Yeast extract

Staphylococcus aureus (S. aureus, DSM 799) was obtained
from the German
Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig,
Germany). Aggregatibacter actinomycetemcomitans (A. ac, MCCM 2474) was obtained
from the Microbial Culture Collection Marburg (Marburg, Germany).
The bacteria were stored at −80 °C as glycerol stocks
and precultured for every experiment. S. aureus was cultivated in tryptone soy broth (Oxoid Limited, Hampshire,
UK) supplemented with 10% yeast extract (Carl Roth GmbH + Co. KG,
Karlsruhe, Germany) continuously shaken under aerobic conditions at
37 °C for 16 h. A. ac was cultivated
in Todd Hewitt broth (Oxoid Limited) supplemented with 10% yeast extract
continuously shaken under microaerophilic conditions at 37 °C
for 24 h.

To analyze bacterial attachment and biofilm formation on nanotopographies,
three individual precultures were centrifuged and resuspended in phosphate-buffered
saline. They were adjusted to an optical density at 600 nm of 0.001
or 0.2 for S. aureus or A. ac, respectively. Test specimens (N = 18 per structure and strain) were incubated with bacterial suspensions
using each preculture in triplicates for 5 h at 37 °C and with
continuous shaking under aerobic (in the case of S.
aureus) or microaerophilic (5% CO₂, in
the case of A. ac) conditions. After
this initial attachment, half of the specimens (N = 9 per structure and strain) were processed for microscopy as described
below.
On the other specimens, the bacterial suspension was
removed and replaced with fresh medium: tryptone soy broth supplemented
with 10% yeast extract and 50 mM glucose (Carl Roth GmbH & Co.
KG) for S. aureus or Schaedler broth
(Oxoid Limited) supplemented with 10 μg/mL vitamin K (Oxoid
Limited) for A. ac. To allow for
biofilm formation of the adhered cells, specimens were further incubated
for a total of 24 h at 37 °C under aerobic conditions and continuous
shaking in the case of S. aureus and
under static microaerophilic conditions (5% CO₂) in the
case of A. ac.