Standardization of XTT reduction assay for evaluating Candida biofilms

2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay has been used widely to measure and compare Candida biofilm development. However, XTT reduction is dependent on cellular metabolic activity and, its use to evaluate mature stage biofilms may lead to inaccuracies since the metabolic activity of the latter tends to slow down and enter a quiescent phase with maturation. OBJECTIVE: The improvement of the XTT reduction assay by using glucose supplements in the XTT formulation. METHODS: Candida albicans ATCC 90028 was used for adhesion phase, and 24, 48 and 72 hours biofilm in 96 well microtiter plates. The standard XTT formula was modified with the addition of 12.5, 25, 50, 100 and 200 mM glucose supplements and, these formulations were used to evaluate Candida biofilm metabolic activity at 90, 180 and 270 minutes. The control was XTT without glucose supplements. RESULTS: XTT assay supplemented with 200 mM glucose yielded the most accurate and reproducible readings. This result well correlated with biofilm development at the adhesion phase and, at 24, 48 and 72 hours. On the contrary, the use of XTT formulation devoid of glucose yielded results that did not correlate with the increased biomass over time. Biofilm growth yield after 180 min incubation, evaluated with the 200 mM glucose supplemented XTT, produced the most reproducible reading in independent assays. CONCLUSION: We have refined the traditional XTT assay used to evaluate biofilm metabolic activity by glucose supplementation of XTT. The current modification minimizes variation inherent in the traditional assay and yield reproducible and more accurate data.

ACKNOWLODGEMENT: This study was supported by CAPES BEX 4621/069 and CNPq 140070/2006-0.