Methods in Molecular Biology (2020) 1980: 55–61
DOI 10.1007/7651_2017_102
© Springer Science+Business Media New York 2017
Published online: 26 January 2018
Monitoring Microalgal Neutral Lipid Accumulation
with Nile Red
Katariina Natunen
Abstract
The widely applied Nile red (NR) method allows near real-time monitoring of microalgal neutral lipid
accumulation. When added to a culture sample, optimally, the fluorescent dye NR penetrates the microalgal
cell wall staining the intracellular neutral lipids, and the measured fluorescence is linearly correlated to the
neutral lipid concentration. Here I describe an optimization protocol for determining the optimal staining
parameters for each new microalgal species, followed by a basic NR staining protocol to be applied for
monitoring of microalgal neutral lipid accumulation.
Keywords: Dye, Fluorescence, Lipids, Microalgae, Nile red, Solvents
1 Introduction
Traditional lipid quantification methods, e.g., lipid extraction and
methylation followed by gas chromatographic analysis, are expensive and time-consuming making them unsuitable for online monitoring the accumulation of the refinable, neutral storage lipids in
fast-growing microalgae cultures. Nile red (NR) (9-diethylamino5-benzo[α]phenoxazinone) is a fluorescent dye that stains both the
polar cell wall lipids and the neutral storage lipids in microalgae
cells. With the NR method, near real-time monitoring is possible
due to quick fluorescence measurements that can be carried out in
situ from living microalgae cultures. Due to its solvatochromic properties (i.e., the position, shape, and intensity of its absorption and
emission bands depend on the polarity of its environment [1, 2]), it
is possible to selectively measure neutral lipids by choosing the right
wavelengths for fluorescence measurement.
The functionality of the already established NR method is
optimally based on a linear relationship between the NR fluorescence intensity and the neutral lipid concentration [3]. Since the
1980s, the NR staining method has been widely applied for detecting and quantifying microalgal neutral lipids. Different means to
measure NR fluorescence from stained microalgal cultures have
included spectrofluorometry and fluorometry [4–6], fluorescence
55
DOI 10.1007/7651_2017_102
© Springer Science+Business Media New York 2017
Published online: 26 January 2018
Monitoring Microalgal Neutral Lipid Accumulation
with Nile Red
Katariina Natunen
Abstract
The widely applied Nile red (NR) method allows near real-time monitoring of microalgal neutral lipid
accumulation. When added to a culture sample, optimally, the fluorescent dye NR penetrates the microalgal
cell wall staining the intracellular neutral lipids, and the measured fluorescence is linearly correlated to the
neutral lipid concentration. Here I describe an optimization protocol for determining the optimal staining
parameters for each new microalgal species, followed by a basic NR staining protocol to be applied for
monitoring of microalgal neutral lipid accumulation.
Keywords: Dye, Fluorescence, Lipids, Microalgae, Nile red, Solvents
1 Introduction
Traditional lipid quantification methods, e.g., lipid extraction and
methylation followed by gas chromatographic analysis, are expensive and time-consuming making them unsuitable for online monitoring the accumulation of the refinable, neutral storage lipids in
fast-growing microalgae cultures. Nile red (NR) (9-diethylamino5-benzo[α]phenoxazinone) is a fluorescent dye that stains both the
polar cell wall lipids and the neutral storage lipids in microalgae
cells. With the NR method, near real-time monitoring is possible
due to quick fluorescence measurements that can be carried out in
situ from living microalgae cultures. Due to its solvatochromic properties (i.e., the position, shape, and intensity of its absorption and
emission bands depend on the polarity of its environment [1, 2]), it
is possible to selectively measure neutral lipids by choosing the right
wavelengths for fluorescence measurement.
The functionality of the already established NR method is
optimally based on a linear relationship between the NR fluorescence intensity and the neutral lipid concentration [3]. Since the
1980s, the NR staining method has been widely applied for detecting and quantifying microalgal neutral lipids. Different means to
measure NR fluorescence from stained microalgal cultures have
included spectrofluorometry and fluorometry [4–6], fluorescence
55