## 6 Detection, Identification, and Differentiation Tools for Mycobacteria
Another culture-dependent method that has been applied to the identification of mycobacteria is matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS). For this purpose, bacterial extracts or intact microorganisms are used which, in the particular case of mycobacteria, require the inactivation and disruption of the mycolic acid-rich cell wall [324]. Those molecules are ionized, transferred into a gas phase and the resulting ions are separated according to their mass/charge ratio [324]. Such extracts produce different mass spectra that can be used for identification [324]. This technique was initially developed targeting the identification of members of MTC for the clinical approach but has more recently been expanded to the identification of NTM [324; 325; 326], while databases for spectra analyses are growing. The mycobacterial cell wall is a critical structure for mycobacteria that has also been exploited for diagnostic purposes. Multiple chromatographic techniques have been employed since the 1980s aiming the analysis of mycolic acids to identify patterns characteristic of each _Mycobacterium_ species [327; 328; 329], switching from thin-layer chromatography [327; 328] to high-performance liquid chromatography (HPLC) [329]. Fluorescence in situ hybridization (FISH) assays designed for mycobacteria target mostly the 165 rRNA or Internal Transcribed Spacer (ITS) genes with both DNA and peptide nucleic acid (PNA) probes [330]. Most reported FISH assays target MTC and/or NTM in the context of clinical specimens from sputum samples [330]. The use of the MN Genus-MTBC FISH commercial kit, which contains a probe for an internal 165 rRNA sequence that the _Mycobacterium_ genus shares with _Nocardi_ genus, and an MTC-specific probe that is complementary to a 235 rRNA 5' sequence, has been evaluated. [PERSON] and coworkers (2017) reported the correct identification of 44 reference _Mycobacterium_ species using this approach, except for _M. wolinskyi_, confirming that the panel of six strains belonging to the MTC specifically reacted to the MTC-specific probe [331]. In the same work, while testing the MTBC-MAC FISH commercial kit, only the six MTC members among the 44 reference species reacted to the MTC-specific probe, and the 14 reference MAC members reacted to MAC-specific probe [331]. PNA probes have been developed for MTC members as well as for NTM, in which a sensitivity of 71.4% and specificity of 100% was obtained in clinical specimens [332]. In another study, a specific PNA probe targeting the 165 rRNA of MAA hybridized with MAP besides its initial target, being successful in MAA identification in both biofilm and potable-water samples, however, the struggle around background fluorescence was also reported [333]. Most emerging techniques (e.g., MALDI-TOF MS, FISH, or flow cytometry) are culture-dependent and do not minimize mycobacteria isolation issues. Flow cytometry is a widely used technique in the context of physiology and immune response studies, usually not focused on environmental matrices which present an inherent complexity that barrier the development of protocols for detection and identification of mycobacteria. It has been applied to mycobacteria, essentially restricted to research works mostly focused on the immune response [334], antibiotic resistance, and physiology [335]. By using high specificity probes, similar to those used for FISH, it could provide the advantage to simultaneously identify _Mycobacterium_ species and, if fixation processes are not used, then it might provide the opportunity to evaluate physiological parameters, like cell wall integrity or metabolic activity. Moreover, the FISH technique coupled with flow cytometry allows a high-throughput characterization on a single-cell level based on the fluorescence resulting from in situ hybridization, allowing the combination of both taxonomic identification and rapid and correct quantification of microbial communities.