3.1 Optimization
Protocol for Nile Red
Staining Method
The optimization protocol described below is one of the many ways
to find out the optimal NR staining parameters. The protocol is
iterative; thus, the steps below might have to be repeated until
optimal results are achieved. They also need to be carried out
separately for every new species and measuring equipment.
1. Verify that your microalgae culture has accumulated neutral
lipids by determining the lipids, e.g., with gas chromatography
(GC) [12] or by other means (see Note 6).
2. Choose a starting NR concentration (e.g., 1 μL/mL final
concentration) and incubation time (e.g., 10 min) for measurements (see Note 7). Add NR into 3 mL of culture sample in a
cuvette, and after incubating (see Note 1), place the sample into
a spectrofluorometer, and determine the optimal wavelengths
for NR measurements by measuring excitation-emission fluorescence matrices (EEMs). Use an excitation range of approx.
450–550 nm and emission range of approx. 550–600 nm (see
Note 8).
3. Optimize NR concentration and staining time with a series of
kinetic fluorescence measurements, i.e., different volumes of
NR stock solutions are added to culture samples, and the
evolvement of relative fluorescence values is followed as a function of time.
4. Place a cuvette with 3 mL of fresh (diluted) culture sample into
spectrofluorometer. Add x μL of NR to the sample and follow
the fluorescence for 20–40 min at the wavelengths determined
in step 2 (see Note 9).
5. Repeat step 4 with a range of different additions of NR, e.g.,
0.08–1 μg/mL (i.e., x ¼ 1–12 μL per 3 mL sample). Choose the
concentration/staining time combination that produces the
highest and/or most stable fluorescence values (see Note 11).
6. Repeat step 2 and see whether optimal excitation-emission
wavelengths change with these new parameters.
7. If the culture has been verified to contain lipids (see Step 1), but
no NR fluorescence is produced in spite of trying out different
NR concentrations, one explanation is that the dye does not
penetrate the rigid cell wall of the microalgae species [6]. The
staining might be enhanced with adding a solvent to the algal
sample, which makes the cell wall more porous for the dye to
enter the cell, without breaking the cell [6, 11] (see Note 12).
A commonly applied solvent is DMSO.
8. Test the effect of the following DMSO concentrations: 5, 10,
20, and 30% (v/v). Use the same volume of culture with every
DMSO concentration: The highest concentration, 30% (v/v)
DMSO, equals 1 mL DMSO in 3 mL sample. Thus, also with
lower DMSO concentrations, use 2 mL of culture and compensate
Lipid staining with Nile Red
57
Protocol for Nile Red
Staining Method
The optimization protocol described below is one of the many ways
to find out the optimal NR staining parameters. The protocol is
iterative; thus, the steps below might have to be repeated until
optimal results are achieved. They also need to be carried out
separately for every new species and measuring equipment.
1. Verify that your microalgae culture has accumulated neutral
lipids by determining the lipids, e.g., with gas chromatography
(GC) [12] or by other means (see Note 6).
2. Choose a starting NR concentration (e.g., 1 μL/mL final
concentration) and incubation time (e.g., 10 min) for measurements (see Note 7). Add NR into 3 mL of culture sample in a
cuvette, and after incubating (see Note 1), place the sample into
a spectrofluorometer, and determine the optimal wavelengths
for NR measurements by measuring excitation-emission fluorescence matrices (EEMs). Use an excitation range of approx.
450–550 nm and emission range of approx. 550–600 nm (see
Note 8).
3. Optimize NR concentration and staining time with a series of
kinetic fluorescence measurements, i.e., different volumes of
NR stock solutions are added to culture samples, and the
evolvement of relative fluorescence values is followed as a function of time.
4. Place a cuvette with 3 mL of fresh (diluted) culture sample into
spectrofluorometer. Add x μL of NR to the sample and follow
the fluorescence for 20–40 min at the wavelengths determined
in step 2 (see Note 9).
5. Repeat step 4 with a range of different additions of NR, e.g.,
0.08–1 μg/mL (i.e., x ¼ 1–12 μL per 3 mL sample). Choose the
concentration/staining time combination that produces the
highest and/or most stable fluorescence values (see Note 11).
6. Repeat step 2 and see whether optimal excitation-emission
wavelengths change with these new parameters.
7. If the culture has been verified to contain lipids (see Step 1), but
no NR fluorescence is produced in spite of trying out different
NR concentrations, one explanation is that the dye does not
penetrate the rigid cell wall of the microalgae species [6]. The
staining might be enhanced with adding a solvent to the algal
sample, which makes the cell wall more porous for the dye to
enter the cell, without breaking the cell [6, 11] (see Note 12).
A commonly applied solvent is DMSO.
8. Test the effect of the following DMSO concentrations: 5, 10,
20, and 30% (v/v). Use the same volume of culture with every
DMSO concentration: The highest concentration, 30% (v/v)
DMSO, equals 1 mL DMSO in 3 mL sample. Thus, also with
lower DMSO concentrations, use 2 mL of culture and compensate
Lipid staining with Nile Red
57