To compare our data reported above, we set up this model for pneumococcal biofilm. Pneumococcal cells grown to early stationary phase were harvested, washed and inoculated 1:10 to approximately 5 × 107 CFU/ml into diluted or undiluted medium in microtiter wells [24]. To permit extension of the experiment for several days half of the spent medium was exchanged twice daily with fresh medium. In this setup the utilisation of diluted fresh medium did not reduce significantly LY2835219 manufacturer pneumococcal attachment (data not shown) and

variation of medium form TSB to BHI yielded approximately the same results (data not shown). Due to the high inoculum cells didn’t go through exponential phase of growth, but maintained constant cell density in the liquid phase (data not shown). In this series of experiments the biofilm formation was quantified through spectrophotometic analysis of crystal violet stained biofilm cells. This readout was chosen since pneumococci tended to form aggregates on the well bottom

(see below) and sonication at AZD8186 mouse sub-lethal doses was not sufficient to ensure their disggregation, rendering viable counts a non reliable parameter (data not shown). A biofilm formed in such conditions could be maintained for up to 5 days, with little changes due to dilution of the medium (data not sown), in accordance with what has been reported by others [24]. To test the impact of competence in this model we analysed the same series of wt and comD and comC mutants as above. As shown in Figure 3A, the wt strain produced significantly more biofilm than the two competence mutants at 24 h. Supplementation of the medium with synthetic CSP complemented the phenotype of reduced biofilm formation in the comC mutant. When analysing the biofilm formation after 48 hours of incubation, we observed an identical trend (Figure 3b). Figure 3 Impact of competence in the stationary phase type microtiter biofilm model. In this model, biofilm formation was evaluated by both crystal violet GANT61 chemical structure staining and analysis at the spectrophotometer.

The FP23 strain (non-capsulated TIGR4) was compared with its isogenic mutants in comD (FP231) and comC (FP259). The comC mutant FP259 was also assayed with addition of synthetic CSP to the medium (striped bars). The experiment MycoClean Mycoplasma Removal Kit was performed in BHI and read after 24 (panel A) or 48 hours (panel B) of incubation at 37°C. The differences in biofilm formations between the wt and the comC and comD mutants and between FP259 with and without CSP were statistically significant (p < 0.005). Data are from triplicate experiments. To explain these differences microscopy was performed. The images reported in Figure 4A show biofilm formed by the TIGR4 strain and the comC and comD mutants (Figure 4B and 4C). The addition of CSP to the comC mutant increase the number of cells attached (Figure 4D). More striking was the observation that wt cells formed microcolony-like aggregates on the well bottom, which increased in size and number over time (data not shown).