and one Gram-positive) were used in this study, two of them were isolated from biodeteriorated mural paintings. The cells were harvested from pure liquid cultures at different growth times. After that, a common FISH protocol was applied using two commercial probes and 80% ethanol/PBS (v/v) as fixative and the resultant fluorescent cells were analysed. The RNA extraction was carried out simultaneously using specific protocols for bacteria and for yeast cells. Good signals were obtained by epifluorescence microscopy independently of the physiological state and of the microorganisms analysed (yeast or bacteria). This indicates the possible application of FISH technique for detecting these microorganisms in CH. The results obtained by flow cytometry show that the fluorescent intensities as well as the percentage of the initial fluorescent cells are strongly dependent of the physiological state. It has also been observed that the percentage of cells that become fluorescent, after the application of the FISH protocol, is much higher for yeast than for bacteria. However, none clear differences were found in terms of fluorescence intensities. For Gram-negative bacteria it was found a direct correlation between the intensities of the FISH signals and the RNA content per cell