FLOW CYTOMETRIC ANALYSIS OF ORAL ACTINOBACILLUS

研究表明，物种特异性免疫荧光抗体标记 细菌为复杂的厌氧培养提供了一种替代方法 定量鉴定特定牙周病原菌。 细菌荧光抗体荧光标记分析 显微镜很费力，而且会产生关于荧光的主观数据。 每个样本中只有几百个细胞的强度。流式细胞仪 当细胞流过激光时测量单个细胞的荧光 敏感的光学和电子学，每个电池有数千个电池 第二。最近的报告表明，流式细胞术提供了另一种选择 测定大量细菌的细菌细胞荧光的方法。 利用激光激发荧光和灵敏光电倍增管 用流式细胞仪测量荧光的试管允许使用更低的 导致非特异性降低的标记抗体效价 贴标签。这项研究将使用标记所有测试的单抗 伴放线放线杆菌(AA)菌株的研究 优化流式细胞术的免疫荧光标记方法 细菌细胞混合物中AA细胞的检测。间接和直接 免疫荧光标记技术将使用FITC进行比较 结合抗体。流式细胞术的敏感性和特异性 优化的流式细胞术检测再生障碍性贫血细胞分析 内部混合物将与使用相同混合物的分析进行比较 荧光显微镜。控制流动中的变量的效果 细胞学分析技术或在细菌细胞上测量AA 将定义混合物中的单元格。细菌细胞变量为 所研究的包括细胞保存技术、细胞浓度或大小、 和细胞生长周期。使用核酸染色和添加一种 将对内部珠子标准进行测试，以确定其中任何一种是否改善了分析。 最后，将使用流式细胞术分析来评估再生障碍性贫血的改变。 体外抗生素产生的细胞形态、DNA或蛋白质含量 曝光。这些单元格的改变将与 菌落形成单位。这项研究定义的技术将提供 测量更大人群中AA感染的新调查工具。

Studies show that species-specific immunofluorescent antibody labeling of bacteria provides an alternative method to complex anaerobic culturing for the quantitative identification specific periodontopathogenic bacteria. Analysis of bacterial fluorescent antibody labeling with fluorescent microscopy is laborious and yields subjective data on fluorescence intensity for only several hundred cells in each sample. Flow cytometers measure individual cell fluorescence as cells flow past a laser with sensitive optics and electronics at a rate of thousands of cells per second. Recent reports suggest that flow cytometry provides an alternative way to measure bacterial cell fluorescence of large numbers of bacteria. The use of lasers to excite fluorescence and sensitive photomultiplier tubes to measure fluorescence by flow cytometers permits use of lower titers of labeling antibody which results in a reduction of non-specific labeling. This study will use monoclonal antibodies that label all tested strains of Actinobacillus actinomycetemcomitans (Aa) to define an immunofluorescent labeling protocol which optimizes flow cytometric detection of Aa cells within bacterial cell mixtures. Indirect and direct immunofluorescent labeling techniques will be compared using FITC conjugated antibodies. The sensitivity and specificity of flow cytometric analyses using optimized flow cytometric technique for detecting Aa cells within mixtures will be compared to analysis of the same mixtures using fluorescence microscopy. Effects that controlled variables in flow cytometric analysis techniques or in bacteria cells have on measuring Aa cells within mixtures will be defined. Bacteria cell variables to be studied include cell preservation techniques, cell concentration or size, and cell growth cycle. Use of nucleic acid staining and addition of an internal bead standard will be tested to see if either improves analyses. Finally, flow cytometric analyses will be used to assess alterations in Aa cell morphology, DNA or protein content produced by in-vitro antibiotic exposure. These cell alterations will be compared to changes in numbers of colony forming units. The techniques defined by this study will provide new investigative tools to measure Aa infection in larger populations.