R., Murillo I., Payot A. susceptibility tests seem the most straightforward. INTRODUCTION Our team developed previously an atomic force microscopy (AFM)Cbased assay to assess the effects of chemicals on the viability of bacteria (motion of Gestodene bacteria (motion of attached bacteria (motions were monitored by recording 12-s-long movies (1000 frames) taken at a magnification of 400 (Fig. 1). By periodically recording these movies, temporal behavior of the cells was characterized as a function of different chemical and physical stimuli (Fig. 1, A and B). To track the cellular motions of single cells, we used a cross-correlation image registration algorithm (displacements of individual cells (typically 20 cells) are calculated using the cross-correlation algorithm. (D) For each cell, the displacement per frame is calculated, and this distribution is represented by a combined violin and box plot. (E) The displacement per frame for all cells for a condition/sampling point is represented as a combined violin plot and box plot. (F) The mean of the total displacements of 20 cells is calculated for each condition/sampling point and represented in a box plot. RESULTS First, we compared single-cell nanomotions of cells that were grown in the presence of nutrients [by growing them in yeast extract, peptone, and dextrose (YPD) growth medium] to cells that were in a nutrient-free physiological phosphate-buffered saline (PBS) buffer. Single-cell displacements were recorded every hour during 4 hours (Fig. 2, A and B, and fig. S1, A and B). Actively growing single cells showed a large distribution of displacements. The distribution of the displacements is not symmetric, and this reflects the nonrandom behavior of the cells (as could also be observed from the displacements graphs in Fig. 3), i.e., cells can make jumps from time to time. This motion behavior is also reflected in the shape of the violin plots that represents the displacements distribution. In this set, a few cells (one to three) display a very small displacement distribution and can be classified as inactive. In contrast, significantly more inactive cells were present in the absence of nutrients, especially after 3 to 4 4 hours of incubation (Fig. 2B and fig. S1B). This behavior is also reflected in the grouped displacements violin plots (Fig. 2, A and B, bottom) and the total displacements boxplots (fig. S1, C and D). In these last plots, the adaptation of the cells to the new growth condition can clearly be observed, i.e., a DKFZp781B0869 significant increase of the total displacement after 1 hour, in contrast with the measurements obtained in PBS. Open in a separate window Fig. 2 Effect of the nutritional environment and the temperature on the cellular nanomotions of yeast cells.(A) The distribution of the displacements per frame of 20 BY4742 cells growing in YPD growth medium after 2 hours (top). Time evolution of the merged distributions of the displacements for 20 cells (bottom). (B) The distribution of the displacements per frame of 20 BY4742 cells present in PBS after 2 hours (top). Time evolution of the merged distributions of the displacements for 20 cells (bottom). (C) Time evolution of grouped displacements in growth medium. Effect of the temperature on the displacement distribution (20 cells) for BY4742 and DSY294. (D) Effect of the heat on the total displacement of BY4742 and DSY294. Wilcoxon test: ****< 0.0001; ***< 0.001; **< 0.01; ns, not significant. Open in a separate windows Fig. 3 Monitoring of life-death transition Gestodene by observing cellular nanomotions of dying candida cells in the presence of ethanol or antifungal.Effect of (A) ethanol (70%, v/v) and (B) caspofungin (100 g/ml) within the displacements of DSY294, DSY562, DSY4606, and BY4742 cells during 12 s (1000 frames, 83 fps). Blue dots represent the position of the cell without ethanol or caspofungin treatment; the orange dots symbolize the positions after the treatment. The cellular nanomotions were also compared to Gestodene the motions of silica beads recorded in the same conditions (fig. S2, A to D). The distributions of the displacements were symmetric. The magnitudes of the motions were much reduced compared to living cells and were of the same order of lifeless cells (fig. S2E). To assess the effect of the heat within the nanomotion pattern of candida, we monitored the cellular Gestodene oscillations at different temps in the range of 13 to 35C (Fig. 2, C and D, and fig. S3). Each strain is characterized by a different distribution of grouped displacement distributions. For both candida strains, a maximal activity was recognized.