Current concepts of plaque development and microbial ecology are based largely on indirect investigations using cultivation, in vitro adherence, metabolic (nutritional), ultrastructural and animal approaches. Only limited information on plaque ecology is available from direct observations of in situ grown plaque specimens. A critical gap in our information is that of the spatial relationships among specifically identified plaque microbes in early, developing and mature plaque as determined directly on plaque specimens. The proposed research program is designed to develop the means to provide this information. The program is a collaborative effort involving biological and physical scientists at the University of Minnesota and 3M Company. Monoclonal antibodies specific at the genus, species and, in some cases, serotype levels will be prepared against oral Streptococcus, Actinomyces, Veillonella, Lactobacillus, Neisseria, Hemophilus, Bacteroides, Fusobacterium, Capnocytophaga, Actinobacillus and Treponena. Antibodies will be distinguised by (i) tagging with transition elements via chelate conjugates and (ii) labeling with sized colloidal gold, silver and metal oxides. In situ grown plaque specimens will be recovered and reacted directly (initial or early plaque) or sectioned (developing and mature plaque) and reacted with a panel of differentially tagged monoclonal antibodies specific for the particular microbes of interest. Antibody stained specimens will then be examined in the scanning electron microscope and subjected to electron microprobe X-ray emission analysis to map locations of the various microbes through the metal-antibody conjugates. Computerized data analysis will be used to false-color image the specimen and to determine microbe- microbe associations, nearest neighbors, and microcolony sizes and frequencies. These methods will provide direct qualitative and quantitative analysis of plaque microbial composition and spatial relationships for the first time and will yield a data base to directly test several current notions of plaque ecology derived from indirect approaches. The overall results of this investigation will be applicable to studies of other surfaces colonized by complex mixtures of bacteria and will open a new era of microecological studies of the human oral flora.
