2.2 Bacterial sampling
In the first half of March 2020, 40 nest boxes (20 at each study site)
were randomly chosen for bacterial sampling. About half of the nest
boxes at each study site had been occupied by one of the two tit species
(great tit or blue tit) in the previous year for breeding, while the
other half had remained empty that year (5 occupied by blue tits, 6 by
great tits and 4 by blue tits, 6 by great tits in the parkland and in
the forest, respectively). Using disinfected latex gloves, an entrance
hole of each nest box was swabbed with circular movement for 30 s with a
sterile cotton swab previously moistened with sterile phosphate-buffered
saline (PBS, pH 7.2; Adlab, Poland). Subsequently, a restricted area of
ca. 2.5 cm2 of an internal rear wall of every nest box
in the middle of its height was swabbed in the same manner. A sample per
entrance hole and another sample per inner wall of each nest box were
taken (in a standardized manner with respect to time and area).