5. 5–10 ml tubes or vials used during staining.
6. Spectrofluorometer vials or 96-vial microplates (depending on
spectrofluorometer type).
7. Automatic pipettes with adjustable volume and their tips.
8. Freeze dryer.
9. Sterile phosphate buffer, pH 7.4 (see Note 1).
3 Methods
3.1 Relative
Fluorescence
Measurements
1. Dissolve 1 mg of BODIPY 505/515 dye (4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene) into 10 ml of DMSO
(99.5%) (see Notes 2 and 8).
2. Store the stock solution at controlled low temperature and protected from light (in coated dark bottle) for up to 30 days.
3. For optimal evaluation of dye concentration, a range of concentrations should be used for testing: 0.04, 0.07, 0.10, 0.30, 0.50,
0.70, 0.90, 1.1, 1.5, 2.0, and 2.6 μg ml
À1 ; incubation time—
10 min at 25
C (see Notes 3–6).
4. For optimization of staining duration, add 2 μl of BODIPY 505/515
DMSO solution to 3 ml of microalgal culture, and measure fluorescence intensity after 1, 2, 3, 5, 10, 15, and 20 min of incubation in
the dark at 25
C (see Notes 3–6).
5. Fill the vials or tubes with appropriate equal volume of the microalgae culture of an optimal cell density (5 Â 10
4
–1 Â 10
6 cell/ml).
Five replicates of each measurement are recommended to obtain
statistically reliable results.
6. Add, e.g., 5 μl of prepared dyes to 6 ml of microalgal culture to
obtain 0.08 μg ml
À1 final concentration of the dye (use the optimal
dye concentration obtained for testing microalgae species).
7. Incubate stained microalgal cells for 1–20 min (select duration
from optimization of testing species) in the dark at 25
C (see
Notes 5 and 6).
3.2 Calibration Curve
for Quantitative Lipid
Evaluation
1. Use freeze-dried biomass of microalgae with the determined
lipid content by conventional gravimetric method to obtain
linear correlation between fluorescence intensity read and neutral lipid content. Freeze-dried algal samples are stable for relatively long-term storage periods and could be used repeatedly up
to 6 months.
2. Resuspend freeze-dried cells in a sterile phosphate buffer solution to a known biomass concentration.
Microalgae Lipid Staining with Fluorescent BODIPY Dye
49
6. Spectrofluorometer vials or 96-vial microplates (depending on
spectrofluorometer type).
7. Automatic pipettes with adjustable volume and their tips.
8. Freeze dryer.
9. Sterile phosphate buffer, pH 7.4 (see Note 1).
3 Methods
3.1 Relative
Fluorescence
Measurements
1. Dissolve 1 mg of BODIPY 505/515 dye (4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene) into 10 ml of DMSO
(99.5%) (see Notes 2 and 8).
2. Store the stock solution at controlled low temperature and protected from light (in coated dark bottle) for up to 30 days.
3. For optimal evaluation of dye concentration, a range of concentrations should be used for testing: 0.04, 0.07, 0.10, 0.30, 0.50,
0.70, 0.90, 1.1, 1.5, 2.0, and 2.6 μg ml
À1 ; incubation time—
10 min at 25
C (see Notes 3–6).
4. For optimization of staining duration, add 2 μl of BODIPY 505/515
DMSO solution to 3 ml of microalgal culture, and measure fluorescence intensity after 1, 2, 3, 5, 10, 15, and 20 min of incubation in
the dark at 25
C (see Notes 3–6).
5. Fill the vials or tubes with appropriate equal volume of the microalgae culture of an optimal cell density (5 Â 10
4
–1 Â 10
6 cell/ml).
Five replicates of each measurement are recommended to obtain
statistically reliable results.
6. Add, e.g., 5 μl of prepared dyes to 6 ml of microalgal culture to
obtain 0.08 μg ml
À1 final concentration of the dye (use the optimal
dye concentration obtained for testing microalgae species).
7. Incubate stained microalgal cells for 1–20 min (select duration
from optimization of testing species) in the dark at 25
C (see
Notes 5 and 6).
3.2 Calibration Curve
for Quantitative Lipid
Evaluation
1. Use freeze-dried biomass of microalgae with the determined
lipid content by conventional gravimetric method to obtain
linear correlation between fluorescence intensity read and neutral lipid content. Freeze-dried algal samples are stable for relatively long-term storage periods and could be used repeatedly up
to 6 months.
2. Resuspend freeze-dried cells in a sterile phosphate buffer solution to a known biomass concentration.
Microalgae Lipid Staining with Fluorescent BODIPY Dye
49