The Aliarcobacter isolates from fecal samples were cultivated in Arcobacter broth (Oxoid GmbH, Wesel, Germany) which was supplemented with three different antibiotics (cefoperazone, amphotericin, and teicoplanin (CAT), Oxoid GmbH). The broth was then spread on plates (Mueller–Hinton agar/CAT/5% defibrinated bovine blood, Sifin GmbH, Berlin, Germany). The incubation criteria for each step were: 48–72 h, 30 °C and microaerophilic atmosphere (5% O2, 10% CO2, and 85% N2). Suspicious colonies were further cultivated and then identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as described before [32 (link),33 (link)]. IVD Bacterial Test Standard and Biotyper 3.1 software were used (Bruker Daltonik GmbH, Bremen, Germany). DNA was purified using the High Pure PCR Template Preparation Kit (Roche Diagnostics, Mannheim, Germany) following the manufacturer’s instructions, and the species identification was confirmed with a multiplex PCR assay [34 (link)].
Ivd bacterial test standard
Manufactured by Bruker
Sourced in Germany
The IVD Bacterial Test Standard is a laboratory equipment product offered by Bruker. It is designed to provide a reliable and consistent standard for the testing and evaluation of in vitro diagnostic (IVD) systems used for the detection and identification of bacterial pathogens.
Automatically generated - may contain errors
Lab products found in correlation
Trifluoroacetic acid, by Merck Group (3 mentions)
Biotyper 3, by Bruker (2 mentions)
Sinapinic acid, by Merck Group (2 mentions)
Arcobacter broth, by Thermo Fisher Scientific (1 mentions)
High pure pcr template preparation kit, by Roche (1 mentions)
α cyano 4 hydroxycinnamic acid, by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
Ethanol, by Carl Roth (1 mentions)
Acetonitrile, by Carl Roth (1 mentions)
Mtp 384 target plate polished steel tf, by Bruker (1 mentions)
Ultraflex instrument, by Bruker (1 mentions)
Alpha cyano 4 hydroxycinnamic acid, by Merck Group (1 mentions)
Ammonium bicarbonate, by Merck Group (1 mentions)
Micro 90 concentrated cleaning solution, by Merck Group (1 mentions)
Red phosphorus, by Honeywell (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
4 protocols using ivd bacterial test standard
1
Isolation and Identification of Aliarcobacter from Fecal Samples
2
MALDI-TOF Mass Spectrometry for Bacterial Identification
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Bacteria were cultured from single colonies as described above. They were suspended in 300 μL of water and precipitated with 900 μL of ethanol (96% vol/vol; Carl Roth GmbH, Karlsruhe, Germany). After centrifugation for 5 min at 10,000 × g, the supernatant was removed and the pellet was resuspended in 50 μL of 70% (vol/vol) formic acid (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). Then, 50 μL of aceto-nitrile (Carl Roth GmbH) was added, mixed, and centrifuged for 5 min at 10,000 × g. The supernatant was transferred into a new tube, 1.5 μL of the supernatant was spotted onto an MTP 384 Target Plate Polished Steel TF (Bruker Daltonik GmbH, Bremen, Germany), and air-dried. The dried extract was overlaid with 2 μL of a saturated solution of α-cyano-4-hydroxycinnamic acid (Sigma-Aldrich Chemie GmbH) in a solution of 50% acetonitrile and 2.5% trifluoroacetic acid (Sigma-Aldrich Chemie GmbH). Air-drying at room temperature followed. Spectra were acquired with an Ultraflex instrument (Bruker Daltonik GmbH) in the linear positive mode in the range of 2,000 to 20,000 Da; acceleration voltage was 25 kV. The instrument was calibrated with the IVD Bacterial Test Standard (Bruker Daltonik GmbH). Mass spectra were acquired with 500 shots. Analysis was done with the Biotyper 3.1 software (Bruker Daltonik GmbH).
3
MALDI-TOF Mass Spectrometry Sample Preparation
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Sinapinic acid (SA), alpha-cyano-4-hydroxycinnamic acid (CHCA), trifluoroacetic acid (TFA), and ammonium bicarbonate (ABC) were purchased from Sigma-Aldrich (Steinheim, Germany). Acetonitrile (ACN) was purchased from Penta (Prague, Czech Republic). Water was double distilled using a quartz apparatus from Heraeus Quarzschmelze (Hanau, Germany). IVD bacterial test standard (BTS) was purchased from Bruker Daltonik GmbH (Bremen, Germany).
4
Preparation of Mass Spectrometry Samples
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Sinapinic acid, trifluoroacetic acid, and Micro-90® concentrated cleaning solution were purchased from Sigma-Aldrich (Prague, Czech Republic). Acetonitrile (purity for isotachophoresis) was purchased from Merck (Darmstadt, Germany). Red phosphorus was purchased from Riedel de Haën (Hannover, Germany). Water was double distilled using a quartz apparatus from Heraeus Quarzschmelze (Hanau, Germany). All other reagents were of analytical grade purity. IVD bacterial test standard (BTS) was purchased from Bruker Daltonik GmbH (Bremen, Germany).
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