Growth temperature, media composition impact Salmonella biofilms.

The complex structure of pathogenic biofilms extensively depends on growth conditions. Most descriptions of biofilm structure are largely qualitative, due to the lack of quantitative analysis tools.

With this in mind, USDA-ARS scientists undertook a quantitative comparison of Salmonella biofilms using digital image analysis (DIA). DIA was applied to images generated from confocal scanning laser microscopy (CSLM). The CSLM-DIA technique is a simple and flexible means of quantifying biofilm structure. CSLM and DIA made it possible to discretely count the microcolonies within each layer. The techniques quantified patterns of biofilm development, offering data on distinct combinations of conditions and trends related to changes in media and temperature.

S. Michigan was grown in tryptic soy broth (TSB) at either full strength or a 5% solution (TSB.05). Cultures in either medium were grown for 48 hours at 28 C or 37 C. Then the cultures were statically incubated in covered glass-bottomed petri dishes, which are appropriate for CSLM.

The researchers stained the biofilms for CSLM using SYTO 9 and propidium iodide. Images were taken in 1-micron optical sections, beginning from the glass-biofilm interface and moving upward. The images were digitally analyzed to remove noise; to distinguish individual cell clusters within and between image layers; and to automatically count cell clusters.

The investigators combined the resulting data for each layer to generate a cell cluster distribution profile for each set of growth conditions, Each profile was modeled using a four-parameter Weibull equation. The equation was able to establish a variety of quantitative values describing the biofilm.

All growth conditions gave a region of maximum cell cluster density between 3.93 and 4.78 microns above the substrate. However, the maximum cell cluster densities within this region for TSB/28 C (514.5 clusters) and TSB/37 C (327.4) were more comparable to each other than to those for TSB.05/28 C (1910.8) or TSB.05/37 C (996.5). TSB.05/28 C biofilms were also more compact than the other three growth conditions.

Further information. Brendan Niemira, USDA-ARS Eastern Regional Research Center, Food Safety Intervention Technologies Research, 600 E. Mermaid Lane, Wyndmoor, PA 19038; phone: [phone]; fax: [phone]; email: [e-mail].