The identification of the bacterial and fungal colonies was carried out using matrix-assisted laser desorption ionization time-of-flight mass spectrometry MALDI-TOF–MS as previously described23 (link),24 (link). For bacteria, each colony was deposited in duplicate onto a MALDI-TOF MSP 96 target plate (Bruker Daltonics, Leipzig, Germany), and 2 μL of matrix solution (saturated solution of alpha-cyano-4- hydroxycinnamic acid in 50% acetonitrile and 2.5% trifluoroacetic acid) was added to each spot and allowed to dry for 5 min and then analysed by Microflex spectrometer (Bruker Daltonics) using the software MALDI BioTyper 3.0 (Bruker Daltonics). For the identification of fungi, each colony was incubated in 1 mL of 70% ethanol for 10 min and then centrifuged at 1300g for 5 min. The pellet was treated with 20 μL of acetonitrile and formic acid (v.v) at 100% and 70%, respectively. This mixture was then centrifuged at 1300g for 5 min, and 1.5 μL of the supernatant was deposited on a MALDI-TOF–MS target and allowed to dry before 1.5 μL of matrix was deposited on each spot, allowed to dry for 5 min and then analysed by par Microflex spectrometer (Bruker Daltonics) using the software MALDI BioTyper 3.0 (Bruker Daltonics).
Microflex spectrometer
Manufactured by Bruker
Sourced in Germany
The Microflex spectrometer is a compact, high-performance mass spectrometer designed for a wide range of analytical applications. It utilizes matrix-assisted laser desorption/ionization (MALDI) technology to provide accurate mass determination and identification of a variety of sample types, including proteins, peptides, and small molecules. The Microflex spectrometer offers robust performance, ease of use, and reliability for laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Lc 20at pump, by Shimadzu (7 mentions)
6230 tof lc ms spectrometer, by Agilent Technologies (7 mentions)
Sil 20a autosampler, by Shimadzu (7 mentions)
Analyzer feed system, by Merck Group (7 mentions)
Cbm 20a communications bus module, by Shimadzu (7 mentions)
Maldi biotyper software, by Bruker (6 mentions)
Mtp 96 maldi tof target plate, by Bruker (5 mentions)
Spd 20a, by Shimadzu (5 mentions)
Mtp 96 maldi tof ms target plate, by Bruker (4 mentions)
Columbia agar, by bioMérieux (4 mentions)
Maldi biotyper 3, by Bruker (4 mentions)
Maldi biotyper database software 2, by Bruker (3 mentions)
Colombia agar, by bioMérieux (3 mentions)
Maldi tof ms, by Bruker (2 mentions)
Maldi biotyper 2, by Bruker (2 mentions)
Cd3od, by Merck Group (2 mentions)
Flexcontrol 3, by Bruker (2 mentions)
Trifluoroacetic acid solution, by Merck Group (1 mentions)
Msp 96 target ground steel, by Bruker (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
39 protocols using microflex spectrometer
1
Bacterial and Fungal Identification by MALDI-TOF MS
2
MALDI-TOF-MS Bacterial Identification Protocol
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The matrix-assisted laser-desorption/ionization time-of-flight mass spectrometer analysis (MALDI-TOF-MS) (Bruker Daltonics, Bremen, Germany) was carried-out as previously described [27] (link). Briefly, a pipette tip was used to pick one isolated bacterial colony from a culture agar plate, and to spread it as a thin film on a MTP 384 MALDI-TOF target plate (Bruker Daltonics, Leipzig, Germany). Twelve distinct deposits were done from twelve different colonies of strain Bisph2. Each smear was overlaid with 2 μl of matrix solution in 50% acetonitrile, 2.5% tri-fluoracetic-acid, and allowed to dry for 5 min. Measurements were performed with a Microflex spectrometer (Bruker). Spectra were recorded in the positive linear mode for the mass range of 2.000 to 20.000 Da. A spectrum was obtained after 675 shots at a variable laser power. The twelve spectra were imported into the MALDI BioTyper software (version 2.0, Bruker) and analyzed by standard pattern matching against the main spectra of 6.213 bacteria including 36 spectra from validly published Enterobacter species that were used as reference data in the BioTyper database. The method of identification includes the m/z 3.000 to 15.000 Da. For every spectrum, 100 peaks at most were taken into account and compared with spectra in the database.
3
MALDI-TOF MS Bacterial Identification
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Bacterial cells were spotted on a sample spot of a MALDI target plate (MSP 96 target, ground steel; Bruker Daltonics, Billerica, MA, USA) and were overlaid with HCCA matrix solution (saturated solution of α-4-cyano-hydroxycinnamic acid; Bruker Daltonics, Billerica, MA, USA) in 50% acetonitrile (Sigma-Aldrich, St. Louis, MO, USA) and 2.5% trifluoroacetic acid solution (Sigma-Aldrich, St. Louis, MO, USA). Mass spectra profiles were acquired using a Microflex spectrometer (Bruker Daltonics, Billerica, MA, USA). The molecular ions were measured automatically in linear positive ion mode with the instrument parameters optimized for a range of 2,000–20,000 m/z. The software packages flexControl 3.0 (Bruker Daltonics, Billerica, MA, USA) and flexAnalysis 3.0 (Bruker Daltonics, Billerica, MA, USA) were used for mass spectra recording and processing. Spectra identification and analysis were carried out using the MALDI Biotyper 3.0 (Bruker Daltonics, Billerica, MA, USA). The identification was performed by comparing the obtained spectra with those in the MALDI Biotyper 3.0 library (version 3.2.1.1).
4
Bacterial Identification via MALDI-TOF MS
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Individual bacterial colonies were collected every day for 4 days, and then each colony was identified by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Microflex Spectrometer; Bruker Daltonics, Bremen, Germany) as previously described77 (link). The obtained MALDI-TOF MS spectra were imported into MALDI Biotyper 3.0 software (Bruker Daltonics) and analysed against the reference bacterial spectral database. The MALDI Biotyper RTC software interprets the results according to predefined values, i.e., values between 2.00 ≤ species identified ≤ 3.00; of 1.70 ≤ probably identified ≤ 1.99 and 0.00 ≤ no identification ≤ 1.69. The unidentified colonies (with values from 0.00 to 1.99) were sequenced using the complete 16S rRNA gene.
5
Culturomics and MALDI-TOF MS for Bacterial Identification
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The sample was cultured using the 18 culture conditions of culturomics [20] (link). The colonies were obtained by seeding on solid medium, purified by subculture and identified using MALDI-TOF MS [18] (link), [21] (link). Colonies were deposited in duplicate on a MTP 96 MALDI-TOF MS target plate (Bruker Daltonics, Leipzig, Germany), which was analysed with a Microflex spectrometer (Bruker). The 12 spectra obtained were matched against the references of the 7567 bacteria contained in the database by standard pattern matching (with default parameter settings), with MALDI BioTyper database software 2.0 (Bruker). An identification score over 1.9 with a validated species allows identification at the species level, and a score under 1.7 does not enable any identification. When identification by MALDI-TOF MS failed, the 16S rRNA was sequenced [22] (link). Stackebrandt and Ebers [23] suggest similarity levels of 98.7% and 95% of the 16s rRNA sequence as a threshold to define, respectively, a new species and a new genus without performing DNA-DNA hybridization.
6
Identification of Bacterial Colonies via MALDI-TOF
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Identification of colonies was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) Microflex spectrometer (Bruker Daltonics, Leipzig, Germany) as previously described, and the spectrum was compared with our database (which includes the Bruker database and our own collection) [10] (link), [12] (link). In case of nonidentification, i.e. if the spectrum did not find a match in the database (score <1.7), we proceeded to spectrum verification. If the spectrum was without background noise, and after exclusion of culture contamination, 16S rRNA was sequenced as previously described [10] (link). In case of a sequence similarity value lower than 96%, the species is considered to be a new genera without performing DNA-DNA hybridization, as suggested by Stackebrandt and Ebers [13] .
7
Isolation and Identification of Skin Microbiome
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Samples were obtained by swabbing a 10 cm2 area of skin from the forehead and hands of volunteer healthy women. The samples were collected using sterile swabs soaked in the Culture Top® transport medium (C-top Ae-Ana, Eurobio, Les Ulis, France). The study was validated by the ethics committee Sud-Est IV (reference ID-RCB: 2019-A01508-49). Each sample mixed with the transport media was serially diluted, and 50 µL of each dilution was incubated under aerobic conditions at 31 °C after being seeded (DS) in Columbia Agar (bioMérieux, Marcy l’Etoile, France). To identify the strains, a Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry protein analysis was carried out in triplicate using a Microflex spectrometer (Bruker Daltonics, Bremen, Germany). Whole genome sequencing (WGS) was performed as described below. The strain spectra were imported into the MALDI BioTyper software (version 3.0, Bruker, Bremen, Germany) and analyzed using standard pattern matching with the default parameters. Our database (https://www.mediterranee-infection.com/acces-ressources/base-de-donnees/urms-data-base/ , accessed on 4 September 2023) was then incremented with the spectra of this new bacterial species.
8
Bacterial Identification using MALDI-TOF MS
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Bacterial species were directly identified from each bacterial colony using matrix-assisted laser desorption ionization-time mass spectrometry (MALDI-TOF MS) (Bruker Daltonics, Bremen, Germany) [58 (link)] following the same protocol as previously described by Seng et al. [59 (link)] with a Microflex spectrometer (Bruker Daltonics, Leipzig, Germany). A score of >2 allowed identification at the species level, and a score of <1.7 was considered as insufficient for the identification at the species level. In this case, 16S rRNA gene was amplified and the amplicon was sequenced. Briefly, DNA extraction was performed using EZ1 DNA kits (Qiagen, Courtaboeuf, France), according to the manufacturer’s protocol. Amplification and sequencing were performed as described in the study by Dahmana et al. [60 (link)] using 16S universal primers [61 (link)]. The obtained electropherograms were assembled and edited using the ChromasPro1.7.7 software (Technelysium Pty Ltd., Tewantin, Australia), and then the sequences obtained were compared with those available in the GenBank database by NCBI BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi ).
9
Culturomics Reveals Fecal Bacterial Diversity
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In order to explore as exhaustively as possible the bacterial diversity of the faecal sample, the culturomics concept was used to culture this sample using 18 culture conditions [8] (link). The purified colonies obtained were identified using MALDI-TOF MS as described previously [13] (link), [14] (link). Colonies were deposited on a MTP 96 MALDI-TOF MS target plate (Bruker Daltonics, Leipzig, Germany), which was analysed with a Microflex spectrometer (Bruker Daltonics). The spectra obtained were matched against the references of the 7567 bacteria contained in the database by standard pattern matching (with default parameter settings) with MALDI BioTyper database software 2.0 (Bruker Daltonics). An identification score over 1.9 with a validated species allowed the identification at the species level, and a score under 1.7 did not enable any identification. The 16S rRNA gene was amplified and sequenced as previously described [15] (link). The obtained 16S rRNA sequence was compared to those in GenBank (http://blast.ncbi.nlm.nih.gov.gate1.inist.fr/Blast.cgi ) to determine the percentage of sequence similarity with the closest bacteria. A new species or genus was defined by a similarity level of the 16S rRNA sequence under 98.65% or 95% respectively [16] (link).
10
Culturomics-based Bacterial Identification Using MALDI-TOF
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Using the 18 culture conditions of the culturomics concept, the fecal sample was cultivated, and the obtained colonies were identified by MALDI-TOF as described below [5] (link). Proteomic analysis of our strain was carried out with MALDI-TOF as previously described [10] (link), [11] (link). A Microflex spectrometer (Bruker Daltonics, Leipzig, Germany) was used with a MTP 96 MALDI-TOF target plate (Bruker) on which 12 individual colonies were deposited. Twelve spectra were thus obtained, imported into MALDI BioTyper 2.0 software (Bruker) and analysed by standard pattern matching (with default parameter settings) against the main spectra of 7567 bacteria. Comparison with the BioTyper database spectra enabled the identification and discrimination of the analysed species from those in the database in accordance with the obtained score: a score >2 with a validated species enabled the identification at the species level, and a score <1.7 did not enable any identification. After a failed identification of the colony with a clean spectrum, it was identified by sequencing the 16S ribosomal RNA as previously described [18] (link). A threshold of 98.7% similarity level was determined to define a new species without performing DNA-DNA hybridization [19] .
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