Bacterial Profiling: Abstinence Effects

To assess changes in the bacterial profiles following 2 days and 2 h of abstinence, 100 µL of each specimen was inoculated on a selection of sterile agars (blood agar base no. 2, malt extract agar, trypticase soy agar; Merck, Darmstadt, Germany) and incubated under aerobic conditions at 36 ± 2 °C for 24 h. Grown bacterial colonies were counted and re-inoculated and incubated again under aerobic conditions at 37 ± 1 °C for 24 h [22 (link)].
Pure bacterial colonies were identified with the matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) Biotyper mass spectrometry (Brucker Daltonics, Bremen, Germany), previously described by Tvrdá et al. [22 (link)]. Briefly, purified cultures re-suspended in distilled water were treated with 99.8% ethanol (Sigma-Aldrich, St. Louis, MO, USA) and air-dried. The pellet was mixed thoroughly with a solution comprising acetonitrile (Sigma-Aldrich, St. Louis, MO, USA), 70% formic acid (Sigma-Aldrich, St. Louis, MO, USA), centrifuged and transferred to the MALDI identification plate. Dried specimens were covered with a working solution of MALDI matrix and assessed with the Microflex LT instrument and the flexControl software (version 3.4). Obtained data were processed with the MALDI Biotyper Bruker Taxonomy database (Bruker Daltonics, Bremen, Germany) [22 (link)].