Error bars represent standard deviations from three independent experiments. In order to compare
the ability of the XTT and qRT-PCR assays to accurately quantify changes in viable mature biofilms, the biomass of biofilms grown for 48 hours was mechanically reduced and remaining AZD7762 price biofilm cells were assessed with the two assays. The XTT assay showed that removal of 25-50% of the biofilm mass resulted in a detectable decrease in OD450 values, compared to intact biofilm. However, there were no significant differences in the XTT signals resulting from removal of different biofilm amounts, thus the XTT signal reduction was not commensurate to the reductions in biomass. This shows that the XTT assay cannot accurately quantify
changes in mature biofilms (Figure 5A). In contrast, the qRT-PCR assay showed excellent agreement with reduction in the biofilm mass since 25%, 33% and 50% biofilm removal resulted in an average of 25.8%, 35.4% Bioactive Compound Library and 49.8% reduction in the logarithmic EFB1 transcript copy numbers, respectively (Figure 5B). This confirms the ability of the real-time RT-PCR assay to accurately measure reduction in biofilm metabolic activity in mature biofilms. Figure 5 Comparison of the XTT and qRT-PCR assays in assessing biomass reduction in mature biofilms. Biofilms were seeded at 105 cells per 30 mm2 of well surface area and were incubated for 48 h. Prior to assessment, biofilms were either left intact (0), or were mechanically reduced by 25%, 33% or 50%, followed by the XTT assay (A) or qRT-PCR assay (B). Error bars represent SD of triplicate experiments. Student t-test p values are shown on the graph for each set of comparisons. Neutrophils exhibit potent candidacidal activities in vitro [26, 27] and interact with SN-38 Candida biofilms forming Methamphetamine on mucosal tissues in vivo . However there is a paucity of information regarding the outcome of the interactions of neutrophils with biofilm organisms
. One of the difficulties in studying these interactions in vitro is the shortage of quantitative assays to accurately assess neutrophil-inflicted damage in mature biofilms. Therefore, we compared the ability of the two assays to detect and quantify damage inflicted to early and mature biofilms by HL-60 cells, a human neutrophil-like cell line. When HL-60 cells interacted with early (3 h) biofilms, significant biofilm damage (up to 80%) could be detected at 10:1 effector to target ratio, regardless of the assay used to measure viable biofilm changes (Figure 6A,B). Significant dose response differences to the number of effectors could also be detected with both assays in early biofilms. Thus there was close agreement between the two assays when early biofilms were tested.