EFFECT OF ABRUPT TEMPERATURE SHIFTS ON THE KINETIC BEHAVIOR OF VERY SMALL POPULATIONS (2-10 CELLS) OF Salmonella enterica ser. Typhimurium

摘要

A time-lapse microscopy method was applied to study the effect of abrupt temperature shifts on the kinetic behavior of very small populations of Salmonella enterica ser. Typhimurium. A z-motorized microscope (Olympus BX61) equipped with a x100 objective (Olympus) and a high-resolution device camera (Olympus DP71) was used for monitoring growth of single cells. The quality of the images was improved by developing an auto focus procedure with an Extended Depth of Focus (EDF) system using the ScopePro module of ImageProPlus software. Each final image was a result of 10-20 images captured in different z-axis planes. The EDF system allowed the combination of the z-stack images of multi-level focal planes into a single in-focus image. The above procedure provided high quality images of bacterial colonies in which cells can be counted either manually or by the image analysis software. The output of each experiment is a video showing the changes in the cell numbers with time for each colony originated from a single cell. In this study the colonial growth of 81 Salmonella enterica ser. Typhimurium single cells on tryptone soy agar was monitored. The temperature was initially set at 25°C and decreased to 15°C after a few divisions (1-5) of the cells. Images were taken at 5-min intervals for twenty hours after inoculation. After counting, the numbers of cells vs time for each colony, after the temperature shift, were fitted to the primary model of Baranyi and Roberts for the estimation of the "additional lag" caused by the shift and the growth rate. The results showed a significant variability in the kinetic behavior after the temperature downshift. The "additional lag" ranged from 0 to 1.26 h (mean=0.291 h) with a coefficient of variation (CV) of 116.7%. The specific growth rate (mean=0.326 h~(-1)) after the shift showed less but still significant variability with CV=36.1%. The above distributions of the kinetic parameters were used to simulate the effect of abrupt temperature shifts at various population levels. The Monte Carlo simulations showed that the variability is masked at high population levels. The results of the present study provide useful information for understanding the colonial growth of single cells under changing temperature environment and can be used for the improvement of risk assessment studies.