Visualizing the Growth and Division of Rat Gut Bacteria by D-Amino Acid-Based Labeling and FISH Staining

Rat is a widely used mammalian model for gut microbiota research. However, due to the difficulties of individual culture of many of the gut bacteria, much information about the microbial behaviors in the rat gut remains largely unknown. Here, to characterize the growth and division of rat gut bacteria, we apply a chemical strategy that integrates the use of sequential tagging with D-amino acid-based metabolic probes (STAMP) with fluorescence hybridization (FISH) to rat gut microbiota. Following sequential gavages of two different fluorescent D-amino acid probes to rats, the resulting dually labeled gut bacteria provides chronological information of their cell wall synthesis. After taxonomical labeling with FISH probes, most of which are newly designed in this study, we successfully identify the growth patterns of 15 bacterial species, including two that have not been cultured separately in the laboratory. Furthermore, using our labeling protocol, we record cells growing at different growth stages of a complete cell division cycle, which offers a new scope for understanding basic microbial activities in the gut of mammalian hosts.