Selective discrimination and lasting tracking of live bacteria are primary steps for microbiology research and treatment of bacterial infection. However, conventional detection methods, such as the gold standard of Gram staining, are being challenged under actual test conditions. Herein, we provided a novel method, namely, three excitation peaks and single-color emission carbon quantum dots (T-SCQDs) for the rapid (5 min) peptidoglycan-targeting discrimination of Gram-positive bacteria and lasting tracking (24 h) through one-step staining. Bacterial viability testing indicates that T-SCQDs can achieve nondestructive identification of Gram-positive bacteria within 50–500 μg mL–1. Interestingly, the fluorescence imaging system suggests that T-SCQDs can also selectively distinguish the type of colonies based on fluorescence intensity. Furthermore, T-SCQDs were successfully used to visually distinguish Gram-positive bacteria from the microbial environment of A549 cells by confocal fluorescence microscopy. These properties endow T-SCQDs with excellent functions for the diagnosis of infection and other biological applications.

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