Paramecium bursaria is a ciliate species that has a symbiotic relationship with Chlorella spp. This study aimed to elucidate the dynamics of digestive vacuole (DV) differentiation in P. bursaria, using yeast stained with a pH indicator. Previously, DV differentiation in P. bursaria has been classified into eight periods based on fixed-cell observations. However, to understand the behavior and physiology of P. bursaria in its natural state, it is essential to observe living cells. This study presented a novel method using Cornig® Cell-Tak™ to immobilize living P. bursaria cells, which enabled long-term observation of the same cell from the same direction. This technique allowed for real-time observation of DV differentiation, including the relationship between changes in the internal pH of DV and the diameter of DV, yeast budding from the DV membrane by a single cell into the cytoplasm, and separation of a DV containing multiple yeasts into two DVs. This study provides new insights into the dynamic process of DV differentiation in P. bursaria. These findings contribute to a better understanding of the cellular mechanisms underlying the symbiotic relationship between the two organisms and shed light on the complex process of intracellular digestion in ciliates.