title: A quantitative real-time RT-PCR assay for mature C. albicans biofilms.
abstract: BACKGROUND
  Fungal biofilms are more resistant to anti-fungal drugs than organisms in planktonic form. Traditionally, susceptibility of biofilms to anti-fungal agents has been measured using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide (XTT) assay, which measures the ability of metabolically active cells to convert tetrazolium dyes into colored formazan derivatives. However, this assay has limitations when applied to high C. albicans cell densities because substrate concentration and solubility are limiting factors in the reaction. Because mature biofilms are composed of high cell density populations we sought to develop a quantitative real-time RT-PCR assay (qRT-PCR) that could accurately assess mature biofilm changes in response to a wide variety of anti-fungal agents, including host immune cells.
  
  
  RESULTS
  The XTT and qRT-PCR assays were in good agreement when biofilm changes were measured in planktonic cultures or in early biofilms which contain lower cell densities. However, the real-time qRT-PCR assay could also accurately quantify small-medium size changes in mature biofilms caused by mechanical biomass reduction, antifungal drugs or immune effector cells, that were not accurately quantifiable with the XTT assay.
  
  
  CONCLUSIONS
  We conclude that the qRT-PCR assay is more accurate than the XTT assay when measuring small-medium size effects of anti-fungal agents against mature biofilms. This assay is also more appropriate when mature biofilm susceptibility to anti-fungal agents is tested on complex biological surfaces, such as organotypic cultures.
authors.affiliation: Division of Periodontology, School of Dental Medicine, University of Connecticut, Farmington, Connecticut, USA.
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Xie
authors.forename: Angela
authors.initials: A
authors.lastname: Thompson
authors.forename: Helena
authors.initials: H
authors.lastname: Kashleva
authors.forename: Anna
authors.initials: A
authors.lastname: Dongari-Bagtzoglou
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology*; D018441:Biofilms / Q000187:drug effects* / Q000254:growth & development*; D002176:Candida albicans / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development*; D008826:Microbial Sensitivity Tests / Q000379:methods*; D050296:Microbial Viability / Q000187:drug effects; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013778:Tetrazolium Salts / Q000378:metabolism
chemical_list: D000935:Antifungal Agents; D013778:Tetrazolium Salts; C059087:2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-((phenylamino)carbonyl)-2H-tetrazolium hydroxide