The questions raised by the direct measurement of microorganisms on solid culture media at different development stages were already addressed in earlier works. In 2000, Maquelin et al.3 demonstrated for the first time the possibility to discriminate at the species level between four species by direct measurement of Raman spectra from 6-h old microcolonies on the solid culture medium. The possibility to cluster the data in definite classes based on microcolonies’ Raman spectra was demonstrated through hierarchical clustering analysis (HCA). In 2002, Maquelin et al.4 conducted a full classification study on 6-h old microcolonies of five yeasts (42 strains) belonging to the Candida genus, demonstrating the possibility to identify at the species level with a high prediction accuracy ranging from 97% to 100%. Classification was performed using a rather complex methodology based on the use of four linear discriminant analyses (LDA), each based on a separate model, requiring to run a posteriori an HCA. An orthogonalization procedure had been used to correct for medium and water contributions. This step, deemed necessary by the author, requires 60 min of acquisition time on bare medium, presumably a one-time procedure for a given medium. A 97% average correct identification level was reported. In 2003, Maquelin et al.5 conducted a more extensive study, building a larger reference database of bacteria and yeasts commonly detected in bloodstream infections, collected from positive hemocultures and grown 6 to 8 h on the solid culture medium and then directly analyzed by Raman spectroscopy. This study resulted in a correct classification level of 92.2%, after the analysis of 115 strains grouped in 11 classes (some of those classes included more than one species and 17 strains were excluded from the comparison because the phenotypic identification yielded a species not included in the database). Lowest identification rates were observed at 80% for Enterobacter aerogenes and Enterobacter cloacae. The total acquisition time reported per sample (50 spectra per sample for 10 replicates from five colonies) was 25 min not including the orthogonalization step. All data processing was conducted on first derivative spectra, as a way to remove the fluorescence background, and the classification analysis was conducted according to a leave-one-strain-out cross-validation method, what we call a “stringent” mode. In the three studies mentioned above, a procedure6 based on vector algebra was used to subtract unwanted signals from the medium (although a highly confocal optical setup was being used to minimize signal contribution originating from the culture medium and variations in water content).