Rapid expansion of the probiotics industry created a substantial need for fast, sensitive, comprehensive, and low-cost QC approaches. Here, we introduce a culture-free strategy combining phenotype and genotype measurements at single-cell resolution called Single-Cell Identification, Viability and Vitality tests, and Source-tracking (SCIVVS). The fingerprint region of D2O-probed single-cell Raman spectrum (SCRS) enables species-level identification of probiotic cells with 93% accuracy, with a reference SCRS database from 21 widely-used probiotic species. Simultaneously, the C-D band accurately quantifies viability, metabolic vitality and intercellular heterogeneity. For source tracking, cells of interest can be individually sorted and coupled with downstream whole-genome sequencing, producing genome assemblies from single cells with up to 99.4% genome coverage. Finally, we validated the integrated SCIVVS workflow with automated SCRS acquisition where the entire workflow before sequencing takes 5 hours only. SCIVVS is over 20-fold faster while costing 90% less than conventional probiotics QC protocols, and is capable of one-step, automated measurement of cellular vitality and heterogeneity. SCIVVS provides a new technological and data framework has the potential to become a new
industrial standard.
