Abstract: 【Abstract】 Objective To optimize the laboratory methods for isolation, culture, and preservation of Neisseria gonorrhoeae （N. gonorrhoeae）. Methods Five quality control bacterial strains （N. gonorrhoeae strain 1, N. gonorrhoeae strain 2, Neisseria mucosa strain 3, Enterococcus faecalis strain 4, and mixed bacterial strain 5） were separately cultured using a self-made gonococcal selective medium and 7 commercialized gonococcal selective media （gonococcal isolation plates, gonococcal T-M selective medium, gonococcal chocolate agar plates, gonococcal selective medium, modified Thayer-Martin ［MTM］ agar plates, disposable gonococcus ［GC］ agar medium, and gonococcal agar plates）, and their cultivation performance was evaluated. Sixteen strains of N. gonorrhoeae were cultured both inside and outside a candle jar to screen for CO?-dependent strains. The preservation performance of 4 self-made gonococcal preservation solutions, including calf serum with 10% dimethyl sulfoxide, calf serum with 10% glycerol, brain-heart infusion broth with 20% glycerol, and trypticase soy broth with 30% glycerol, was evaluated. The survival of N. gonorrhoeae in freeze-dried and non-freeze-dried states was observed. Results The growth performance of the 5 quality control strains varied across different commercialized gonococcal culture media. Concretely, N. gonorrhoeae strain 1 formed large and numerous colonies on both the self-made culture medium and MTM agar plates, which outperformed the other 6 culture media; the growth performance of N. gonorrhoeae strain 2 on the 7 commercialized culture media was inferior to that on the self-made culture medium; all 7 commercialized culture media had inhibitory effects on the growth of Neisseria mucosa strain 3, among which the self-made culture medium, gonococcal chocolate agar plates, MTM agar plates, disposable GC agar medium, and gonococcal agar plates could completely inhibit its growth; the gonococcal T-M selective medium could completely inhibit the growth of Enterococcus faecalis strain 4; mixed bacterial strain 5 showed better separation performance on the self-made culture medium, gonococcal isolation plates, gonococcal T-M selective medium, gonococcal chocolate agar plates, MTM agar plates, and disposable GC agar medium. Under CO2-enriched conditions, all 16 strains of N. gonorrhoeae exhibited good growth performance; however, the growth of 11 strains was markedly inhibited without CO2. No significant differences were observed in the preservation performance of the 4 preservation solutions at -70°C, and confluent colonies could be observed in all preservation solutions after 12 months of strain preservation; at -20 ℃, the trypticase soy broth with 30% glycerol showed the best preservation performance, with a few viable strains remaining after 6 months, while the calf serum with 10% dimethyl sulfoxide performed worst, with partial strains remaining viable after 2 weeks. Non-freeze-dried N. gonorrhoeae survived for varying duration at different temperatures （4 ℃, -18 ℃, -29 ℃, -70 ℃, and liquid nitrogen）; no N. gonorrhoeae strains survived by day 4 when stored at 4°C; freeze-dried N. gonorrhoeae remained viable with the presence of confluent colonies for 6 months at 4 ℃. Conclusion The self-made gonococcal selective medium demonstrated superior cultivation and isolation performance compared to commercialized gonococcal selective media; a small amount of CO? could promote the growth of N. gonorrhoeae; ultralow temperature and freeze-drying preservation could increase the survival time of N. gonorrhoeae, with freeze-drying at 4 ℃ being the most cost-effective long-term preservation method.

Key words: Neisseria gonorrhoeae, Isolation culture, CO2-dependent type, Preservation solution, Preservation method