Mtp 96 maldi tof ms target plate

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.

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).

In the context of the rebirth of culture in microbiology, our sample was cultured using the 18 culture conditions of culturomics [17] (link), [18] (link). Colonies were obtained by spreading samples on petri dishes. They were then purified by subculture and identified by MALDI-TOF MS [19] (link), [20] (link). Some colonies were put down in duplicate on a MTP 96 MALDI-TOF MS target plate (Bruker Daltonics, Leipzig, Germany), which was analyzed 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 2.0 software (Bruker). An identification score of >1.9 with a validated species allows identification at the species level, and a score of <1.7 does not enable identification. When identification by MALDI-TOF MS failed, the 16S rRNA was sequenced [21] (link). Stackebrandt and Ebers [22] suggest similarity levels of 98.7% of the 16S rRNA sequence as a threshold to define a new species without performing DNA-DNA hybridization.