Uginosa cells did not survive incubation in this lot of serum, as MedChemExpress Calciferol determined by plating. These cells exhibited no detectable pre-rRNA synthesis upon 4-IBP price nutritional stimulation. Genomic DNA signals were about as high in P. aeruginosa as they were in A. baumannii, indicating the persistence of intact cells and/or cell-free P. aeruginosa DNA (Figure 2C). The observation that serum-inactivated P. aeruginosa cells do not synthesize pre-rRNA upon nutritional stimulation was consistent with previous observations made on hypochloriteinactivated A. hydrophila cells in water [18]. Viable P. aeruginosa cells derived from the second lot of serum exhibited robust pre-rRNA production upon nutritional stimulation (below); therefore, the lack of pre-rRNA production in spiked serum was not due to a unique inability of P. aeruginosa cells to produce pre-rRNA in response to nutritional stimulation. These observations illustrate the utility of ratiometric pre-rRNA analysis. The `0′- hour (pre-stimulation) measurements of gDNA in Figure 2 resemble standard PCR-based diagnostic tests, in that material taken directly from samples was subjected to PCR to detect the DNA of a targeted species, regardless of viability. These would have appeared strongly positive for A. baumannii and P. aeruginosa, and more weakly positive for S. aureus. Such findings would have been inconsistent with plating results, which showed that few if any viable P. aeruginosa survived incubation in serum. Ratiometric pre-rRNA results were more consistent with plating, with P. aeruginosa yielding a negative result and the other two species yielding positive results by this method.second set of experiments was conducted in which a second lot of human serum was inoculated with a smaller number of cells (,1E5/mL) and acclimated in serum for shorter periods (as little as four hours). In these experiments viable A. baumannii, P. aeruginosa, or S. aureus cells were present in each replicate at every time point tested (4, 24, and 168 hours), according to plating results. It is not known whether the improved survival of P. aeruginosa in this experiment was due to differences in seeding density, serum lot, or both. In agreement with plating results, prerRNA production evealed by increased P:G ratios in stimulated samples relative to unstimulated samples ndicated viable cells were present in all replicates tested. For A. baumannii and P. aeruginosa, nutritional stimulation strongly induced pre-rRNA production in each biological replicate, increasing P:G ratios .10-fold, after only four hours in serum (Figure 3A and 3B). After seven days (168 hours) in serum, nutritional stimulation induced pre-rRNA synthesis that was less robust in both organisms, although still significant (P:G+/ P:G2.1) for each replicate. S. aureus appeared to require longer incubation in serum to thoroughly acclimate and drain its prerRNA 24786787 pools (Figure 3C). However, 24 hours of acclimation to serum resulted in sufficient drainage of pre-rRNA pools to yeild a .10-fold increase in pre-rRNA upon nutritional stimulation. Thus, the results in Figure 2 did not depend on high cell density and/or extended (7 days) acclimation to serum.Pre-rRNA Analysis of a Slow-growing Mycobacterium SpeciesThe three species in Figures 2 and 3 share the physiological property of rapid aerobic growth. With the requisite viability, all exhibited rapid pre-rRNA upshift (1? generation times) in response to nutritional stimulation after incubation in sub-optimal.Uginosa cells did not survive incubation in this lot of serum, as determined by plating. These cells exhibited no detectable pre-rRNA synthesis upon nutritional stimulation. Genomic DNA signals were about as high in P. aeruginosa as they were in A. baumannii, indicating the persistence of intact cells and/or cell-free P. aeruginosa DNA (Figure 2C). The observation that serum-inactivated P. aeruginosa cells do not synthesize pre-rRNA upon nutritional stimulation was consistent with previous observations made on hypochloriteinactivated A. hydrophila cells in water [18]. Viable P. aeruginosa cells derived from the second lot of serum exhibited robust pre-rRNA production upon nutritional stimulation (below); therefore, the lack of pre-rRNA production in spiked serum was not due to a unique inability of P. aeruginosa cells to produce pre-rRNA in response to nutritional stimulation. These observations illustrate the utility of ratiometric pre-rRNA analysis. The `0′- hour (pre-stimulation) measurements of gDNA in Figure 2 resemble standard PCR-based diagnostic tests, in that material taken directly from samples was subjected to PCR to detect the DNA of a targeted species, regardless of viability. These would have appeared strongly positive for A. baumannii and P. aeruginosa, and more weakly positive for S. aureus. Such findings would have been inconsistent with plating results, which showed that few if any viable P. aeruginosa survived incubation in serum. Ratiometric pre-rRNA results were more consistent with plating, with P. aeruginosa yielding a negative result and the other two species yielding positive results by this method.second set of experiments was conducted in which a second lot of human serum was inoculated with a smaller number of cells (,1E5/mL) and acclimated in serum for shorter periods (as little as four hours). In these experiments viable A. baumannii, P. aeruginosa, or S. aureus cells were present in each replicate at every time point tested (4, 24, and 168 hours), according to plating results. It is not known whether the improved survival of P. aeruginosa in this experiment was due to differences in seeding density, serum lot, or both. In agreement with plating results, prerRNA production evealed by increased P:G ratios in stimulated samples relative to unstimulated samples ndicated viable cells were present in all replicates tested. For A. baumannii and P. aeruginosa, nutritional stimulation strongly induced pre-rRNA production in each biological replicate, increasing P:G ratios .10-fold, after only four hours in serum (Figure 3A and 3B). After seven days (168 hours) in serum, nutritional stimulation induced pre-rRNA synthesis that was less robust in both organisms, although still significant (P:G+/ P:G2.1) for each replicate. S. aureus appeared to require longer incubation in serum to thoroughly acclimate and drain its prerRNA 24786787 pools (Figure 3C). However, 24 hours of acclimation to serum resulted in sufficient drainage of pre-rRNA pools to yeild a .10-fold increase in pre-rRNA upon nutritional stimulation. Thus, the results in Figure 2 did not depend on high cell density and/or extended (7 days) acclimation to serum.Pre-rRNA Analysis of a Slow-growing Mycobacterium SpeciesThe three species in Figures 2 and 3 share the physiological property of rapid aerobic growth. With the requisite viability, all exhibited rapid pre-rRNA upshift (1? generation times) in response to nutritional stimulation after incubation in sub-optimal.
