Single bacteria identification by Raman spectroscopy.

We report on rapid identification of single bacteria using a low-cost, compact, Raman spectroscope. We demonstrate that a 60-s procedure is sufficient to acquire a comprehensive Raman spectrum in the range of 600 to 3300 cm⁻¹. This time includes localization of small bacteria aggregates, alignment on a single individual, and spontaneous Raman scattering signal collection. Fast localization of small bacteria aggregates, typically composed of less than a dozen individuals, is achieved by lensfree imaging over a large field of view of 24 mm². The lensfree image also allows precise alignment of a single bacteria with the probing beam without the need for a standard microscope. Raman scattered light from a 34-mW continuous laser at 532 nm was fed to a customized spectrometer (prototype Tornado Spectral Systems). Owing to the high light throughput of this spectrometer, integration times as low as 10 s were found acceptable. We have recorded a total of 1200 spectra over seven bacterial species. Using this database and an optimized preprocessing, classification rates of ~90% were obtained. The speed and sensitivity of our Raman spectrometer pave the way for high-throughput and nondestructive real-time bacteria identification assays. This compact and low-cost technology can benefit biomedical, clinical diagnostic, and environmental applications.

Direct identification of clinically relevant bacterial and yeast microcolonies and macrocolonies on solid culture media by Raman spectroscopy.

Decreasing turnaround time is a paramount objective in clinical diagnosis. We evaluated the discrimination power of Raman spectroscopy when analyzing colonies from 80 strains belonging to nine bacterial and one yeast species directly on solid culture medium after 24-h (macrocolonies) and 6-h (microcolonies) incubation. This approach, that minimizes sample preparation and culture time, would allow resuming culture after identification to perform downstream antibiotic susceptibility testing. Correct identification rates measured for macrocolonies and microcolonies reached 94.1% and 91.5%, respectively, in a leave-one-strain-out cross-validation mode without any correction for possible medium interference. Large spectral differences were observed between macrocolonies and microcolonies, that were attributed to true biological differences. Our results, conducted on a very diversified panel of species and strains, were obtained by using simple and robust sample preparation and preprocessing procedures, while still confirming published results obtained by using more complex elaborated protocols. Instrumentation is simplified by the use of 532-nm laser excitation yielding a Raman signal in the visible range. It is, to our knowledge, the first side-by-side full classification study of microorganisms in the exponential and stationary phases confirming the excellent performance of Raman spectroscopy for early species-level identification of microorganisms directly from an agar culture.