Preparation of the obtained fungal isolates for MALDI-TOF MS identification was performed as previously described [63 (link)], according to Bruker’s recommendations. Identification of the observed fungal genera was performed using a Microflex LT mass spectrometer (Bruker Daltonik, Bremen, Germany) using the MALDI Biotyper software package (version 3.0) with the Filamentous Fungi Library 1.0 (Bruker Daltonik). Each sample’s mass spectrum was compared to the reference mass spectra in the Bruker MALDI-TOF fungal database (Bruker Daltonik), and the arbitrary unit score logarithmic values (ranging between 0 and 3) were determined. The Bruker interpretation criteria were used based on the consistency categories (A (species consistency), B (genus consistency), and C (no consistency)) based on the MALDI-TOF log-score values, which were interpreted as follows: scores ≥ 2.0 confirmed both the genus and species of the fungi (a high species probability was determined at scores ranging between 3.000 and 2.300), scores of ≥1.7 but <2.0 were acceptable for only genus identification, while scores < 1.7 were unreliable.
Maldi biotyper software package
Manufactured by Bruker
Sourced in Germany
The MALDI Biotyper software package is a microorganism identification tool that utilizes matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology. The software analyzes mass spectral fingerprints of microorganisms to provide rapid and reliable identification.
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4 protocols using maldi biotyper software package
1
Fungal Identification by MALDI-TOF MS
2
Anaerobic Isolation and Identification of Clostridium botulinum
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Fifty microliters of TPGY were plated on egg yolk agar (EYA) produced as previously described [22 (link)] and incubated for 48 h at 37 °C in an anaerobic cabinet (Shel Lab, Cornelius, OR, USA) with an atmosphere composed of 5% hydrogen, 5% carbon dioxide and 90% nitrogen. Single colonies with different macroscopic morphology and/or different lipase/lecithinase reactions, were collected and streaked on 2 different plates of Blood Agar Base No.2 (BAB2) (Oxoid, Hampshire, UK) and 2 plates of EYA. One plate for each medium was incubated in aerobic and one in anaerobic conditions at 37 °C for 48 h. Colonies growing only in anaerobic conditions were then identified by MALDI TOF MS (Biotyper Microflex LT, Bruker Daltonics, Bremen, Germany), using the MALDI Biotyper software package (version 3.0, Bruker Daltonics, Bremen, Germany) and an “in house” database created with C. botulinum reference and field strains [23 (link)]. As specified by the manufacturer, a score value of <1.7 indicated that identification was unreliable; scores between 1.7 and 2.0 that identification was reliable at the genus level; scores between 2.0 and 2.3 that it was reliable at the genus level and probable at the species level; scores higher than 2.3 indicated highly probable species identification.
3
MALDI-TOF MS identification of bacterial isolates
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Preparations of bacterial isolates for MALDI-TOF MS measurement were done as previously described (Croxatto et al., 2012 (link)). The on-target extraction preparation method was used to identify all the isolates to establish a standardized rapid routine identification process. Briefly, one fresh colony material was picked and smeared onto a 384 polished steel MSP target (Bruker Daltonik GmbH, Germany) using a toothpick, then one μL of 70% formic acid was added onto the bacterial spot and allowed to air dry afterward add one μL of 10 mg/mL a-cyano-4-hydroxy-cinnamic acid (HCCA, Bruker Daltonik GmbH, Germany) matrix solution (50% acetonitrile; 2.5% trifluoroacetic acid; 47.5% distilled water) and allowed to air dry. The acquisition and analysis of mass spectra were then performed by an Autoflex TOF/TOF mass spectrometer (Bruker Daltonik GmbH, Germany) under the linear positive mode, MALDI Biotyper software package (version Compass) with the reference 7,854 database entries and default parameter settings was used. The Bruker Bacterial Test Standard (BTS) was used for calibration according to the manufacturer’s instructions. Two colony/sample material preparations were analyzed for each isolate, and the higher-scoring identification result was taken.
4
Identification of PGPR Strains by MALDI-TOF MS
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PGPR strains were identified by MALDI-TOF MS according to the procedure developed by Lasch et al. (2008) (link). A wire loop of cell material was picked from Agar plates and transferred into 20 μl of water and mixed with 80 μl of pure TFA (Uvasol grade; Merck, Darmstadt, Germany). The suspension of bacteria was incubated under gentle mixing for 30 min at room temperature until cells were completely solubilized. Then 10 μl of the suspension were diluted 1:10 with bidistilled water adjusting a final TFA-concentration of 8%. Now the samples get turbid, because a part of the solubilized material becomes insoluble and precipitates. Two μl of the turbid suspension were mixed with 2 μl of a saturated solution of the CCA matrix for MALDI-TOF mass spectrometric analysis. Mass spectra were generated in linear mode in the mass range of 1–10 kDa. The effects of 3 × 3000 laser shots were accumulated. Data acquisition and analysis were carried out using the MALDI-Biotyper software package provided by Bruker (Bremen, Germany) and a Matlab-based software (Microbe MS). For MALDI-TOF MS identification analysis, samples were processed, as outlined in Lasch et al. (2008 (link), 2015) (link). For evaluation the obtained mass spectra were smoothed and baseline-corrected.
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