Thesis-PDF - IAP/TU Wien

free, manipulations were done under a sterile hud (UV-light sterilized) over an
open gas flame.
Experiments with whole cells carried out in Vienna used algal cells from the
same strain as in Pisa and were kept at room temperature and under natural lighting.
On average every 4 months a new incubation was carried out. Experiments
investigating cell parts were using a cell part solution prepared and kindly provided
by Laura Barsanti of the Gualtieri group.
Figure 5.1: Receptacles of 4 to 5 days old algal cultures of Euglena gracilis
under constant illumination.
5.1.2 Cell Parts Solution
The following protocol resumes the steps to be followed to obtain the solution of cell
parts that was used for the experiments. This protocol aimed at the extraction and
purification of intact photoreceptor proteins in the first place, but was discovered to
be also useful as starting point for AFM investigation of crystalline cell parts. The
protocol attempts to clean the solution by either removing or dissolving unwanted
skeletal features (like pellicle parts) and membrane tissue as far as possible.
Cell Collection
For the cell collection 4-5 days old cultures are used. The cells, contained in their
culture medium at a concentration of 7 − 8 ∗ 10 6 cells/ml, are harvested at low
centrifugal force. Then the cells are resuspended in 750 ml of a 25 mM sodium
acetate solution (ph 7.0) and cooled to 4 ◦ C in an ice bath.
66