In all participating centres, the MALDI-TOF system used was a Biotyper microflex LT (Bruker Daltonics). The software used was FlexControl v3.0 with the Mycobacteria Library v4.0. The spectra were obtained in the positive linear mode, over a mass/charge (m/z) ratio of 2000–20,000 Da and the accelerating voltage was 20 kV. The samples were measured in automatic mode using a nitrogen laser at 40 shots per second, with a total of 240 laser shots collected per spot. The log(score) cut-offs used were those recommended by the manufacturer: < 1.60 as not reliable identification, 1.60–1.79 as low confidence identification and a log(score) ≥ 1.80 as high confidence identification.
Biotyper microflex lt
Manufactured by Bruker
Sourced in Germany
The Biotyper Microflex LT is a compact and automated MALDI-TOF mass spectrometry system designed for the rapid identification of microorganisms. The system utilizes a robust and reliable Microflextm linear time-of-flight (TOF) mass analyzer to provide reliable and accurate identification of a wide range of bacterial and fungal species.
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7 protocols using biotyper microflex lt
1
MALDI-TOF Mycobacteria Identification Protocol
2
MALDI-TOF Mass Spectrometry Protocol
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A MALDI Biotyper Microflex LT controlled by Compass flexControl software (version 3.4; Bruker Daltonics, Billerica, Massachusetts, USA) was used to generate mass spectra for all isolates. Mass spectra were acquired using automatic mode and default settings (2000 to 20 0000 Da; linear positive mode; 240 laser shots). For each isolate, twelve spectra (four spots of each isolate extracted were measured 3 times) were recorded.
3
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.
4
Bacterial Strain Identification and Isolation for Specificity Testing
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Bacterial strains used for specificity testing (Table 2; Table 3) were cultured on suitable agar media and incubated at appropriate temperatures and durations. Species verification was performed with MALDI‐TOF (Biotyper Microflex LT; Bruker Daltonics). Isolates of uncertain taxonomic status were classified and confirmed as non‐Y. ruckeri by whole‐genome‐based analyses (Figure S1 ) as described previously (Riborg et al., 2022 (link)).For spiking experiments, Y. ruckeri CC1 strain NVI‐10705 was cultured in Tryptic Soy Broth at 22°C with shaking until mid‐log phase, from which a dilution series was prepared with sterile phosphate‐buffered saline (PBS) chilled on ice and enumerated by plating on 5% bovine blood agar (BA) in triplicate. For the challenge trials, Y. ruckeri NVI‐10705 was grown in Brain Heart Infusion Broth at 15°C with shaking for 20 hr, harvested by centrifugation and re‐suspension in PBS, followed by enumeration on a cell counter (Casy Inovatis; Roche Diagnostics) and by plating of a 10‐fold dilution series on BA. All BA plates were incubated at 22°C for 2 days prior to counting.
5
Rapid Bacterial Identification via MALDI-TOF MS
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Colonies from the replica plates that did not phenotypically resemble S. aureus were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) with a microflex LT Biotyper (Bruker Daltonics, Bremen, Germany) according to manufacturer’s instructions. Briefly, different colonies were applied onto the MALDI-TOF MS target and overlaid with 1 μl matrix solution, which is a saturated solution of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 2.5% trifluoroacetic acid. The target was then air-dried at room temperature and analyzed in the Biotyper. A log score is calculated by the Biotyper software by comparing data of the unknown organism with data of reference organisms for identification of the unknown organism (log score ≥ 2—reliable species identification (ID); log score > 1.7 < 2 reliable genus ID; log score < 1.7 no reliable ID).
6
Quantification and Identification of cGMP by HPLC and MALDI-TOF-MS
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The fraction corresponding to the target signal at 10.8 min, observed by HPLC, was collected employing a fraction’s collector and analyzed by MALDI-TOF-MS (matrix assisted laser desorption ionization-time of flight mass spectrometry) from Bruker Microflex LT Biotyper (Bruker, Bremen, Germany). For the analysis, 1 μL of the fraction collected at 10.8 min retention time was spotted onto a polished steel target plate, air-dried at room temperature, and overlaid with 1 μL of matrix solution (alpha-cyano-4-hydroxycinnamic acid, diluted in 50% acetonitrile and 2.5% trifluoroacetic acid, followed by air-drying). The mass spectrum for cGMP was analyzed using Flex Control software to verify its presence, based on its molecular weight.
Additionally, qualitative identification of cGMP was performed with Immunostick cGMP visual assay to selected samples of interest. To identify the presence of cGMP in each sample’s fraction, an immunochromatographic strip (OPERON S.A.), containing monoclonal antibodies specific for cGMP, was introduced into the collected fraction, following the manufacturer’s specifications.
Additionally, qualitative identification of cGMP was performed with Immunostick cGMP visual assay to selected samples of interest. To identify the presence of cGMP in each sample’s fraction, an immunochromatographic strip (OPERON S.A.), containing monoclonal antibodies specific for cGMP, was introduced into the collected fraction, following the manufacturer’s specifications.
7
Optimizing Agar Concentration for AST
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First, an optimum agar concentration was determined using four strains: P. aeruginosa ATCC 27853, a colistin-susceptible Acinetobacter calcoaceticus-baumannii complex (ACB) strain, a colistin-resistant ACB strain, and mcr-1-harboring Klebsiella aerogenes. Assays were run in triplicate. Species were identified using a Microflex LT Biotyper (Bruker Daltonics, Leipzig, Germany). MHA was modified by reducing the agar granule concentration from 100% (17 g/L) to 30% (5.1 g/L) of the concentration in commercial MHA with 10% intervals (Becton, Dickinson, & Company, Sparks, MD, USA). The optimum concentration was determined based on the least agar concentration that was manageable in the laboratory. Lower the agar concentration, more fragile is the AST determination. The final agar concentration was reduced to 30%.
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