Fig. 1:

An overview for using the CBD for the purpose of microscopy and 3D visualization of microbial biofilms. A. To begin, fresh subcultures of the microbial strain were grown on the appropriate agar medium. B. Using a cotton swab, colonies from a fresh second subculture were suspended in broth medium to match a 1.0 McFarland standard. This was diluted 30-fold in broth to create the inoculum for the CBD. C. The peg lid of the CBD was either inserted into a microtiter plate (containing 150 μL of inoculum in each well) or a corrugated trough (with 22 mL of inoculum inside). The inoculated devices were placed in a humidified incubator on a gyrorotary shaker or platform rocker, respectively. D. After cultivation, biofilms were rinsed with saline to remove loosely adherent cells. E. Pegs were removed from the CBD using pliers and the biofilms then were enumerated by viable cell counting. A second set of pegs was removed for examination by microscopy. There is an option to expose biofilms to an array of antimicrobial agents or other test conditions and then to remove a second set of pegs for microscopy. F. For SEM, pegs were first fixed and then dehydrated, which was carried out using 1 of 2 protocols. G. The fixed samples were mounted on stubs using epoxy resin, dried, and then sputter coated with gold-palladium. H. The biofilms were then examined by SEM, and the resulting images were contrast enhanced. I. For CLSM, pegs were immersed in the appropriate stain and then placed in 2 drops of 0.9% saline on a glass coverslip. J. Images of the biofilms were captured using CLSM, and the instrument software was used to generate 2D averages of image z-stacks. K. The z-stacks were imported into amira™ for advanced image processing and 3D visualization.