Purpose: To study biofilms of L. monocytogenes using cultivation and microscopic techniques.
Methods: L. monocytogenes was inoculated (7 log CFU) in 24-wells polystyrene microplate containing sterilized stainless steel coupons (AISI 304, 1.57 cm2) and BHI broth. It was incubated (25 °C, 72 h, 60 rpm) and adhered cells were enumerated every 24 h, by removal and washing of coupons, sonication, surface plating on BHI agar (37 °C/24 h) and counting. Biofilm formation was also evaluated by fluorescence microscopy using acridine orange to stain total DNA. Additionally, L. monocytogenes was cultivated (25 °C/192 h) in BHI broth in eight-chamber covered glass slide. At times 24, 96 and 192 h, biofilms were stained with LIVE/DEAD BacLightTM kit and observed with confocal laser microscope.
Results: Population of L. monocytogenes adhered to coupons after 72 h/25 °C was ca. 7 log CFU/cm2. Under fluorescence microscopy, after 24 h, isolated cells and microcolonies of L. monocytogenes were observed adhered to the coupons, in the presence of extracellular matrix. Larger cell aggregates were seen at 48 h, while after 72 h there were channels and also and signs of dispersion (holes). Images of confocal microscopy of glass slides taken at 24 h, revealed a monolayer of viable bacterial cells and some hollows. At 96 h, the surface was patchy covered, but non-viable cells were abundant and voids were also observed. Several layers of viable cells covered the slide surface after 192 h, and holes of different sizes were observed.
Significance: L. monocytogenes form biofilms either in glass or stainless steel, and microscopic analysis revealed important aspects of biofilm architecture that may contribute to understand the persistence of the organism in nature.