Abstract:[Objective] To develop a stable and controllable method for isolating and cultivating the eukaryotic microorganism Entodinium caudatum from the rumen of ruminants, which would lay a foundation for the germplasm banking and provide sufficient experimental materials for researching the physiological function of rumen ciliates. [Methods] First, a rumen cannula was used to collect rumen fluid from cows in Wuhan, and E. caudatum was gradually enriched and isolated from the rumen fluid by micro-strainer filtration through different sizes of nylon mesh. Subsequently, the enriched E. caudatum was cultured in anaerobic culture bottles loaded with the modified SP medium. After purification, a single culture of the strain was established. The species was identified by morphological observation and the phylogenetic analysis based on the 18S rRNA gene. Finally, the generation time of the E. caudatum was calculated based on the half-transfer cultivation method. [Results] A single culture of E. caudatum was isolated from the rumen fluid of Holstein cows. The modified SP medium used in this study was composed of SP salt solution, supernatant of the rumen fluid without protozoa, cysteine hydrochloride, antibiotics, starch, and grass powder. In the modified SP medium, the obtained culture grew and proliferated steadily, reaching the highest density of 37 000 cells/mL on day 16 from the initial inoculation density of 320 cells/mL. Morphological features and molecular data indicated that the culture obtained in this study was E. caudatum, named E. caudatum strain WH. The generation time of E. caudatum strain WH was 19.0 h. [Conclusion] This study has successfully developed an in vitro cultivation method for E. caudatum strain WH, a prevalent eukaryotic microorganism from the rumen of ruminants. This work lays a technical and theoretical basis for the germplasm banking and in-depth research on the physiological functions of rumen ciliates.
