For all subsequent experiments, we labeled the holdfasts with 100 μg/ml lectin for 15 min. Atomic force microscopy (AFM) In order to obtain a clean surface as a substrate for AFM imaging, glass coverslips were soaked in a solution of 6 % (w/v) Nochromix (GODAX Laboratories, Inc.) in concentrated H2SO4 for 1 hour and then rinsed thoroughly with deionized water. A drop of culture containing synchronized swarmer cells was placed on a clean coverslip for 5 min. The unattached cells were rinsed off with oxygenated fresh PYE and the attached cells were then grown at 30 °C over various time intervals to allow Selleckchem LY2606368 for holdfast growth. The coverslip was then
blow-dried gently with compressed N2 gas so that the attached cells fell over to the side, getting stuck and dried onto the glass surface. The dried cells and their holdfasts, also dried on the glass surface, were I-BET151 nmr imaged using ZD1839 solubility dmso a Nanoscope IIIa Dimension 3100 (Digital Instruments, Santa Barbara, CA) atomic force microscope using contact mode in air. Results Distribution
of holdfast fluorescence intensity at various ages Fluorescein-WGA labeling confirmed the previous report that young swarmer cells start secreting holdfast within minutes following their attachment [12]. Figure 1 shows phase contrast and fluorescence images of cells at various ages. Holdfasts were clearly visible for attached cells as young as 7.5 min old. The intensity increased with age but the difference between holdfasts of 27.5 and 37.5 min old cells became insignificant. Analysis of the fluorescence intensity of labeled holdfast showed a wide AZD9291 supplier variation in intensity at each time point (Figure 2). This result suggests that the holdfasts of different cells grow at different rates, and that the final sizes of the holdfast vary significantly from cell to cell. Interestingly, the intensities
of the holdfasts fell into two groups, marked as I and II in Figure 2. Examples of each group of cells at age of 27.5 min are shown in the inset of Figure 2c. Holdfasts of group I have very weak intensities, less than one tenth of those in group II on average. Approximately 10% of holdfasts fell into group I. This intriguing result was reproducible among several experiments. Since the cells from each experiment came from clonal populations, it is unclear what causes the bimodal distribution in holdfast fluorescence intensity. Figure 1 Holdfast secretion level at different ages, detected by labeling with 100 μg/ml fluorescein-WGA-lectin for 15 min on ice, (a) 7.5 ± 2.5 min, (b) 17.5 ± 2.5 min, (c) 27.5 ± 2.5 min, and (d) 37.5 ± 2.5 min. Top panel shows phase contrast images, middle panel fluorescence images, and bottom panel the combined phase and fluorescence images.