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^® BacLight^TM; 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.