9. Put the new glass slide under the microscope and search for the
cell of interest.
10. Repeat steps 4À9 until you are left with only the cell of interest.
11. Suck up the whole content, and put it into a 5 ml well that you
have prefilled with growth medium.
12. Repeat steps 4À12 until you have established several clones of
the same species or several species that you are interested in
getting in culture.
13. Place the well plate under the light which should be set to a
16 h light: 8 h dark cycle. The temperature should preferably
be close to the temperature of the collected water (step 1).
14. Inspect the well plate visually on a daily basis, and when the
culture turns visible colored, place a small volume under the
microscope in order to check for contamination by unwanted
algal species or heterotrophic organisms.
15. If no contamination can be seen, take a subsample with a
pipette, and inoculate the 50 ml cultivation bottle that has
been prefilled with cultivation medium. This is your base culture (see Note 5).
16. The base culture needs to be monitored regularly (weekly) and
reinoculated into fresh medium when growth slows down. The
base culture can also be used for setting up larger cultures (see
Note 6).
3.2 Monitoring
Growth
1. Take a small subsample from your cultivation, and put it onto a
sedimentation chamber with or without a grid (the optimal
size of the sedimentation chamber will depend on the concentration of cells).
2. Count the cells in a grid or fields of view and enumerate >400
cells.
3. Calculate the number of cells per ml based on the grids
counted; if you use fields of view, you have to first calculate
the area in the field of view based on the microscope objective
that you are using (see Note 7).
4. After monitoring cell concentration over time, the growth rate
(μ) can be calculated according to μ ¼ ln(cell t /cell 0 )/dt, where
cell 0 and cell t are the cell concentration at time 0 and t, respectively, and dt is the difference in time between time 0 and t.
5. For determining dry weight (DW), prepare glass fiber filters by
combusting them at 450
C for 4 h, and weigh them. You need
to keep track of individual filters as their weight may vary up to
20%.
6. Apply the filter in a filtration stand, apply suction, and filter a
known volume of culture onto the filter.
Basic Methods for Isolating and Culturing Microalgae
37
cell of interest.
10. Repeat steps 4À9 until you are left with only the cell of interest.
11. Suck up the whole content, and put it into a 5 ml well that you
have prefilled with growth medium.
12. Repeat steps 4À12 until you have established several clones of
the same species or several species that you are interested in
getting in culture.
13. Place the well plate under the light which should be set to a
16 h light: 8 h dark cycle. The temperature should preferably
be close to the temperature of the collected water (step 1).
14. Inspect the well plate visually on a daily basis, and when the
culture turns visible colored, place a small volume under the
microscope in order to check for contamination by unwanted
algal species or heterotrophic organisms.
15. If no contamination can be seen, take a subsample with a
pipette, and inoculate the 50 ml cultivation bottle that has
been prefilled with cultivation medium. This is your base culture (see Note 5).
16. The base culture needs to be monitored regularly (weekly) and
reinoculated into fresh medium when growth slows down. The
base culture can also be used for setting up larger cultures (see
Note 6).
3.2 Monitoring
Growth
1. Take a small subsample from your cultivation, and put it onto a
sedimentation chamber with or without a grid (the optimal
size of the sedimentation chamber will depend on the concentration of cells).
2. Count the cells in a grid or fields of view and enumerate >400
cells.
3. Calculate the number of cells per ml based on the grids
counted; if you use fields of view, you have to first calculate
the area in the field of view based on the microscope objective
that you are using (see Note 7).
4. After monitoring cell concentration over time, the growth rate
(μ) can be calculated according to μ ¼ ln(cell t /cell 0 )/dt, where
cell 0 and cell t are the cell concentration at time 0 and t, respectively, and dt is the difference in time between time 0 and t.
5. For determining dry weight (DW), prepare glass fiber filters by
combusting them at 450
C for 4 h, and weigh them. You need
to keep track of individual filters as their weight may vary up to
20%.
6. Apply the filter in a filtration stand, apply suction, and filter a
known volume of culture onto the filter.
Basic Methods for Isolating and Culturing Microalgae
37