Microflex maldi tof ms

After homogenisation, one loop from each sample was spread with triple plating onto tryptic soy agar (TSA) (Oxoid, Hampshire, UK) which contained the azo dye Reactive Black 5 (RB5) reagent (Sigma, Germany) [30 (link)]. RB5 was mixed from its stock solution in the freshly autoclaved TSA to the final concentration 0.1 g/L. One series of plates were incubated under anaerobic conditions on 23 °C and 30 °C for 3 days, and another series of plates were incubated under aerobic conditions. A purple or yellowish discoloration of the dark blue RB5 around the colonies indicated the electrogenic potential of an isolate. Positive isolates were identified with MALDI-TOF MS (Microflex, Bruker Daltonics). For that purpose, candidate colonies were lifted with a sterile toothpick and spread on the instruments’ sample test plate. For analyses, samples were treated with formic acid and analysed in parallel. Results were compared to the databank.

The bacterial strain was identified using conventional methods based on the culture and morphological characters, standard biochemical tests [34 ,35 ], sequencing of a region of the 16S rRNA gene, and the Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry method, performed using MALDI-TOF MS Microflex (Bruker Daltonics, Bremen, Germany), based on the automatic analysis of mass spectra of protein molecules specific for the microorganism species.
Phylogenetic analysis of the strain was performed using the Neighbor-Joining method [36 (link)]. The evolutionary distances were computed using the Maximum Composite Likelihood method [37 (link)] and were in the units of the number of base substitutions per site. Codon positions included were 1st + 2nd + 3rd + Noncoding. All ambiguous positions were removed for each sequence pair (pairwise deletion option). Phylogenetic and molecular evolutionary analyses were conducted using the MEGA software package version X [38 (link)].

This study was conducted in the Region of Skåne in southern Sweden, comprising 1.3 million persons. The region is served by a single microbiological laboratory that has satellite blood culture cabinets (BACTEC FX, Becton Dickinson, Franklin Lakes, USA) in the five largest hospitals, where blood cultures are put into the cabinets at all hours. Five smaller hospitals are without blood culture cabinets. Due to probable and unpredictable delays before vials from these hospitals were incubated, cases from hospitals without blood culture cabinets were not included. Cultures were incubated for a maximum of 120 h. Species identification in positive blood cultures was performed using Microflex MALDI-TOF MS (Bruker Daltronics, Bremen, Germany), with the software FlexControl and MBT Compass 4.1 and the reference database MBT Compass Library DB-7854.

The cephalothorax of the adult mosquitoes was selected for MALDI-TOF MS analysis. Briefly, each mosquito was examined under the stereomicroscope and its cephalothorax was carefully removed from the rest of the body (legs, wings, abdomen), and transferred to a 1.5 ml Eppendorf tube containing 20 μl of 50% formic acid. The cephalothorax was manually grinded with formic acid with the help of a fused tip. A solution containing 60% acetonitrile (ACN) and 0.3% tri-fluoroacetic acid (TFA) obtained from Sigma-Aldrich was prepared, and 7.5 μl of it was added to separate tubes. To this solution, 5 μl of the mosquito homogenate was added and mixed thoroughly. One microliter of this final solution was spotted onto the 96-well stainless steel plate, air dried, and overlaid with 1 μl of HCCA matrix (α-cyano-4-hydroxycinnamic acid) prepared by dissolving the matrix powder into a solution containing 50% ACN, 47.5% molecular-grade water and 2.5% TFA. Once the plate was completely dry, it was inserted into the port of Microflex MALDI-TOF MS (Bruker Daltonik GmbH, Bremen, Germany) to take the reading. Each specimen was spotted onto four different wells to check reproducibility. A blank tube with no mosquito parts in it underwent all the processing steps each time. E. coli ATCC 25922 was also spotted along with each plate to run as a control.

The chemical composition of propolis was determined using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The propolis sample consisted of 50% diluted propolis in methanol covered with 0.1020 M of α-cyano-4-hydroxy cinnamic acid (HCCA) in 1:4 (v/v) H₂O/acetonitrile. A 1.0 µL diluted aliquot of the sample was first spotted on the plate and covered with 2.0 µL of matrix. The mass spectra were obtained using a bench-top Microflex MALDI-TOF MS from Bruker Daltonics^® (Bremen, Germany) equipped with a pulsed nitrogen laser at 355 nm wavelength. The spectra were recorded from 40 to 2000 Da (positive mode), and from 100 to 400 Da (positive mode) using FlexControl 3.3 software (ion source 1: 19 kV; ion source 2: 15.5 kV; lens voltage: 9.45 kV; laser frequency: 60 Hz; pulsed ion extraction (PIE) delay: 120 ns). Other parameters are shown in Table 2. Mass gates of 400 m/z (positive mode) were set for all experiments. Individual mass spectra from each spot were acquired by averaging 350 laser shots. Data acquisition was set to automate, and the “random walk” movement was activated at 10 shots per raster during the sequence. The peak lists and intensities were calculated using the peak-picking centroid algorithm in FlexAnalysis 3.3 software.