SGER: Identification of Spores of Bacillus Anthracis and Other Species With Optical Sizing and Fluorescence

该奖项支持对芽孢杆菌属细菌的孢子和冻干细胞的一些物理性质的仔细研究。将确定孢子的大小和形状之间的关系，包括物种特性、湿度和制备方法的影响。计划中的工作建立在初步结果的基础上，这些结果表明，这些特性可能为快速准确地确定环境中孢子的存在及其物种特征提供基础。在这个新项目中，将使用自动显微镜系统和相关的图像分析软件来比较不同种类芽孢杆菌的孢子和冻干细胞，并研究孢子和细胞对相对湿度（R.H.）阶跃变化的响应。将测量滑石、高岭石、盐和其他非细菌颗粒的粉末进行比较。要检查的孢子，包括无毒的炭疽芽孢杆菌菌株，将从比以前检查的更多的芽孢杆菌种类中获得。为了比较r.h.变化的反应时间，将使用计算机控制的显微操作器将孢子和细胞转移到微室内的显微镜载玻片上。过去的核磁共振（NMR）技术研究表明，由于水偶极子靠近带电的蛋白质分子，孢子中的大部分水是分层的。显微方法可以在rh变化的几秒钟内检测到颗粒大小的变化；这一结果将为孢子内的水是如何以及在哪里构成提供新的见解。研究结果还可能有助于了解二氧化氯等气体净化剂在相对湿度低于70%至80%时的有限有效性。除了孢子大小的研究外，还将检查芽孢杆菌孢子自身荧光的强度和发射光谱。这些特性似乎也因物种而异，可以补充大小和对湿度变化的反应，作为孢子存在的诊断工具。孢子的自身荧光被认为受到孢子外孢子组成的影响，其中包括一种叫做BclA的交联胶原样蛋白质，这种蛋白质在营养细胞中不存在。

This award supports careful investigation of some physical properties of spores and lyophilized cells of bacteria in the genus bacillus. Relationships between the size and shape of spores, including the effects of species identity, humidity and method of preparation, will be determined. The planned work builds on preliminary results indicating that these properties may provide the basis for rapid and accurate determination of the presence of spores in the environment and of their species identity. In this new project, an automated microscope system and associated image analysis software will be used to compare spores and lyophilized cells of different species of bacillus, and to study response of spores and cells to step-function changes in relative humidity (R.H.). Powders of talc, kaolinite, salt, and other nonbacterial particles will be measured for comparison. Spores to be examined, including those of an avirulent strain of B.anthracis, will be obtained from a larger number of bacillus species than examined previously. To compare the response time to changes in R.H., a computer-controlled micromanipulator will be used to transfer spores and cells to a microscope slide inside a micro-chamber. Past work with nuclear magnetic resonance (NMR) techniques has shown that much of the water in spores is layered, due to orientation of water dipoles in proximity to charged protein molecules. The microscopic method can detect particle size changes within a few seconds of a change in R.H.; the results should provide new insight into how and where the water inside spores is structured. The results may also provide insight into the restricted effectiveness of gas decontaminants such as chlorine dioxide at R.H. below 70 to 80%. In addition to the investigation of spore size, the intensity and emission spectrum of autofluorescence by bacillus spores will be examined. These properties also appear to vary by species and may complement sizing and response to humidity changes as a diagnostic tool for the presence of spores. The autofluorescence of spores is thought to be influenced by the makeup of the spore exosporium, which includes a cross-linked, collagen-like protein called BclA absent in vegetative cells.