〔Abstract〕 Objective To explore the type and control measure of black dot-like contaminants in cell cultures.Methods The growth state of bacteria was investigated under an inverted microscope; Their morphological characteristics were analyzed by Gram and auramine O staining as well as electron microscopy; 16S rDNA gene sequencing was used to analyze bacterial species; Drug sensitivity test was used to screen antibiotics against the bacteria;Cryopreserved SH-SY5Y cells were resuscitated by cell culture supernatant of RAW264 cells.Results Inverted microscopic real-time observations showed that black dot-like substances had two growth states: static and moving.They were negative for Gram staining while positive for auramine O staining. Electron microscopy revealed that they were short rod-shaped bacteria with a polar flagellum during moving phase. 16S rDNA gene sequencing showed that these bacteria werephenylobacterium zucineum HLK1.Ceftriaxone, carboxycillin and imipenem were screened by drug sensitivity test to have inhibitory effects on the bacteria, but cell culture experiments showed that they could not remove the bacteria from SH-SY5Y cells. Contaminated cells could not be cryopreserved for a long time, but resuscitation with RAW264. 7 cell culture supernatant significantly improved the survival rate of cells.Conclusion The black dot-like contaminants in cell cultures are a special type of oligotrophic bacterium with strong viability that can invade the cells and cannot be cleared with antibiotic treatment. RAW264. 7 cell culture supernatant seems contain some substances against bacteria, and resuscitating frozen cells with RAW264. 7 cell culture supernatant may significantly improve the survival rate of cells.