1.0 mL of bacterial culture was centrifuged at 15.000 rpm for 5 min to pellet the cells. The supernatant was discarded, the cells were washed  in 1 mL of water and twice in 1 mL of 0.1% trifluoroacetic acid (TFA).

After each step, the cells were pelleted by centrifugation at 15.000 rpm for 5 min. The pellet was dissolved by vortexing for 2 min in 50-100 ul of 0.1% TFA.

Finally 0.5 mL of bacteria suspension containing approximatively 106-107 cells/mL were deposited onto a MALDI sample probe, mixed with 0.5 ml of matrix (a-cyano-4-hydroxycynnamic acid 10 mg/mL in 50% ACN, 50% H2O, containing 500 fmol/mL of cytochrome c as internal standard) and dried under ambient conditions. For each bacterial species, three different cultures, growth under the same experimental conditions but in different  days, were analysed.