5×107 and 1.9×106 CFU/ml of the fresh and 2-weeks old ALG-00-530, respectively. Controls were exposed to MS broth without bacteria. Fish were monitored at 12 h intervals for abnormal behavior, loss of appetite and mortality. Moribund fish were sampled for F. columnare and putative colonies were confirmed using following standard protocols [20]. Growth curve To compare the growth potential of fresh and starved cultures 20 μl of a 24 h, 1-month, and 3-month-old cultures

of strain ALG-00-530 (obtained as described above) were inoculated into microtiter plates containing fresh MS medium (80 μl) and allowed to grow at 28±2°C for 24 h. Cell optical density (OD595) was measured at regular intervals using a Synergy HT microplate reader (Bio-TEK, USA). Immediately after each reading, 100 μl of the LIVE/DEAD mixed dyes were added to each well and fluorescence was quantified at 528 nm (green) selleck chemicals and 590 nm (red). Four independent

replicates were carried out per culture. Revival of starved cultures To better understand how the starved cells transitioned into a rich-nutrient environment, we monitor the ultrastructural changes in five-month old ALG-00-530 cultures when they were exposed to different levels of nutrients present in MS medium. Starved cells were inoculated (1:100 Smoothened Agonist dilution) into the following media: MS, 10 times diluted MS (MS-10), MS containing salts and tryptone but not yeast extract (MS-T), MS containing salts and yeast extract but not tryptone (MS-Y), and MS containing salts but not organic nutrient (MS-S). The experiment was carried out in triplicate. Tubes were incubated at 28°C with gentle shaking for 78 h. Cell morphology was analyzed at regular intervals by using light microscopy and SEM as previously described. Cell optical density (OD595) was measured as proxy for bacterial growth (see above). Statistical analysis Colony forming unit counts were converted to base 10 logarithms to fit the model assumption of normal distribution. One-way analysis of variance (ANOVA) was used to determine the differences in F. columnare CFU/ml from the short-term survival study.

Welch’s ANOVA (allowing for unequal variance) was used to determine differences of bacillus versus ‘coiled’ forms. If either ANOVA Metabolism inhibitor or Welch’s ANOVA was statistically significant (P value < 0.05), Tukey’s method and Scheffe’s method were applied to perform post hoc, pair-wise comparisons at α = 0.05 for the means of log F. columnare counts or the Dunnett’s T3 test (allowing unequal variance) as post hoc, pair-wise comparisons for ‘bacilli/coiled’ forms at α = 0.05. Mortality data were compared by ANOVA using the Duncan’s multiple range test. Calculations were done using the OriginPro version 8.5 (OriginLab Co., Northampton, MA). Results Survival under starvation conditions Table 1 shows the culturability of the four F. columnare strains when subjected to two weeks of starvation conditions in ultrapure water.