[2] Molecular tools for study of biofilm physiology

This chapter describes methods for the handling and analysis of microbial behavior of organisms in biofilm communities at both microscopic and macroscopic levels. Only methods and reporter systems that can be applied without disturbing the spatial organization of the organisms in the biofilm are presented. The in situ methods described in this chapter can be used for more than just identifying or tracing cells or genes in biofilms. By combining promoters that respond to specific environmental signals with appropriate marker genes, it may be possible to tag specific organisms and use these as monitor systems to estimate local chemical composition directly in the biofilms. Changes in environmental conditions will also have significant effects on the physiological state of the organisms. Such shifting conditions may result in several responses, such as altered growth rates, stress response, starvation, or even cell death. Most of these responses can be visualized directly using specific promoter–reporter fusions. The ribosome number is a reliable indicator of growth rate in bacteria growing in balanced growth and has been used as a standard for growth rates in biofilm-embedded bacteria as well.