Cells on the lower surface of the Transwell membrane were stained with crystal violet solution and observed under a phase contrast microscope (n = 5 per group).
After culturing for 36 hours, cells on the lower surface of the membrane were stained with 0.1% crystal violet and photographed using the Carl Zeiss Axio Observer microscope.
Following an incubation period (90 min at 37 °C, 5% CO2), the cells that have migrated through the membrane were stained with trypan blue and manually counted.
After culturing for 24 hours, cells on the lower surface of the membrane were stained with 0.1% crystal violet and photographed using the Carl Zeiss Axio Observer microscope.
Cells migrating across the membrane were stained with blue-purple stain (Fig. 3A) and counted (Fig. 3B).
Migrating and invading cells on the lower surface of the membrane were stained with Hematoxylin and eosin.
The invading cells on the bottom surface of the membrane were stained using Hema 3 system (Fisher Scientific, Middletown, VA).
Cells which invaded across the membrane were stained with hematoxylin and eosin stain and counted (Fig. 4A B).
Cells that passed through the Matrigel-coated membrane were stained with Diff-Quik (Sysmex, Kobe, Japan) and photographed.
As no PNA probe was used for E. coli detection, for biofilm samples containing this bacterium, the polycarbonate membrane were stained with 60 µl of 4'-6-Diamidino-2-phenylindole (DAPI; 100 µg/ml) for 10 m in the dark.
After incubation, the non-invading cells were removed from the upper surface of the membrane by gentle scrubbing, and the cells on the lower surface of the membrane were stained with crystal violet.
