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

University Of California-Berkeley, Berkeley CA

Investigators

Abstract

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.