Improvement of XTT assay performance for studies involving Candida albicans biofilms.
2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay has been used to study Candida biofilm formation. However, considering that the XTT reduction assay is dependent on cell activity, its use for evaluating mature biofilms may lead to inaccuracies since biofilm bottom cell layers tend to be relatively quiescent at later stages of biofilm formation. The aim of this study was to improve XTT reduction assay by adding glucose supplements to the standard XTT formulation. Candida albicans ATCC 90028 was used to form 24-, 48- and 72-h biofilms. The oxidative activity at 90, 180 and 270 min of incubation was evaluated. The control consisted of standard XTT formulation without glucose supplements, and was modified by the addition of 50, 100 and 200 mM of glucose. The XTT assay with 200 mM glucose showed more accurate and consistent readings correlating with biofilm development at 24, 48 and 72 h. Biofilm growth yield after 180 min incubation, when evaluated with the 200 mM glucose supplemented XTT, produced the most consistent readings on repetitive testing. It may be concluded that glucose supplementation of XTT could minimize variation and produce more accurate data for the XTT assay.
da Silva
Wander José
WJ
Department of Prosthodontics and Periodontology, Dental School of Piracicaba, State University of Campinas, Piracicaba, SP, Brazil.
Seneviratne
Jayampath
J
Parahitiyawa
Nipuna
N
Rosa
Edvaldo Antonio Ribeiro
EA
Samaranayake
Lakshman Perera
LP
Del Bel Cury
Altair Antoninha
AA
D018441:Biofilms / Q000254:growth & development*; D002176:Candida albicans / Q000254:growth & development*; D015169:Colony Count, Microbial / Q000379:methods*; D003470:Culture Media / Q000737:chemistry*; D005947:Glucose; D007202:Indicators and Reagents / Q000737:chemistry*; D010084:Oxidation-Reduction; D015203:Reproducibility of Results; D013778:Tetrazolium Salts / Q000737:chemistry
D003470:Culture Media; D007202:Indicators and Reagents; D013778:Tetrazolium Salts; C059087:2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-((phenylamino)carbonyl)-2H-tetrazolium hydroxide; D005947:Glucose