Protocol for comparing ribosomal levels in single bacterial cells at different growth stages using rRNA-FISH

Ribosome quantification in single cells is typically achieved through fluorescence tagging of ribosomal proteins. Here, we present a protocol for comparing ribosomal levels in bacteria at different growth stages using fluorescence in situ hybridization of rRNA (rRNA-FISH), eliminating the need for genetic engineering of the strain of interest. We detail the steps for preparing bacterial samples, staining with fluorescent probes, and acquiring data using flow cytometry and microscopy. Furthermore, we provide guidelines on controlling for proper labeling through signal localization analysis. For complete details on the use and execution of this protocol, please refer to Ciolli Mattioli et al.¹.