Journal
JOURNAL OF MICROBIOLOGICAL METHODS
Volume 91, Issue 2, Pages 321-328Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.mimet.2012.09.001
Keywords
Biodiesel; Neutral lipids; Nile red; Oleaginous yeast; Kinetic reading; Black microplate
Categories
Funding
- NIH Fogarty International Center [U01TW008160]
- NIH Office of Dietary Supplements
- National Science Foundation
- Department of Energy
- USDA Agricultural Food Research Initiative of National Food and Agriculture, USDA [35621-04750]
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A rapid and inexpensive method for estimating lipid content of yeasts is needed for screening large numbers of yeasts samples. Nile red is a fluorescent lipophilic dye used for detection and quantification of intracellular lipid droplets in various biological system including algae, yeasts and filamentous fungi. However, a published assay for yeast is affected by variable diffusion across the cell membrane, and variation in the time required to reach maximal fluorescence emission. In this study, parameters that may influence the emission were varied to determine optimal assay conditions. An improved assay with a high-throughput capability was developed that includes the addition of dimethyl sulfoxide (DMSO) solvent to improve cell permeability, elimination of the washing step, the reduction of Nile red concentration, kinetic readings rather than single time-point reading, and utilization of a black 96-well microplate. The improved method was validated by comparison to gravimetric determination of lipid content of a broad variety of ascomycete and basidiomycete yeast species. (C) 2012 Elsevier B.V. All rights reserved.
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