TY - JOUR
T1 - How to us Nile Red, a selective fluorescent stain for microalgal neutral lipids
AU - Alemán-Nava, Gibrán S.
AU - Cuellar-Bermudez, Sara P.
AU - Cuaresma, María
AU - Bosma, Rouke
AU - Muylaert, Koenraad
AU - Ritmann, Bruce E.
AU - Parra, Roberto
PY - 2016
Y1 - 2016
N2 - The use of Nile Red for rapid monitoring of the neutral lipid content in microalgae has gained interest over the last decade, since neutral lipids are feedstock for renewable transportation fuel. In this review, we discuss the main considerations needed to make an NR protocol reliable for staining neutral lipids in microalgae. Cell wall permeability must be enhanced by using stain carriers: DMSO (5% v/v to 25% v/v), glycerol (0.1 to 0.125 mg/mL), or EDTA (3.0 to 3.8 mg/mL). Temperatures between 30 and 40 °C facilitate the diffusion of NR through the cell wall without incurring excess quenching. Good NR-lipid interaction requires using a low NR/cell ratio; the NR concentration must be between 0.25 μg/mL and 2.0 μg/mL, and the cell concentration > 5 × 104 cells/mL. In order to have the maximum and stable NR fluorescence, it is necessary to scan the excitation/emission wavelengths for up to a 40-min of incubation time. We outline a five-step method to customize the Nile Red protocol to a specific strain: 1) Evaluate the strain's suitability by checking for the presence of neutral lipid, 2) Select of the best excitation/emission wavelength, 3) Optimization of incubation time, stain carrier, dye concentration, and temperature, 4) Prepare single-strain algal cultures with different lipid contents to calibrate NR fluorescence with neutral-lipid content, and 5) Correlate NR fluorescence intensity to neutral lipid content for the same strain. Once the protocol is customized, the NR method allows for rapid and reliable monitoring of neutral lipid content of a microalgae strain.
AB - The use of Nile Red for rapid monitoring of the neutral lipid content in microalgae has gained interest over the last decade, since neutral lipids are feedstock for renewable transportation fuel. In this review, we discuss the main considerations needed to make an NR protocol reliable for staining neutral lipids in microalgae. Cell wall permeability must be enhanced by using stain carriers: DMSO (5% v/v to 25% v/v), glycerol (0.1 to 0.125 mg/mL), or EDTA (3.0 to 3.8 mg/mL). Temperatures between 30 and 40 °C facilitate the diffusion of NR through the cell wall without incurring excess quenching. Good NR-lipid interaction requires using a low NR/cell ratio; the NR concentration must be between 0.25 μg/mL and 2.0 μg/mL, and the cell concentration > 5 × 104 cells/mL. In order to have the maximum and stable NR fluorescence, it is necessary to scan the excitation/emission wavelengths for up to a 40-min of incubation time. We outline a five-step method to customize the Nile Red protocol to a specific strain: 1) Evaluate the strain's suitability by checking for the presence of neutral lipid, 2) Select of the best excitation/emission wavelength, 3) Optimization of incubation time, stain carrier, dye concentration, and temperature, 4) Prepare single-strain algal cultures with different lipid contents to calibrate NR fluorescence with neutral-lipid content, and 5) Correlate NR fluorescence intensity to neutral lipid content for the same strain. Once the protocol is customized, the NR method allows for rapid and reliable monitoring of neutral lipid content of a microalgae strain.
KW - Biodiesel
KW - Microalgae
KW - Neutral lipids
KW - Nile Red
KW - Protocol
U2 - 10.1016/j.mimet.2016.07.011
DO - 10.1016/j.mimet.2016.07.011
M3 - Article
AN - SCOPUS:84978647682
SN - 0167-7012
VL - 128
SP - 74
EP - 79
JO - Journal of Microbiological Methods
JF - Journal of Microbiological Methods
ER -