Given these facts we sought to critically examine the limitations of the XTT assay in measuring metabolic changes in mature biofilms and develop a molecular assay based on PCR for biofilm viability estimates that learn more would overcome these limitations. Results We first tried to optimize the XTT assay for a wide range of Candida cell densities, which would represent different stages of biofilm growth. As shown in Figure 1A-B overall, a linear relationship between the OD450 signal and yeast cell number was observed only when yeast did not exceed 1 × 105 cells per well. Above this cell density, significant changes in yeast cell

number (2-fold or greater) resulted in very small or undetectable differences in OD450 values. This suggests that the XTT assay would be of limited value in mature biofilms, since C. albicans biofilms are frequently started by seeding ≥1 × 105 yeast cells per well, in 96 well plates, and grown for 48h or longer for biofilms to mature [2, 6, 28]. Figure 1 Effect of XTT assay parameters in the assessment of C. albicans metabolic activity. Overnight planktonic cultures of C. albicans yeast cells were seeded at 103-5 × 105 cells per well (30 mm2 well surface area)

and XTT assay was performed as described. (A) Relationship between OD450 and Candida cell C646 purchase density at two different XTT concentrations. (B) Effect of CoQ concentration on the linearity range. A representative of three independent experiments is shown. Increasing

the concentration of XTT up to 2 mg/ml (since XTT maximum solubility in water is 2.5 mg/ml) did not result in a change in OD450 when Adenosine triphosphate the seeding yeast cell number was equal to or lower than 1 × 105 cells per well (Figure 1A). With yeast cell numbers higher than 1 × 105 cells per well, increasing the concentration of XTT resulted in higher OD450 values, which extended the linearity range only up to 2 × 105 cells per well. This suggests that XTT solubility and final concentration are limiting factors in this reaction, especially when large numbers of yeast cells are used to start biofilms. We also investigated if varying the concentration of the electron-coupling agent CoQ (8-350 μM) would allow us to extend the linearity range of the XTT signal. XTT conversion rates were slower at lower concentrations of CoQ, generating flat slopes (Figure 1B). However, we found that increasing the concentration of CoQ would not increase the linearity range (Figure 1B). Reading the plates at 490 nm as opposed to 450 nm or increasing the XTT reaction time to 3 hours still did not improve the linearity range (data not shown), since reaction time in higher cell densities (>106 cells/well) was typically very fast (less than 10 min). Collectively, these data suggest that the XTT assay cannot be adequately optimized to accommodate the cell numbers present in mature biofilms.