## 3 Results  
Microscopical analysis of curdlan producing _Agrobacteriumium_ cells reveals curdlan capsule. The strain ATCC31749 routinely produces curdlan when grown in liquid minimal salts with limiting nitrogen for longer than 3 days, whereas a knock-out (KO) strain of _crdA_ produced no detectable curdlan (<0.01 g/L). This 3-day delay in curdlan production fits with the knowledge that curdlan producing _agrobacteriumium_ cultures undergo a two stage development: an initial stage involving cell growth, and a later stage capable of producing curdlan provided that nitrogen is limiting and a suitable carbon source is present [27]. The 3-day growth period was also necessary for the appearance of flocs of variable size which were visible to the naked eye in cultures of the curdlan producing strain, but not in those of the _crdA_ mutant. From this observation, it is reasonable to suspect that curdlan production was associated with floc formation. From a 7-day culture, the cells were examined using phase contrast microscopy. Two prominent morphological forms of cells were observable in the curdlan producing cultures. These were a thicker rod form averaging 1.5 \(\times\) 3.0 \(\upmu\)m, located in flocs (cell aggregates) and a slender rod form, averaging 0.5 \(\times\) 2.0 \(\upmu\)m that was planktonic (Figure 2A). Only the slender rod form was observed in cultures of the _crdA_ mutant. When stained with the (1\(\rightarrow\)3)-\(\beta\)-glucan-specific aniline blue fluorochrome (ABF) [60; 61] only the larger form exhibited strong yellow/green fluorescence in a defined zone around the cell periphery (Figure 2B). These observations suggest that curdlan is deposited as a capsule and that production of the capsule is restricted to cells located in flocs. Since curdlan production commences primarily in the post-growth stage of fermentation, it is likely that floc formation is mostly through the recruitment of capsulated cells, rather than through growth of new cells within a floc. Electron microscopy of curdlan capsules. The physical form of native curdlan on the _agrobacteriumium_ cell surface was investigated by TEM of uranyl acetate stained preparations. At low magnification (9600\(\times\)), flocs from the curdlan producing culture were heavily stained, as might be expected for encapsulated cells. Occasional cells appeared to contain incomplete capsules (Figure 2C). In contrast, the aggregates of cells from the _crdS_ mutant culture were quite difficult to find (being very rare) and not heavily stained (Figure 2D). The fibrillary nature of the capsular material is evident in sections of embedded curdlan producing cells stained with lead citrate-uranyl acetate (Figure 2E) when compared with the naked surface of the _crdS_ mutant cells (Figure 2F). The use of electron microscopy (Figure 2C-F) confirmed the deposition of curdlan as a capsule--defined as a polysaccharide layer that lies outside the cell envelope. In contrast, bacteria incapable of producing curdlan had a 'clean' surface. These analyses confirmed curdlan as a discreet capsule found only on the surface of curdlan producing cells. Formation and organization of flocs. As noted above, the curdlan producing form was rarely planktonic. Rather, the curdlan capsule was usually confined to cell aggregates or flocs. This observation lead to the hypothesis that abundant flocs ought to appear in curdlan producing cultures, but not in cultures incapable of curdlan production. Floc formation was assessed using a particle counter capable of detecting flocs as particles. (The particle counter detects flocs as particles using a cut-off of 10 and 40 \(\upmu\)m, meaning that all flocs outside of this range, both larger and smaller, are excluded). When grown in liquid minimal salts for 3 d on a rotary shaker, ATCC31749 contained a proportion of particles larger than 20 \(\upmu\)m in diameter, while the _crdS_ disruptant contained mostly particles with a diameter less than 20 \(\upmu\)m (Figure S1).