Comparison of three assays for the quantification of Candida biomass in suspension and CDC reactor grown biofilms.
A common assay to measure yeast metabolic activity in biofilms is based on the reduction of the tetrazolium salt XTT {2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide} to a colored formazan. However, a recent report, also confirmed by our own findings about the shortcomings of the chromogenic XTT assay, has prompted us to investigate alternative methods for yeast biomass quantification. To this end, two fluorogenic assays using fluorescein diacetate (FDA) and SYTO 9 as well as the XTT assay were comparatively evaluated with regard to the linear range of Candida albicans and Candida parapsilosis cell number-response curves, precision and intra- and interspecies variability. Reading of fluorescence and absorbance was carried out in a multilabel microtiter plate reader. All three assays were adequate for the determination of planktonic yeast biomass, but the FDA and SYTO 9 assays present practical advantages. When applied to the quantification of yeast biofilm biomass obtained in the CDC biofilm reactor, the FDA assay proved superior.
Honraet
Kris
K
Laboratory for Pharmaceutical Microbiology, University of Ghent, Harelbekestraat 72, B-9000, Ghent, Belgium.
Goetghebeur
Els
E
Nelis
Hans J
HJ
D018441:Biofilms / Q000254:growth & development; D018533:Biomass; D019149:Bioreactors; D002175:Candida / Q000254:growth & development* / Q000302:isolation & purification / Q000502:physiology; D002176:Candida albicans / Q000254:growth & development / Q000302:isolation & purification / Q000502:physiology; D015169:Colony Count, Microbial / Q000379:methods*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009172:Mycology / Q000379:methods*; D009930:Organic Chemicals; D013778:Tetrazolium Salts / Q000378:metabolism
D005452:Fluoresceins; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C103389:SYTO 9; D013778:Tetrazolium Salts; C059087:2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-((phenylamino)carbonyl)-2H-tetrazolium hydroxide; C018506:diacetylfluorescein