coli and A. baumannii, incubated with ampicillin and imipenem respectively as described previously, were mixed with 950 μl of methanol:acetic-acid (3:1), one drop being spread onto glass slides and air-dried. The slides were immersed in methanol:acetic-acid (3:1) 5 min and air-dried again. Then, they were incubated with increasing ethanol baths (70-90-100%), -20°C, 5 min each, and air-dried. DNA was denatured by immersion in 75% formamide/2 × SSC, pH7, 67°C, 90 sec and then the slides were immersed in increasing ethanol baths (70-90-100%),

-20°C, 5 min each, and air-dried. Whole genome DNA probes to label the total DNA from E. coli and from A. baumannii were prepared. DNA from each microorganism RGFP966 molecular weight was isolated using standard procedures, and was labelled with biotin-16-dUTP, using a nick translation kit, according to the manufacturer’s instructions (Roche Applied Science,

San Cugat del Vallés, Spain). The DNA probes were mixed at 4.3 ng/μl in the hybridization buffer (50% formamide/2 × SSC, 10% dextran sulfate, 100 mM calcium find more phosphate, pH 7.0) (1 × SSC is 0.015 M NaCitrate, 0.15 M NaCl, pH 7.0). The probes in hybridization buffer were denatured by incubation at 80°C for 8 min and were then incubated on ice. The DNA probe solutions (15 μl) were pipetted onto the denatured and dried slides, click here covered with a glass coverslip (22 × 22 mm) and incubated overnight at 37°C, in the dark, in a humid chamber. The coverslip was removed, and the slides were washed twice in 50% formamide/2 × SSC, pH 7.0, for 5 min, and twice in 2 × SSC pH 7.0, for 3 min, at 37°C. The slides were incubated

with blocking solution (4 × SSC, 0.1% Triton X-100, 5%BSA) Osimertinib purchase for 5 min, covered with a plastic coverslip, in a humid chamber, at 37°C. This solution was decanted, and the bound probe was detected by incubation with streptavidin-Cy3 (Sigma Chem, St Louis, MN, USA) in 4 × SSC, 0.1% Triton X-100, 1%BSA (1:200), covered with a plastic coverslip, in a humid chamber at 37°C. After washing in 4 × SSC, 0.1% Triton X-100, three times, 2 min each, slides were counterstained with DAPI (1 μg/ml) in Vectashield (Vector, Burlingame, CA). Fluorescence Microscopy and Digital Image Analysis Images were viewed with an epifluorescence microscope (Nikon E800), with a 100× objective and appropriate fluorescence filters for FITC-SYBR Gold (excitation 465-495 nm, emission 515-555 nm), PI-Cy3 (excitation 540/25 nm, emission 605/55 nm) and DAPI (excitation 340-380 nm, emission 435-485 nm). In the experiment of dose-response to ampicillin, images were captured with a high-sensitivity CCD camera (KX32ME, Apogee Instruments, Roseville, CA). Groups of 16 bit digital images were obtained and stored as .tiff files. Image analysis used a macro in Visilog 5.1 software (Noesis, Gif sur Yvette, France).