Raman spectroscopy is an emerging method for the identification of bacteria. Nevertheless, a lot of different parameters need to be considered to establish a reliable database capable of identifying real-world samples such as medical or environmental probes. In this review, the establishment of such reliable databases with the proper design in microbiological Raman studies is demonstrated, shining a light into all the parts that require attention. Aspects such as the strain selection, sample preparation and isolation requirements, the phenotypic influence, measurement strategies, as well as the statistical approaches for discrimination of bacteria, are presented. Furthermore, the influence of these aspects on spectra quality, result accuracy, and read-out are discussed. The aim of this review is to serve as a guide for the design of microbiological Raman studies that can support the establishment of this method in different fields.
Keywords: Raman spectroscopy; bacteria; machine learning; sample isolation; single-cell analysis.