genomewide characterization of host-pathogen interactions by ...

Maren Depke
Material and Methods
PATHOGEN GENE EXPRESSION PROFILING
Growth Media Comparison Study
Bacterial cultivation, growth media, and sampling time points
Staphylococcus aureus RN1HG was grown at 37°C with orbital shaking of 200 rpm in
Erlenmeyer bacterial culture flasks. Culture volume did not exceed 1/5 of culture flask volume.
Optical density (OD) was measured at 600 nm (Fig. M.5.1).
Different media were included in this study in an international co-operation in the settings of the
EU-IP-FP6-project BaSysBio (LSHG-CT2006-037469) consortium, e. g. the complete bacterial
culture medium TSB, cell culture medium, minimal medium, and human serum. Here, only results
from samples of the medium “pMEM” will be presented. The contents of the adapted cell culture
medium pMEM (Schmidt et al. 2010) have already been listed above (see Material and
Methods/Host Cell Gene Expression Pattern in an in vitro Infection Model/Bacterial growth
medium, page 51).
Bacterial samples were taken in the exponential growth phase and 2 h (t 2 ) and 4 h (t 4 ) after
entry into stationary growth.
OD600
10.00
1.00
0.10
TSB
pMEM
Fig. M.5.1:
Example for bacterial growth in TSB and pMEM medium.
0.01
0 2 4 6 8 10 12 14 16 18 20 22 24
time / h
Bacterial cell harvest and disruption
At different growth phases, 5 to 15 optical density units of S. aureus RN1HG were harvested
on ice with addition of at least one third volume of Killing Buffer (20 mM Tris pH 7.5, 5 mM
MgCl 2 , 20 mM NaN 3 ). Pellets were flash-frozen in liquid nitrogen and stored at −70°C until cell
disruption.
For cell disruption, the pellet was resuspended on ice in 200 µl Killing Buffer, transferred to
liquid nitrogen cooled Teflon vessels, and disintegrated mechanically at 2600 rpm for 2 min in a
bead mill (Mikrodismembrator S, B. Braun Biotech International GmbH, Melsungen, Germany;
now part of Sartorius AG, Göttingen, Germany). Frozen cell and buffer powder mix was
resuspendend in 4 ml of 50°C pre-warmed lysis solution (4 M guanidine-thiocyanate, 25 mM
sodium acetate pH 5.5, 0.5 % N-lauroylsarcosinate) until the solution appeared clear and
homogeneous. Four aliquots with 1 ml each were intermittently frozen in liquid nitrogen and
stored −70°C.
55