目的:模拟口腔菌斑生态环境,建立能够对生物膜形成过程连续观察的动态研究模型。方法:自制发酵罐和多出口连续培养室。以变形链球菌、血链球菌、乳酸杆菌、内氏放线菌为实验菌株,将羟磷灰石片、玻片等置于连续培养室中,通过调节温度、氧分压、pH值、清除率、循环流速及代谢底物等参数建立模型。以25mM蔗糖和蒸馏水进行系统测试。观察系统中实验菌CFU计数、pH动态变化等各项反应及不同环境下所形成生物膜的微结构变化。所得数据采用SPSS10.0软件包进行统计学分析,选用单因素方差分析的Dunnet t双侧检验,在α=0.05的情况下比较蔗糖加入组与对照组之间的差异。结果:随着蔗糖的给予,连续培养室中pH表现出相似于天然口腔的致龋环境,蒸馏水组pH值始终维持在7.5左右。而蔗糖组pH值呈明显的周期性波动,最低在4.5以下,但4～5h后,其pH值仍能逐步恢复到6.5左右。观察到细菌计数、生物膜形态的时间依赖性。随着处理液的加入,2组生物膜的活菌计数均明显增加,约72h趋于稳定。对120h生物膜,蔗糖组变形链球菌、乳酸杆菌的细菌计数显著高于蒸馏水组(P<0.05);蔗糖组变形链球菌计数最高,而蒸馏水组血链球菌计数最高。荧光显微镜观察发现,蔗糖组大量的胞外多糖基质和细菌成团、成片分布;蒸馏水组虽然也存在细菌和基质增多的时间依赖性,但膜细菌基质团块仍清晰可见。结论:本研究建立的体外模型能稳定控制有关参数,各观察指标均具有良好的重复性。模型的建立,实现了生物膜形成过程的动态观察。 OBJECTIVE: To simulate the environment of oral plaque and establish a dynamic research model that can continuously observe the biofilm formation process. Methods: homemade fermenter and multiple export continuous culture room. Streptococcus mutans, Streptococcus sanguis, Lactobacillus, actinomycetes Neisseria as the experimental strains, hydroxyapatite tablets, slides and other placed in a continuous culture chamber, by adjusting the temperature, partial pressure of oxygen, pH value, clear Rate, circulating velocity and metabolic substrate and other parameters to establish the model. System testing was performed with 25 mM sucrose and distilled water. Observed the system of experimental bacteria CFU count, pH dynamic changes and other reactions and the formation of biofilms under different environments microstructure changes. The data were analyzed by SPSS10.0 package for statistical analysis. Dunnett two-sided test was used to compare the difference between sucrose group and control group when α = 0.05. Results: With the sucrose, the continuous culture room pH showed similar to the natural oral cariogenic environment, distilled water group pH remained at about 7.5. However, the pH value of sucrose group showed obvious periodic fluctuation, the lowest was below 4.5, but after 4 ~ 5h, the pH value could still recover gradually to about 6.5. Bacterial counts were observed, and the biofilm morphology was time-dependent. With the addition of treatment solution, viable count of biofilm in two groups increased obviously and stable in 72h. The bacterial counts of Streptococcus mutans and Lactobacilli in 120h biofilm and sucrose group were significantly higher than that of distilled water group (P <0.05). The counts of Streptococcus mutans in sucrose group were the highest, while the counts of Streptococcus sobrinus in distilled water group were the highest. Fluorescence microscopy showed that a large amount of exopolysaccharide matrix and bacteria were distributed in the sucrose group. Although there was also a time-dependent increase of bacteria and matrix in the distilled water group, the matrix of the bacterial bacterial matrix was still clearly visible. Conclusion: The in vitro model established in this study can stably control the related parameters, and all the observed indexes have good repeatability. The establishment of the model realizes the dynamic observation of the biofilm formation process.