Puradisc

Interstitial water samples were extracted by squeezing water out of inner parts of cores using Carver presses (Manheim, 1966 ) with filtering through prewashed 11 μm cellulose filters (Whatman, Cat.-No. 1001090). Water samples were then filtered through 0.45 μm PES syringe filters (GE Puradisc, Cat-No. 6780.2504) during splitting into aliquots. pH, and concentrations of [Mg_(aq)], sulfate, DIC and DOC were determined onboard as described in Fryer et al. (2018a (link)). All data are available online (Fryer et al., 2018b (link),c (link)).

Sau was inoculated into a 6-well microplate and incubated at 37°C with 5% CO₂ for 4 h after which planktonic cells were removed and biofilms were washed once with sterile PBS. THY was added to the washed Sau biofilms and then these were inoculated with ~1 × 10⁶ cfu/ml of the early-log phase inoculum, prepared as described above, or with supernatants, planktonic cells, biofilms or washed bacteria obtained from 4 h cultures of Spn. These inoculants were prepared as follows: ~1 × 10⁶ cfu/ml of the early-log phase inoculum was inoculated into 6-well plates and incubated for 4 h. Planktonic cells were then removed, centrifuged, and washed twice with PBS. The supernatant was separated and filter sterilized using a 0.45 μm syringe filter (Puradisc, GE Healthcare, UK). Biofilms were harvested as mentioned earlier and washed twice with sterile PBS. In another set of wells, biofilms were detached by sonication, then both biofilms and planktonic cells were collected by centrifugation, and the pellet was washed twice with PBS. The same amount (~1 × 10⁶ cfu/ml) of washed bacteria, planktonic cells, or biofilms were inoculated into preformed Sau biofilms; an aliquot of supernatant (100 μl) was inoculated as well. Inoculated and control cultures were incubated for 2 h at 37°C with 5% CO₂ after which bacteria were counted as described.