Understanding the physiology of bacteria grown in vivo in various hosts has been a difficult task. By using the adhesive properties of the microorganism, it is possible to isolate certain bacteria from their natural environment for further investigations. Pure cultures so obtained do not require growth on an artificial laboratory medium. The chapter describes the method for adhesin-dependent isolation and characterization of bacteria from their natural environment. It is performed by using a streptomycin-treated mouse as a host for a human Escherichia coli strain, producing type 1 fimbriae in vivo. Type 1 fimbriae bind specifically to D-mannose moieties. In this context, experiments were performed in which sepharose beads coupled with D-mannose were used for isolating bacterial cells from a fecal suspension, and/or from cecal mucus and cecal contents. Then, the isolated intact bacterial cells can be characterized by techniques such as electron microscopy, flow cytometry, and in situ hybridization. By electron microscopy, cell shape and surface components can be observed. A flow cytometer can be used for determining rapidly and with high precision the cell size of individual bacterial cells and the cell-size distribution in the isolated population. DNA content can be measured and the number of genomes per cell calculated. By in situ hybridization with labeled ribosomal RNA probes, the rRNA can be measured, which will reflect the metabolic activity of the bacterial cell in the host.
|Titel||Methods in enzymology : Adhesion of Microbial Pathogens|
|Redaktører||Ron J. Doyle, Itzhak Ofek|
|Status||Udgivet - 1995|
|Navn||Methods in Enzymology|