The Use of Flow Cytometry to Study Sporeforming Bacteria
from: Flow Cytometry in Microbiology: Technology and Applications (Edited by: Martin G. Wilkinson). Caister Academic Press, U.K. (2015) Pages: 109-118.
Sporeformers comprise a diverse group of important bacteria and possess two unique traits: the ability to form dormant and resistant spores and their ability to restart metabolism and germinate. Flow cytometry (FCM) offers many possibilities for the study of sporeformers, bringing advantages in terms of speed, novelty and the ability to measure heterogeneity within a population. In this chapter, five categories of FCM studies involving sporeformers (mainly involving species within the genera, Bacillus and Clostridium) have been identified: those based on autofluorescence or light scatter; those involving antibodies or fluorescent in situ hybridization probes; those using reporter proteins in the context of genetic transformation; those involving permeability dyes; those utilizing physiological dyes. In the past, instrumentation was expensive, difficult to operate and the range of fluorescent dyes and antibodies available to the microbiologist was limited. In the last half a decade, however, the number of low-cost, easy-to-run and powerful (≥ two lasers, ≥ six detectors) digital instruments has mushroomed. In a similar vein, the variety of monoclonal antibodies and dyes available to the microbiologist is rapidly increasing. Both developments allow the researcher interested in sporeformers to design more information-rich and daring experiments than ever before. Experiments combining the use of fluorescent reporter proteins, physiological dyes, a permeability marker and a monoclonal antibody are now possible. Using antibodies or fluorescent dyes specific to particular structural components of endospores such as the exosporium or protein coat, FCM may become a very useful tool in the real-time analysis of sporulation or resistance mechanisms. FISH probes could be used for environmental studies. The growing variety of reporter proteins such as GFP or mCherry allows the possibility of simultaneously studying the expression of a number of genes, an experimental approach which may help unravel the sequence of events leading to sporulation. The use of multiplex bead arrays could prove a powerful tool in characterising a species' repertoire of secreted proteins or toxins. Of prime importance in implementing new methods for the study of sporeformers is their comparison with existing gold standard techniques. Even a research tool as powerful as FCM must be shown to yield data as robust and reliable as conventional methods read more ...