Biology Reference
In-Depth Information
8.
Sterile plastic reservoir.
9.
Sterile 200 and 1200
L pipette tips for eight-channel pipettor.
10.
Eight-channel pipettors for transferring 5, 100, and 1000
m
L liquid culture.
m
5.1.2
Methods
1.
To each well of autoclaved 96-deep-well plate, add 800
L-1 mL TAP medium.
For N deprivation and induction of oil accumulation, use TAP medium
containing 1 mM instead of 10 mM ammonium chloride.
2.
The mutant colonies appeared on hygromycin-selected plates are picked with
toothpicks and used to inoculate the culture of 96-well plate (always reserve
the first or last well as wild type control), cover the plate with a sterilized lid.
3.
Place the inoculated 96-well plates in incubator with constant light at 22
C,
pulse-vortex the plates twice a day, let cells grow 3-7 days.
4.
Pipette 100
m
L of 4- or 7-day old cultures to a black 96-well plate using a
multichannel pipettor, measure the cells autofluorescence using a 96-well plate
reader with a filter set at 485-nm excitation and 535-nm emission.
5.
Add 5
m
L Nile red stock solution (1 mg/mL in acetone) to each well of the black
plate (a final concentration of 50
m
g/mL).
6.
Allow 10-15-min incubation to let the dye to enter the cells, measure the cells
fluorescence again with the same filter set.
7.
Calculate the fluorescence ratio (fluorescence recorded in the presence of Nile
red divided by autofluorescence without Nile red staining). Colonies with
significantly higher or lower fluorescence ratios than the ratio of the control are
putative lipid mutants.
8.
Confirm the putative mutants by repeating the screen procedure described above.
m
5.2
CONFOCAL AND TRANSMISSION ELECTRON
MICROSCOPIC OBSERVATION
The Nile red fluorescence staining of oil droplets inmicroalgae yields brilliant yellow
fluorescence, which allows us to examine the size, shape, and number of the oil drop-
lets with confocal or epifluorescence microscopy. Several studies have reported the
confocal microscopy of oil droplets in
Chlamydomonas
(
Moellering & Benning,
2010; Wang, Ullrich, Joo, Waffenschmidt, &Goodenough, 2009
). However, to better
understand the mechanism underlying the oil droplet dynamics and the biogenesis of
oil droplets and their potential interactions with other organelles, ultrastructural anal-
ysis with transmission electron microscopy (TEM) is desirable (see example in
Fig. 5.3
). Below is the TEM sample preparation protocol optimized in our laboratory.
5.2.1
Materials
1.
Phosphate buffer: 0.1 M sodium phosphate (pH 7.2)
2.
Primary fixative: glutaraldehyde: 5% (w/v) in 0.1 M sodium phosphate (pH 7.2)
