Concurrent Quantification of Cellular and Extracellular Components of Biofilms

The overall goal of this procedure is to quantify and compare concurrent three dimensional distributions of three cellular and extracellular biofilm components after treatment with antibacterial agents. This is accomplished by first fabricating and then polishing resin composite specimens. In the second step, the biofilms of interest are grown on the specimens and then treated with antibacterial agents.

Next, the biofilms are stained with fluorochromes for extracellular polymeric substances, nucleic acids and proteins, and then imaged by confocal laser scanning microscopy. In the final step, three dimensional images of the overlaid fluorochromes are reconstructed to facilitate the concurrent visualization of the biofilm components and structural parameters are calculated from the biofilm images. Ultimately, statistical analysis of the biofilm structural parameters such as bio volume and mean biofilm thickness can be performed to compare differences between the treatment and control groups.

The main advantage of this procedure over existing methods is that it uses distinct but interconnected techniques to characterize the distribution of multiple components within the structure of biofilms grown under specific conditions, demonstrating the procedure will be Dr.Fernando Flores, a postdoctoral research fellow, and Ms.Kristen Smart, a research assistant from my laboratory To create the specimens begin by placing one increment of 0.4 or TPH three dental resin composite into custom made stainless steel molds. Next, polymerize the first increment with an LED light curing unit for 40 seconds. After repeating the procedure for the second increment, use a semi-automated grinder polisher to polish the cured specimens to an acceptable final surface finish.

Finally, sterilize the specimens to grow the biofilm. First, inoculate a single colony of S mutans in four milliliters of overnight culture medium, and then incubate the culture at 37 degrees Celsius for 16 to 18 hours to achieve an optical density of at least 0.9. Next, dilute the culture at a one to 100 ratio in 10 milliliters of biofilm growth.

Medium aseptically transfer the sterile resin composite specimens to sterile 12 well plates, and then add 2.5 milliliters of the diluted culture to each of the wells. For mouthwash treatment and control groups, add 2.5 milliliters of sterile un inoculated biofilm growth, medium to the sterility control wells, and then grow the biofilms at 37 degrees Celsius under micro paraphilic conditions on a shaker at 100 RPM. After 24 hours, aseptically aspirate the medium from all the wells and wash each well twice with 2.5 milliliters of sterile PBS aspirating the saline after each wash, then replenish each well with 2.5 milliliters of fresh biofilm growth media and incubate the plate for an additional 24 hours as just demonstrated for a total biofilm growth Duration of 48 hours after the biofilm growth is complete, aspirate the growth media and then dispense 2.5 milliliters of mouthwash into each treatment group well and 2.5 milliliters of sterile ultrapure water into the control group.

Well agitate the plates on an orbital shaker at 150 RPM, and then after 60 seconds, aspirate both the mouthwash and ultrapure water from the wells to stain the exo polysaccharide component of the biofilms. Incubate each biofilm with 50 microliters of LOR conjugate for 30 minutes at room temperature protected from light. Then wash the specimens two times with 2.5 milliliters of TC buffer aspirating after each wash and stain the nucleic acids in each biofilm with a 50 microliter drop of cyto nine for 30 minutes at room temperature protected from light.

After washing the specimens twice, stain the protein components in each biofilm with 50 microliters of Cipro red for 30 minutes at room temperature, protected from light. After washing the specimens three times, transfer them to individual wells of a six well plate containing six milliliters of sterile ultrapure water per well to facilitate their visualization by confocal microscopy to acquire images of each of the component stains within the biofilms of the treatment and control groups. Set the lasers of a confocal laser scanning microscope to the following settings for the detection of exo polysaccharides by Exor conjugate stain, use a 633 nanometer laser and a 659 to 749 nanometer emission band for the detection of nucleic acids by cyto nine stain.

Use a 488 nanometer laser and a 495 to 500 nanometer emission band, and for the detection of proteins by Cipro red stain, use a 543 nanometer laser and a 615 to 651 nanometer emission band. Then using a 63 x dipping lens with a 0.9 numerical aperture and a working distance of 2.2 millimeters. Collect confocal laser scanning microscopy images at 400 hertz using sequential scanning in the between stacks mode to optimize the capture of images of multiple stains within the same biofilm.

After analyzing the confocal laser scanning microscopy images, use the 3D renderer menu option in velocity image analysis software to create a reconstructed 3D image of the overlaid component stains within each biofilm. Next, rotate the 3D image to obtain an optimal view of the biofilm. Then capture a snapshot of the biofilm reconstruction and export the snapshot in a suitable image file format.

Finally, perform a visual analysis of the concurrent distribution of the exo polysaccharide nucleic acid and protein components within the biofilms. In the reconstructed images, calculate biofilm structural parameters such as bio volume and mean biofilm thickness using ISA 3D software in this table, the mean and standard deviation values of biofilm structural parameters calculated using statistical analysis software are displayed. The mean and standard deviation values for the structural parameters of the biofilms treated with mouthwash that differed significantly from those of the biofilms in the control group are highlighted in red mixed models.

Statistical analyses demonstrated that the Biotene PBF mouthwash produced biofilm structures that differed significantly from the control group on both resin composites, whereas Listerine total care mouthwash did not produce significant differences on either resin composite clearly indicating differences in the cellular and extracellular biofilm components remaining after the two mouthwash treatments. The concurrent distribution of exo polysaccharides nucleic acids and proteins within the biofilms can be visualized via 3D reconstructions generated using velocity software. In this figure, a representative reconstruction of control group biofilms grown on 0.4 is shown the blue color was assigned to exo polysaccharide.

Within S mutans biofilms, the green color was assigned to nucleic acids and the red color to proteins. The intervening space may be occupied by water or other biofilm components that have not been fluorescently labeled. In this figure, a representative reconstruction of control group biofilms grown on TPH three resin composites with the colors representing the same components as the previous figure is shown.

This procedure has brought application in a variety of disciplines such as the medical, dental, geological, and marine sciences. Since many substrates and biofilms can be substituted for the ones used here.