Chapter 52 - Listeria monocytogenes: Techniques to Analyze Bacterial Infection in vitro

Abstract

The chapter describes some basic methods in cellular microbiology that have been used routinely to examine the infection process of Listeria monocytogenes in vitro that can be adapted to research the pathogenesis of other intracellular bacteria. Make a 10-fold dilution of the initial bacterial solution by adding 100 μl of this preparation to 900 μl of sterile distilled water contained in an Eppendorf tube. Make another 10-fold dilution by adding 100 μl of the first 10-fold dilution to 900 μl of sterile distilled water contained in a different Eppendorf tube. Repeat this operation twice. Wash the cells twice with l ml of PBS and add l ml of sterile distilled water to each well. Disrupt the cells by pipetting up and down water several times. Make a 10-fold dilution by adding 100 μl of the disrupted cell solution contained in each well to a different Eppendorf containing 900 μl of steriled distilled water. Visualization of bacteria allows evaluation of several parameters, such as the proportion of cells in the inoculated monolayer to which bacteria attach, as well as the proportion of adherent versus invasive bacteria.