Panels D, E, and F show ARS-1 strain. Panels G, H, I show ALG-00-530 strain. Panels J, K, and L display ALG-02-36 strain. Panels A, D, G, and J show cells at day 1 (scale bar 10 μm); panels B, E, H, and K display 7 days starved cells (scale bar 5 μm); panels C, F, I, and L show 14 day starved cells (scale bar 1 μm). Figure 2 shows how the cell morphology shifted from long and thin rods to coiled forms at 14 days. Data on ATCC 23643 strain could not be analyzed due to the matrix that covered the cells making morphotype ascription unfeasible. At day 1, there were not significant differences
between mean percent of bacillus forms eFT508 order observed in ARS-1, ALG-00-530, and ALG-02-36 strains. At day 7, the percent of bacillus forms in ALG-00-530
was significantly lower than in the other two strains. At day 14, 75% or more of all observed cells were coiled forms in all strains. The number of coiled forms at day 14 was statistically GS-1101 research buy identical in all three strains. Figure 2 Percent of bacillus and coiled forms observed over time during starvation in ultrapure water. Bacillus and coiled forms are represented by solid and open symbols, respectively. ARS-1 (■), ALG-00-530 (●), and ALG-02-36 (▲). The ultrastructure of F. Selleck LY333531 columnare under starvation was further investigated using TEM. At day 1, the ultrastructure of ALG-00-530 shows the outer membrane of the cells with formations that appear to be membrane vesicles breaking off the cells (Figure 3A). No clear glycocalyx or capsule was detected in any cell. Fine-granular cytoplasmatic structure and a denser area that typically corresponds with the nucleoid were observed. By contrast, cells starved for 14 days showed greater heterogeneity in their structure with many apparently empty membrane Sodium butyrate vesicles and lysed cells (Figure 3B). The remaining structurally intact cells were curved (some were coiled) and were characterized by an enlarged periplasmic space, a fine granular structure in the periplasm that lack any clearly visible ribosomes, regions of nucleoid compaction (electron-dense areas), and some inclusions.
Figure 3 TEM observations of Flavobacterium columnare ALG-00-530 strain in ultrapure water. Panel A, day 1 after transfer to ultrapure water. Panel B, maintained in ultrapure water for 150 days. Arrows indicate surface blebbing (SB), membrane vesicle (MV), nucleoid (N), cell membrane (CM), outer membrane (OM), periplasmic space (PS), inclusion (I), and nucleoid compaction areas (NC). Scale bars represent 500 nm. Viability of coiled cells By using a ‘dilution to extinction’ strategy, the few bacilli that remained in the microcosm after 14 days of starvation were diluted out until, by probability, all cells present in the dilutions were coiled. Dilutions up to 10-8 yielded positive tubes (three independent dilution experiments were carried out per strain) in all cultures.
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