Static microtiter plate culture system for development of the BLS Bacteria were grown in a static microtiter plate culture system using sterile 24-well polystyrene #selleck screening library randurls[1|1|,|CHEM1|]# plates
(Falcon; BD, Franklin Lakes, NJ) [64, 65]. Tested strains were grown overnight in LB broth. Cells were pelleted, washed, and resuspended in PBS. For analysis of the BLS formed by individual bacterial species, resuspended cells were inoculated in ASM+ to an initial OD600 of 0.02-0.03 and dispensed into the plate wells in 1 ml aliquots. For the analysis of BLS produced by two bacterial species, individual species were prepared and inoculated at an initial OD600 of 0.015. The plates were incubated at 37°C in static (nonshaking) conditions under environmental oxygen (EO2) concentration of 20% (aerobic), 10% (microaerobic), or 0% (anaerobic). Individual GasPak jars with Campy Pak Plus envelopes (BD) or GasPak EZ Anaerobic Pouches (BD) were used to generate the microaerobic and anaerobic EO2
conditions, respectively. Visualization of the BLS This was done using confocal laser scanning microscopy (CLSM) [35, 64]. The BLS were visualized within the wells of the microtiter plates using an Olympus IX71 Fluoview 300 confocal laser scanning microscope (Olympus America, Melville, NY). All images were obtained through a 203/0.40 Ph1 NA objective utilizing a green helium laser (546 nm) or argon laser (510–530 nm). Three-dimensional image reconstructions were performed using NIS-Elements 2.2 (Nikon Instruments, Melville, NY) to visualize the architecture of the BLS. All instrument settings were consistent for each individual experimental selleck kinase inhibitor parameter tested. Quantitative structural analysis of the BLS The number of image stacks obtained from the BLS was based
on the greatest depth of the structures formed Cyclooxygenase (COX) under the test conditions and was the same for all strains/conditions within an experiment (See Tables 1, 2, 3, 4). Each experiment was done in duplicate. Two 10-image stacks were obtained from random positions within each BLS (total 40-image stacks for each strain and/or condition). The 40-image stacks were analyzed using the COMSTAT program [20] for structural features of the BLS: biovolume, estimates the biomass of the BLS; mean thickness, a measure of spatial size of the BLS; roughness coefficient, a measure of how much the thickness of the BLS varies, or the heterogeneity of the BLS; total surface area, space occupied in each image stack; and surface to biovolume ratio, estimates the portion of the BLS exposed to nutrients (biovolume divided by the surface area of the substratum). Values represent the mean ± SEM. Quantification of the bacteria within the BLS The highly viscous ASM+ forms a gelatinous mass in which the bacteria grow. Therefore, at the indicated time points, the mass from each well was transferred to a 1.5 ml microcentrifuge tube and vigorously vortexed to suspend the bacteria.