## 3 Results and Discussion
### Experimental Evaluation of a Paenibacillus_ sp. _Specific Probe_  
Normally, when designing a probe, several experimental conditions are tested for the evaluation and optimization of the probes, however, excellent in silico results do not always translate into excellent experimental results [37]. Therefore, after the design of the probes, it is necessary to proceed to the experimental evaluation, as well as to tests to optimize the stringency conditions, in which the hybridization reaction occurs between the probe and its complementary target (generally located in the rRNA), as well as how to test the probe against non-target organisms from the same ecosystem as the target organism. The stringency of the reaction is adjusted by varying the incubation temperature or the concentration of formamide in the hybridization buffer in concentration steps of 5% (\(v/v\)) [38; 39; 40]. In the experimental evaluation of the hybridization efficiency of the FISH probe, it is still of vital importance to carry out, under the appropriate experimental conditions, different assays: blank (to exclude autofluorescence of the microorganism), positive and negative controls, and the test probe [21; 41]. The results of the experimental evaluation and stringency of the Pab489-Cy3 probe are shown in Figure 1 (flow cytometry (FC) results) and Figure 2 (percentage of fluorescent cells and fluorescence intensity). Comparing the flow cytometry results (Figure 1) of the negative control (EUK516-Cy3) and the Pab489-Cy3 probe, we observed that the probe was able to hybridize with _Paenibacillus_ sp. (target microorganism). It was also possible to observe that, regardless of the formamide concentration (stringency assay), the Pab489-Cy3 probe is always able to hybridize with _Paenibacillus_ sp. cells (green) when compared to the negative control EUK516-Cy3 (yellow). After processing the results, it was possible to calculate the percentage of fluorescent cells and the fluorescence intensity (FI). As we can see in Figure 2, the Pab489-Cy3 probe, regardless of the formamide concentration, hybridizes with the target cells, showing values close to those observed for the positive control (EUB338-Cy3). The Pab489-Cy3 probe shows fluorescence intensity values > 500,000 a.u. for formamide concentrations below 30%, where they reach the maximum percentage of hybridized cells (FI > 1,000,000 a.u.). The positive control probe EUB338-Cy3 has a similar profile to the test probe Pab489-Cy3, with maximum FI when [FA] = 30%. The negative control EUK516-Cy3 showed FI values less than 200,000 a.u. for all formamide concentrations, showing a slightly higher FI value for [FA] = 40%. Regarding the percentage of hybridized cells, we observed that for the probes Pab489-Cy3 and EUB338-Cy3, the values were always above 90%, while for the negative control (EUK516-Cy3), we observed values of percentage of fluorescent cells below 10%, except for [FA] = 40% which showed about 40% fluorescent cells. These results confirmed that, for [FA] < 40%, hybridization of the Pab489-Cy3 probe occurs in the target cells, with no false positives observed. Figure 1: \(Cont.\)  
Figure 1: Flow cytometry (FC) results (fluorescence intensity (FI)/forward scattering (FSC)) of the Pab489-Cy3 probe for the target microorganism (_Panetobacillus_ sp.) at different concentrations of formamide (FA) from 0 to 55%. The analysis by epifluorescence microscopy showed results in agreement with those obtained by flow cytometry, with fluorescent cells being observed for the probes Pab489-Cy3 and EUB338-Cy3, while for the blank (without probe) and negative control samples (EUK516-Cy3) there were no fluorescent cells, regardless of the formamide concentration. In Figure 3, a microphotography of each sample in the absence of formamide is shown, by way of example. Figure 3: Microphotography captured under the epifluorescence microscope in objective amplification of \(100\times\) ([FA] = 0%).