Determination of Microalgal Lipid Content and Fatty Acid for Biofuel Production
Table 3
Nile red assay procedures for the determination of microalgal lipids.
Reagent(s)
Nile red lipid assay procedure
Fluorescence determination
Ref.
Isopropyl alcohol (IPA); Nile red solution; bleach solution; methanol; corn oil (dissolved in 2 : 1 methanol/chloroform).
Suspend lipid extracts in chloroform; dilute extracts with methanol; add diluted samples and corn oil to the microplate to achieve a range; incubate the plate and evaporate the solvents; add IPA and cool the plate; add Nile red solution; add bleach solution to each well; incubate the plate.
Determine fluorescence using a plate reader with excitation set to 530 nm and emission set to 575 nm with a 570 nm cut-off; read the plate; use the first reading after the fluorescence peak for quantitation.
Place microalgal cells in microcentrifuge tubes and put under microwave treatment; mix with DMSO; put under second microwave treatment using the previous conditions; add Nile red solution and incubate the tubes in the dark; pipet the samples into 96 well microplates.
Measure fluorescence using Fluorescence Analyzer with excitation at 535 nm and emission at 580 nm; measure untreated suspension and medium containing Nile red alone as autofluorescence; convert fluorescence to dry weight of lipids.
Stain diluted microalgal culture samples with Nile red using DMSO as carrier; top up the volume and incubate with agitation.
Read the fluorescence using Microplate Reader with excitation set to 520 nm and emission captured at 570 nm; compare the fluorescence to a virgin olive oil standard curve.
Pure triolein; chloroform; isopropanol; Nile red stock (in 100% spectral grade acetone).
Standardized sample fluorescence: add triolein to chloroform and dilute with isopropanol; prepare working standards by bringing intermediate stocks with DI water. Nile red assay: add Nile red to cell suspension or to a lipid standard.
Measure fluorescence using spectrophotofluorometer with excitation set to 475 nm and emission set to 580 nm; express cellular neutral lipid as triolein equivalents.
