Viagram red bacterial gram stain and viability kit

Culture samples were treated using either a 5 μM solution of SYTO® 9 fluorescent stain, Live-Dead® Stain (SYTO® 9 and propridium bromide), or ViaGram™ Red⁺ Bacterial Gram Stain and Viability Kit (SYTOX® Green nucleic acid stain, 4,6-diamidino-2-phenylindole and Texas Red®-X dye–labeled wheat germ agglutinin) in sterile water (Life Technologies). Dark field fluorescence microscopy was performed using a Nikon Eclipse TE2000-S epifluorescence microscope at 20× or 2000× magnification using a high-pressure Hg light source.

Frozen sections of chronic wound tissues were stained using ViaGram Red + Bacterial Gram-Stain and Viability Kit (Life Technologies, Carlsbad, CA) with modifications to the manufacturer's protocol. Briefly, the frozen tissue sections were washed in 1X PBS for 5 minutes at room temperature to remove the OCT. Sections were then incubated in wheat germ agglutinin (WGA) conjugate stock solution for 5 minutes. The WGA solution was drained off the slide followed by the addition of 2.5 µL of the DAPI/Sytox Green working solution for 10 minutes at room temperature. The excess working solution was then removed from the section. Sections were mounted and visualized using a Nikon Microphot-FXA microscope with a Nikon DS-Fi1 digital camera.

In this second part of the microscopy assays, the variability of the resulting morphology of 48 h L34-Pseudomonas spp. dual-species biofilms were assessed. Therefore, three different dual-species biofilms, i.e., L34-CECT110, L34-CECT324, and L34-B52, were cultured and collected as described above. Samples were then stained by means of ViaGram™ Red+ Bacterial Gram Stain and Viability Kit (Life Technologies) following the manufacturer’s instructions. This stain allows in a mixed population the distinction of Gram-positive cells (red-emitting) with a counterstain with DAPI. Finally, samples were visualized under the epifluorescence microscope using a 63× water immersion objective, and the images were acquired as described in the previous section.