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Acanthamoeba polyphaga feeding on Salmonella typhimurium in a simple model biofilm were observed by light microscopy and a detailed record of interactions kept by digital image capture and image analysis. A strain of S. typhimurium SL1344 carrying a fis:gfp reporter construct (pPDT105) was used to assess intracellular growth in A. polyphaga on non-nutrient agar (NNA) plates. Invasion of the contractile vacuole (CV) was observed at a frequency of 1:100–1000 acanthamoebae at 35°C. The salmonellae contained in CVs illustrated significant up-regulation of fis relative to extracellular bacteria, indicating that they were in the early stages of logarithmic growth, and reached numbers of 100–200 cells per vacuole after 4 days. This is the first report of this mode of intracellular growth. Up-regulation of fis was also observed in a proportion of S. typhimurium cells contained within food vacuoles. Filamentation of S. typhimurium and E. coli cells was frequently observed in coculture with A. polyphaga on NNA plates, with bacterial cells reaching lengths of up to 500 µm after 10 days’ incubation at 35°C. A. polyphaga was also seen to mediate bacterial translocation over the agar surface; egested salmonellae subsequently formed microcolonies along amoebal tracks. This illustrated intracellular survival of a fraction of the S. typhimurium population. These phenomena suggest that protozoa such as A. polyhaga may play an important role in the ecology of S. typhimurium in soil and aquatic environments. [ABSTRACT FROM AUTHOR]