Epoxy coated microscope glass slides

The bacterial population within the 48-h multi-species biofilms and in their planktonic fraction was discriminated using the peptide nucleic acid fluorescence in situ hybridization (PNA FISH) method, as previously described [37 (link)]. Briefly, after fixing the biofilm suspension, a PNA probes specific for G. vaginalis (Gard162) and for A. vaginae (AtoITM1) were added to each well of epoxy-coated microscope glass slides (Thermo Fisher Scientific). An additional staining step was done at the end of the hybridization procedure, covering each glass slide with DAPI (2.5 μg/mL). Microscopic visualization was performed using filters capable of detecting the PNA Gard162 probe (BP 530-550, FT 570, LP 591 sensitive to the Alexa Fluor 594 molecule attached to the Gard162 probe), the PNA AtoITM1 probe (BP 470–490, FT500, LP 516 sensitive to the Alexa Fluor 488 molecule attached to the AtoITM1 probe), and DAPI (BP 365–370, FT 400, LP 42). Twenty fields were randomly acquired in each sample. The number of bacteria was counted using ImageJ Software [59 (link)]. To reduce any possible overestimation due to the use of DAPI as the probe efficiency was not 100%, we then applied the equations from Table 2 to obtain more accurate relative values. These assays were repeated three times on separate days.

Prior to microscopic analysis, we assessed if the PM-477 challenge with G. vaginalis and F. vaginae single-species biofilms affected the performance of the PNA FISH method. As such, we determined the efficiency of the PNA probes by using several dilutions of G. vaginalis and F. vaginae single-species biofilm cells without and after 24 h of the PM-477 challenge, correlating the DAPI with PNA FISH counts, as previously described [41 (link)]. In brief, biofilms aliquots were first fixed on epoxy-coated microscope glass slides (Thermo Fisher Scientific). Then, a PNA probe specific for G. vaginalis (Gard162) [43 (link)] or for F. vaginae (AtoITM1) [44 (link)] was added to each well. At the end of the hybridization procedure, an additional staining step was done, covering each glass slide with DAPI (2.5 μg/mL). Microscopic visualization was performed using filters capable of detecting the PNA Gard162 probe (BP 530-550, FT 570, and LP 591 sensitive to the Alexa Fluor 594 molecule attached to the Gard162 probe), the PNA AtoITM1 probe (BP 470-490, FT500, and LP 516 sensitive to the Alexa Fluor 488 molecule attached to the AtoITM1 probe), and DAPI (BP 365-370, FT 400, LP 42). For each sample, thirteen fields were randomly acquired. The number of bacteria was counted using ImageJ software [45 (link)]. These assays were repeated three times on separate days.