Maldi tof biotyper mass spectrometry

Purified colonies were identified with the help of MALDI-TOF Biotyper mass spectrometry (Brucker Daltonics, Bremen, Germany). Each purified culture was mixed with 300 μL of distilled water and 900 μL of 99.8% ethanol (Centralchem, Bratislava, Slovakia) and subsequently centrifuged at 3200 RPM for 2 min. The pellet was left to dry, subsequently resuspended in 30 μL of 70% formic acid (Sigma-Aldrich, St. Louis, MO, USA), acetonitrile (Sigma-Aldrich, St. Louis, MO, USA), and centrifuged again (3500 RPM, 2 min). One microliter of the supernatant was placed on a 96-point MALDI identification plate and dried [12 (link),19 (link),27 (link)]. The sample was then covered with a working solution of the MALDI matrix composed of acetonitrile, ultrapure water, trifluoroacetic acid and cinnamic acid powder (Sigma-Aldrich, St. Louis, MO, USA), as previously described [27 (link)]. Bacterial identification was performed with the Microflex LT instrument and flexControl software version 3.4. Obtained spectra were compared with the MALDI Biotyper Bruker Taxonomy database (Bruker Daltonics, Bremen, Germany) [25 (link)].

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)].

The isolated and purified bacterial cultures were identified using a MALDI-TOF Biotyper mass spectrometry (Brucker Daltonics, Bremen, Germany).
A small amount of a purified culture was re-suspended in 300 μL of distilled water. Subsequently, 900 μL of 99.8% ethanol (Centralchem, Bratislava, Slovakia) was administered and the mixture was centrifuged (920× g, 2 min, 20 °C). The resulting pellet was allowed to dry freely and then mixed vigorously with 30 μL of 70% formic acid (Sigma-Aldrich, St. Louis, MO, USA) and 30 μL of acetonitrile (Sigma-Aldrich, St. Louis, MO, USA). The samples were subsequently centrifuged at 1096× g at 20 °C for 2 min. Then, 1 μL of the supernatant was placed on the MALDI identification plate, allowed to dry freely and subsequently covered with a working solution of MALDI matrix, composed of acetonitrile, ultrapure water, trifluoroacetic acid, and cinnamic acid (Sigma-Aldrich, St. Louis, MO, USA). Identification of the isolates was carried out with a Microflex LT instrument and the flexControl software version 3.4. The spectra obtained by MALDI-TOF were linked with the MALDI Biotyper Bruker Taxonomy database (Bruker Daltonics, Bremen, Germany) [10 (link)].