5 ± 2.5 min, (b) 17.5 ± 2.5 min, (c) 27.5 ± 2.5 min, and (d) 37.5 ± 2.5 min. Note that the intensities fall into two groups, indicated as I and II. Approximately 10% of holdfasts are in group I, whose intensities remain very low. Inset in

(c) is a combined phase and fluorescence image of 27.5 ± 2.5 min old cells, showing a few examples of the two groups of holdfasts with different fluorescence intensities. The fluorescence intensities of two holdfasts indicated by arrows are much weaker than the others. These two cells are identified as group I cells in co-existence with several group II cells. We found that the average fluorescence intensity of holdfasts increased Torin 2 solubility dmso with cell age during the first 30 min but then saturated at a constant level (Figure 3). Since the labeling step was done following different times of holdfast growth, our data suggest either that the attached cells stopped secreting more holdfast after about 30 min, or that the holdfast continued to thicken after 30 min, but if the fluorescein-WGA only bound to the surface of the dense holdfast material the fluorescence intensity would no longer increase noticeably as the holdfast layer continued to thicken. We turned to AFM analysis below in order to distinguish between these possibilities. Figure 3 Growth of holdfast attached to a solid surface measured with fluorescence microscopy. This figure shows the fluorescence intensity of holdfast buy Pifithrin-�� as a function

of cell age. Each data point is the average over two or three samples. 3-mercaptopyruvate sulfurtransferase Error bars are the standard error. The dotted lines are drawn as a guide to the eye. The holdfast spreads to a thin plate at the attachment site Previous studies have used electron microscopy or FITC-WGA labeling to measure holdfasts [13, 14]. While these methods provided useful information about holdfast size, AFM can be used to measure holdfast size in three-dimensions [9, 16]. In order to directly analyze holdfast synthesis by AFM, swarmer cells were synchronized by the plate release method. They were allowed to quickly attach to a glass microscope coverslip. After

the unattached cells were washed away, attached cells were allowed to grow for different amounts of time before drying and imaging by AFM. Figure 4 shows typical AFM images of cells at different ages. The cell body laid down on the surface during the drying procedure and typically only a part of the holdfast was approachable by the AFM tip. In very young cells, the cell body occluded the holdfast. For instance, AFM could not image the holdfast of 7.5 min old cells. The holdfasts of 17.5 and 27.5 min old cells were larger and partially detectable. For cells over 37.5 min old, a thin stalk appeared, so most of the holdfast area became detectable at the tip of the stalk. The edge of the holdfast was clearly discernible in Figure 4e, and was roughly circular. The holdfast became gradually thinner towards the edge, taking the shape of a suction cup.