A rapid colorimetric assay of fungal viability with the tetrazolium salt MTT.
A rapid colorimetric assay for viability of fungi that uses the tetrazolium salt MTT is described. All live species of a wide range of medically important fungi that were tested reduced MTT, with resultant intracellular formation within several hours of clearly visible purple crystals of MTT-formazan. The assay proved particularly useful in measuring viability of individual hyphae; greater than 99% of live Aspergillus fumigatus and Rhizopus oryzae hyphae stained purple when incubated with MTT compared with 0% of hyphae killed by any of a variety of methods. The MTT method for measuring fungal viability closely correlated with a slide-culture technique when killing of A. fumigatus hyphae by graded concentrations of hydrogen peroxide was compared. MTT-formazan was easily extracted from the fungi by alcohol, a procedure allowing spectrophotometric quantification of fungal killing in both cell-free and cellular systems. A linear relation between inocula and MTT reduction of several fungi, including Candida albicans blastospores and A. fumigatus hyphae, was demonstrated. Human leukocytes killed 80.3% of C. albicans blastospores as measured by the MTT assay, compared with 70.5% as measured by dilutions and pour plates. Killing of A. fumigatus hyphae by human leukocytes was also quantitated by using the MTT assay.
S M
SM
Levitz
R D
RD
Diamond
D005658:Fungi / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007962:Leukocytes / Q000276:immunology; D009172:Mycology / Q000379:methods*; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D013172:Spores, Fungal / Q000276:immunology / Q000502:physiology; D013194:Staining and Labeling; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism
D013778:Tetrazolium Salts; D013844:Thiazoles; D006861:Hydrogen Peroxide; C022616:thiazolyl blue