Optimization of laboratory methods for isolation, culture and preservation of Neisseria gonorrhoeae
- VernacularTitle:淋病奈瑟菌实验室分离培养与保存方法的优化研究
- Author:
Xiuqin DAI
1
;
Xiangsheng CHEN
1
;
Yueping YIN
1
Author Information
- Publication Type:Journal Article
- Keywords: Neisseria gonorrhoeae; Isolation culture; CO 2-dependent type; Preservation solution; Preservation method
- From: Chinese Journal of Dermatology 2025;58(9):848-851
- CountryChina
- Language:Chinese
- 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 2-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 CO 2-enriched conditions, all 16 strains of N. gonorrhoeae exhibited good growth performance; however, the growth of 11 strains was markedly inhibited without CO 2. 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 2 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.
