## 3 Results and Discussion
### Experimental Evaluation of a Paenibacillus_ sp. _Specific Probe_
(**A**) Blank sample (without probe); (**B**) negative control (EUK516-Cy3); (**C**) test probe (Pab489-Cy3); and (**D**) positive control (EUB338-Cy3). Figure 2: Percentage of _Paenibacillus_ sp. fluorescent cells and fluorescence intensity (FI) obtained by flow cytometry after RNA-FISH treatment with Pab489-Cy3 probe (test), EUB338-Cy3 probe (positive control) and EUK516-Cy3 probe (negative control) at different concentrations of formamide (FA, 0 to 55%). The percentage of hybridized cells was calculated according to the following formula: (\(\Sigma\) number of cells hybridized with Pab489-Cy3 probe) \(-\) (\(\Sigma\) number of cells hybridized with EUK516-Cy3 probe)) \(\times\) 100/1000. Knowing that formamide is toxic and a carcinogenic compound to humans [42; 43; 44], and that in its absence the Pab489-Cy3 probe presents good hybridization results, the hybridization assays of the Pab489-Cy3 probe with non-target microorganisms (Figure 4) were carried out in the absence of formamide. Experimental tests performed with the Pab489-Cy3 probe in the presence of non-target microorganisms suggests the specificity of this probe for the target microorganism since the populations of cells hybridized with the Pab489-Cy3 probe are like the populations of _Bacillus_ spp., _Arthobacter_ spp. and _R. radiotolerans_ cells hybridized with the EUK516-Cy3 probe (negative control). It should be noted that RNA-FISH was not tested on the spores and therefore the presence of _Paenibacillus_ sp. in the samples studied could be underestimated. By the technique of epifluorescence microscopy, no signal was observed in the cells of non-target microorganisms hybridized with the probe Pab489-Cy3, thus confirming the results observed by flow cytometry. This new probe Pab489 represents a considerable advance in the detection/identification of strains of the genus _Paenibacillus_, especially in comparison with the other methodologies. On the one hand, the classic microbiology methods for the detection/identification of microorganisms imply isolation from the original matrix and allow characterization of only those microorganisms capable of growing, multiplying, and forming colonies in the selected growth medium and conditions [45; 46]. In addition to being conditioned by the use of the appropriate culture medium, culture conditions can affect microbial communities, affecting the complete observation of the ecosystem, since it is estimated that only 1% of microorganisms that exist in nature can be cultivated in the laboratory [47; 48; 22; 46]. On the other hand, molecular biology techniques, based on DNA amplification, which are more specific, more sensitive, and faster than traditional methods, do not allow collection of information about spatial distribution, identity, and morphology, nor in situ analysis [21; 24; 47]. Using the FISH methodology, the analysis of microbial communities is fast and culture-independent, having as the main advantage the specific in situ identification of individual microbial cells [47; 49; 50], presenting a high potential for the study of the biodeterioration of the cultural heritage [51; 52]. Figure 4: Flow cytometry (FC) results (fluorescence intensity (FI)/forward scattering (FSC)) of Pab489-Cy3 (test) and EUK516-Cy3 (negative control) in the presence of target (_Paenibacillus_ sp.) and non-target microorganisms, i.e., two strains of _Bacillus_ sp., (_Bacillus_ sp., 1 and 2), two strains of _Arthobacter_ sp. (_Arthobacter_ sp., 1 and 2) and two strains of _Rubbacher radiololerans_ (_R. radiololerans_ 1 and 2).