Flask cultures.
The first assays were made in 50 mL conical flasks. After 24h of incubation at 37º C, the bottom of the flask was scrapped off with a spatula to extract the biofilm. The bacterial suspension and the biofilm extracted was put in several tubes and centrifuged at 3000 rpm for 10 min, and the supernatant was discarded. After this 2mL of water was added to each tube and the tubes were vortexed. The content of the tubes were gathered in a single tube. The tube was centrifuged again at 3000 rpm and the liquid phase was eliminated. To the bacterial precipitate, 0.5g of glass beads 1 mm i.d. (Bio Spec, #11079110) were added. The sample was cooled to -4ºC and then 1mL of extracting solution, N,N-dimethylacetamide (Fluka) +5% LiCl, (Panreac) was added.The tube was vortexed for 2 min at shorts intervals until a complete dispersal of the biomaterial was observed; then, 1 mL of distilled water was added and the tube was centrifuged at 6000g. The supernatant was passed through a 0.45µ filter (Millipore) to eleminate any bacterial residue. This was the sample analyzed by chromatography.
Tube cultures.
The strain S. epidermidis ATCC35984 was grown in 5 mL of trypticase soy broth during 24h at 37º C. Five tubes were previously supplemented with allicin, and the other five were left as control.
After the incubation period, the tubes were centrifuged at 3500 rpm and the supernatant was discarded. The tubes were washed with 5 mL of water, and the washing process was repeated. Glass beads (1mm i.d.), 0.8 g were put in each tube. Two mL of extractant solution were added to the first tube and it was vortexed to dispersal the biofilm. Then the suspension was poured into a second tube and vortexed again. The extract was passed to a third tube and the process was repeated until the final extract remained in the fifth tube, then 1mL of water was added. The solution was centrifuged and passed through a 0.45µ filter before the chromatographic separation. The same procedure was followed for the control cultures.
