Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

The overall goal of this procedure is to harvest and quantify fluorescently tractable bacteria from an intermediary host for subsequent infection of mammalian host cells. Here Legionella mala expressing GFP are used to infect the natural host protozoan acan amoeba, castellani monolayers. The el mala are then harvested by selective lysis and the concentration of bacteria is then calculated using a formula based on fluorescence emission dilution of protozoan.

Primed bacteria are then used to infect mammalian cell monolayers. Finally, the number of infectious bacteria in the mammalian cells are determined using a combination of fluorescence microscopy, flow cytometry and plating lysates to recover colony forming units. The resulting data demonstrate a pathogenic advantage gained for the bacteria that were cultivated in protozoan cells in the priming stage when compared to the same strain that was cultivated in artificial medium.