and Pediococcus sp used in Quevedo et al [36]; The R symbol of

and Pediococcus sp. used in Quevedo et al. [36]; The R symbol of the DNA probe sequence may be Adenosine or Guanosine, therefore Quevedo et al. [36] used a degenerate base in the sequence of the DNA probe to detect Lactobacillus spp. f The Y symbol of the DNA probe sequence may be Cytidine or Thymidine, therefore Fredricks et al. [6] used a degenerate base in the sequence of the DNA probe to

detect G. vaginalis g Values determined in Machado PF-01367338 molecular weight et al.[26]. FISH hybridization procedure Biomass from a single colony of each strain was diluted and homogenised in sterile water, and then 20 μL were spread on epoxy coated microscope glass slides (Thermo Scientific, USA). For mixed samples (see Table 3), 10 μL of the final suspension from each strain suspension (prepared as previously referred) for the selected mixed sample were spread on glass slides. The slides were air-dried prior to fixation.

Next, the smears were immersed in 4% (wt/vol) paraformaldehyde (Fisher Scientific, United Kingdom) followed by 50% (vol/vol) ARS-1620 clinical trial ethanol (Fisher Scientific, United Kingdom) for 10 min at room temperature on each solution. After the fixation step, the samples were covered with 20 μL of hybridization solution containing 10% (wt/vol) dextran sulphate (Fisher Scientific, United Kingdom), 10 mM NaCl (Sigma, Lazertinib order Germany), 30% (vol/vol) formamide (Fisher Scientific, United Kingdom), 0.1% (wt/vol) sodium pyrophosphate (Fisher Scientific, United Kingdom), 0.2% (wt/vol) polyvinylpyrrolidone (Sigma, Germany), 0.2% (wt/vol) ficoll (Sigma, Germany), 5 mM disodium EDTA (Sigma, Germany), 0.1% (vol/vol) triton X-100 (Sigma), 50 mM Tris-HCl (at pH 7.5; Sigma, Germany) and 200 nM of the PNA probe. Subsequently, the samples on glass slides were covered with coverslips and incubated in moist chambers at the hybridization temperature under analysis (from 50°C to 72°C) during a range of hybridization times (from 230 to 180 min). Next, the coverslips were removed and a washing step was performed by immersing the slides in a pre-warmed washing solution for 30 min at the same temperature of the hybridization step. This solution consisted of 5 mM Tris-base (Fisher Scientific, United Kingdom), 15 mM NaCl (Sigma,

Germany) and 0.1% (vol/vol) P-type ATPase triton X-100 (at pH 10; Sigma, Germany). Finally, the glass slides were allowed to air dry. Table 3 Results of the Lac663 and Gard162 probes specificity test in artificial mixed samples Species in the artificial mixed samples Bacteria strain collection codes Multiplex PNA-FISH assay Lac663 Probe efficiency Gard162 Probe efficiency L. pentosus; CECT 4023T; – ++++ ++++ G. vaginalis 51 L. casei; CECT 5275T; – ++++ ++++ G. vaginalis 101 L. rhamnosus; CECT 288T; – ++++ ++++ G. vaginalis AMD L. crispatus; ATCC 33820T; – ++++ ++++ G. vaginalis ATCC L. delbrueckii sub. delbrueckii; Atopobium vaginae ATCC 9649T; CCUG 38953T +++ – L. acidophilus; ATCC 4356T; CCUG 42099T ++++ – A. vaginae L. gasseri; ATCC 9857T; CCUG 44116T ++++ – A.

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