Universal Fluorescence Light-Up Gram-Staining Technique for Living Bacterial Differentiation

Traditional Gram staining is a standard method used to differentiate Gram-positive (G+) and Gram-negative (G–) bacteria, but this approach contains multiple steps, which require considerable expertise and practice. Furthermore, Gram staining is incompatible with living cells. Based on the cell surface components of pathogenic bacteria, here, we developed a proof-of-concept fluorescence light-up Gram-staining technique using two bio-orthogonal fluorescence turn-on probes TPPB and BPTZ. This Gram-staining technique can selectively label G– bacteria by TPPB, while BPTZ can label both G+ and G– bacteria, offering a clear differentiation of the living G+ and G– bacterial populations with high selectivity and sensitivity. Our approach offers a resourceful tool for specific discrimination of living G+ and G– bacteria in both fundamental and clinical bacteria detection.