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In situ Biofilm Formation by Multi-species Oral Bacteria Under Flowing and Anaerobic Conditions

Department of Periodontics, College of Dentistry, University of Illinois at Chicago, 801 S. Paulina Street (MC 859), Chicago, IL 60612, USA;

View larger version (105K): [in a new window]   Figure 1. Photomicrographs illustrating the stages of biofilm development and the planktonic and biofilm interspecies aggregates. (a) Interspecies planktonic aggregate at 1 hr, showing a single F. nucleatum (Fn) cell in the center with groups of A. actinomycetemcomitans (Aa) and S. gordonii (Sg) cells on either side. (b) Representative light microscopy image of biofilm development at 1 hr and (c) at 3 hrs. Darker areas represent greater biofilm accumulation; (m) indicates micro-colonies comprised of the aggregates; (i) indicates the interstitial spacing (void fraction) between the micro-colonies of the biofilm; (fe) indicates the free-end structure which oscillated in the direction of flow and also trapped passing aggregates. (d) Biofilm aggregates (ba) at 3 hrs with the base film cells (bf). (e) Biofilm aggregate with the structures that were observed bridging adjacent aggregates together, such as the group of distinctly organized A. actinomycetemcomitans cells (Aa) and individual F. nucleatum cells (Fn). By varying the focal plane, we could visualize different layers of the aggregate. However, it was not always possible to focus the entire cluster, due to thickness variability and continuous flow of the culture medium. The unfocused area represents cells protruding into and oscillating in the culture medium. The dark deposits are medium artifacts. (f) Interspecies planktonic aggregate at 24 hrs showing all 3 bacteria surrounded by an amorphous cohesive matrix (m). The aggregates were stained with Live/Dead® BacLightTM; however, no inferences in the viability of the cells were made for these samples, since organization of the aggregates was better visualized after the sample fluorescence had faded.

View larger version (20K): [in a new window]   Figure 2. Surface area coverage by aggregated and individual cells of a multi-species biofilm. SDS (0.1%) was added at the time indicated by the arrow. For each time point in each replicate experiment, 30 images were taken, and the mean area covered by aggregates and the mean area covered by individual cells were calculated. Error bars show standard deviations from 3 independent replicates and illustrate the inherent heterogeneous nature of biofilm formation.

View larger version (28K): [in a new window]   Figure 3. Representative surface plot profiles depicting biofilm accumulation over time and the effect of SDS. The profiles were taken from reference lines applied to the captured image. The vertical axis represents the degree of biofilm accumulation (based on pixel intensity). The horizontal axis represents distance along the horizontal profile. The width of the peaks indicates the width of the microcolonies, and the distance between the edges of the peaks indicates the distance between adjacent microcolonies (µm). The arrow indicates the distance between the micro-colonies (mc). Biofilm at (a) 3 hrs, (b) 24 hrs, (c) 1 hr after SDS application, and (d) 5 hrs after SDS application.