Development of a rapid screen for lipid content

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National Renewable Energy Laboratory

Development of a rapid screen for lipid content.

In an attempt to develop a reproducible, easy-to-use screening procedure to identifiy algal strains

with high lipid contents, Dr. Keith Cooksey (an ASP subcontractor at Montana State University)

suggested that investigators explore the possibility of using the lipophilic dye Nile Red (9-

diethylamino-5H-benzo{a}phenoxazine-5-one) to stain cells. Nile Red was first isolated from

Nile Blue by Greenspan et al. (1985), who showed that Nile Red will fluoresce in a nonpolar

environment and could serve as a probe to detect nonpolar lipids in cells. Nile Red permeates all

structures within a cell, but the characteristic yellow fluorescence (approximately 575 nm) occurs

only when the dye is in a nonpolar environment, primarily neutral storage lipid droplets. Earlier

work within the ASP by Dr. Steve Lien had shown the utility of Nile Blue in microscopically

assessing the lipid content of algal cells (Lien 1981). The active ingredient in these Nile Blue

preparations may in fact have been Nile Red. Parallel efforts to develop a Nile Red staining

procedure were carried out by SERI researchers and ASP subcontractors, notably Drs. Cooksey

and Sommerfeld.

Cooksey et al. (1987) used the diatom Amphora coffeiformis to optimize the Nile Red staining

procedure. The dye was dissolved in acetone and used at a concentration of 1 mg/mL of cell

suspension. In this species, the fluorescence of the dye in live stained cells was stable for only

2-7 min; fluorescence measurements had to be completed rapidly to ensure consistent results. The

kinetics of fluorescence in stained cells varied in different species, presumably due to differences

in the permeability of cell walls to the stain, and differences in how the lipid is stored in the cells,

i.e., as large or small droplets. Fixing the stained cells with formaldehyde or ethanol preserved

the Nile Red fluorescence for 2 h, but cells that were chemically fixed before Nile Red staining

did not exhibit the characteristic yellow fluorescence. When Nile Red fluorescence was measured

in algal cultures over time, the fluorescence increased as the culture became N deficient. The

fluorescence level was linearly correlated with an increase in the total lipid content, determined

gravimetrically, in a growing culture of algal cells. Fractionation of the lipids by silicic acid

column chromatography demonstrated that the increase in lipid was due primarily to an increase

in neutral lipids rather than in the polar lipids or glycolipids, which are found primarily in cell

membranes.

Additional development of the Nile Red screening procedure occurred at SERI and at Milt

Sommerfeld=s laboratory at Arizona State University. The resultant protocol involved taking a

fixed volume of a diluted algal culture (typically 4 mL), adding 0.04 mL of a Nile Red solution

(0.1 mg/mL in acetone), and determining the fluorescence after 5 min using a fluorometer

equipped with the appropriate excitation and emission filters.

Although the use of Nile Red allowed various microalgae to be rapidly screened for neutral lipid

accumulation, interspecies comparisons may be subject to misinterpretation because of the

species-specific staining differences described earlier. Nonetheless, before Nile Red was used,

quantitating lipids from cells was very time consuming. It required the extraction of lipid from

a large number of cells using organic solvents, evaporation of the solvent, and determination of

the amount of lipid by weighing the dried extract. Consequently, the use of Nile Red as a rapid

screening procedure can still have substantial value.

A Look Back at the Aquatic Species Program--Technical Review