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背景:有研究证实白色念珠菌细胞壁外发现有一层分泌的蛋白质,而最近实验发现其具有荚膜结构。目的:通过改良荚膜肿胀试验观察白色念珠菌的荚膜结构。设计:观察对比实验。单位:赣南医学院病原生物学教研室。材料:菌株:中央标准株(菌号:CCCMC1a)由中科院真菌菌种保藏中心提供;国际标准株(菌号:ATCC 14053)由北京大学医院真菌菌种保藏中心提供。2 株临床分离菌株(C1、C2),经中科院真菌菌种保藏中心鉴定为白假丝酵母菌。免疫血清用上述实验菌株全细胞抗原分别免疫家兔(由中山大学动物实验中心提供),常规制备免疫血清,备作荚膜肿胀试验用。方法:实验于 2005-12 在赣南医学院病原生物学教研室完成。①荚膜肿胀实验:将白色念珠菌培养物(C1, C2, CCCMC1a, ATCC)分别涂片,实验组 1 玻片加入相应的兔抗血清,对照组 1 加入正常兔血清,两组都加入 1%美蓝置湿盒,37 ℃,20 min;取出玻片,盖上盖玻片,在油镜下用测微计测定 40 个菌细胞的荚膜厚度,计算平均值。②改良荚膜肿胀实验:将白色念珠菌培养物(C1,C2,CCCMC1a,ATCC)分别涂片,实验组 2 玻片加入相应的兔抗血清,对照组 2 加入正常兔血清,不加美蓝直接置湿盒内,37 ℃,20 min;取出玻片,不盖盖玻片,待其自然干燥,然后用HISS 荚膜染色法进行染色,同样在油镜下用测微计测定 40 个菌细胞的荚膜厚度,计算平均值。主要观察指标:荚膜肿胀实验及改良荚膜肿胀实验各组荚膜厚度平均值。结果:①传统荚膜肿胀实验表现为阳性,实验组 1 白色念珠菌培养物C1, C2, CCCMC1a, ATCC 荚膜厚度分别为 (0.558+0.081),(0.530+0.081),(0.475+0.081),(0.600+0.068)μm,均大于对照组 1[(0.225+0.061),(0.252+0.038),(0.200+0.072),(0.225+0.046)μm,P < 0.01]。②改良荚膜肿胀实验组 2 白色念珠菌培养物 C1, C2,CCCMC1a, ATCC 荚膜厚度分别为 (0.541±0.038),(0.510±0.060),(0.487±0.041),(0.595±0.027) μm,大于对照组 2 [(0.215±0.022),(0.247±0.018),(0.213±0.033),(0.220±0.016)μm,P < 0.01]。③对照组 2 荚膜厚度小于对照组 1(P < 0.01), 实验组 2 荚膜厚度小于实验组 1(P < 0.01)。结论:作为一种定量试验,改良荚膜肿胀试验比传统荚膜肿胀试验更为稳定与准确。
Background: Studies have confirmed that Candida albicans found a layer of secreted protein outside the cell wall, and recent experiments found that it has a capsule structure. OBJECTIVE: To observe the capsular structure of Candida albicans by improving capsular swelling test. Design: observe the contrast experiment. Unit: Gannan Medical College of Pathogenic Biology. Material: Strain: The central standard strain (strain: CCCMC1a) was provided by the fungal strain collection center of Chinese Academy of Sciences. The international standard strain (ATCC 14053) was provided by the fungal strain collection center of Peking University Hospital. Two clinical isolates (C1 and C2) were identified as Candida albicans by the fungal strain collection center of Chinese Academy of Sciences. Immune serum Rabbit immunized with whole cell antigen of the above experimental strains (provided by Animal Experiment Center of Sun Yat-sen University), routinely prepared immune sera were prepared for capsule swelling test. Methods: The experiment was performed at the Department of Pathobiology, Gannan Medical College from December 2005 to December 2005. ① Capsule swollen test: Candida albicans culture (C1, C2, CCCMC1a, ATCC) were smear, the experimental group 1 slide added to the corresponding rabbit antiserum, control group 1 was added to normal rabbit serum, both groups were added 1 % Methylene blue wet box, 37 ℃, 20 min; remove the slide, cover the cover glass, measure the capsular thickness of the 40 bacterial cells with a micrometer under an oil microscope to calculate the average. (2) Modified capsular swelling test: Candida albicans cultures (C1, C2, CCCMC1a, ATCC) were smeared separately, the experimental group 2 slides were added to the corresponding rabbit antiserum, the control group 2 was added to normal rabbit serum without methylene blue direct Set the wet box at 37 ℃, 20 min; remove the slide, cover the slide without cover, to be dried naturally, and then stained with HISS capsule staining, the same under the microscope with a micrometer to measure 40 bacterial cells Capsule thickness, calculate the average. MAIN OUTCOME MEASURES: Capsule swelling test and modified capsule swelling test in each group average capsule thickness. Results: ① The results of traditional capsular swelling test were positive. The thickness of C1, C2, CCCMC1a and ATCC in experimental group 1 were 0.558 + 0.081, 0.530 + 0.081, 0.475 + 0.081, (0.600 + 0.068) μm, all of which were significantly higher than that of the control group (P <0.01). (2) The capsule thickness of C1, C2, CCCMC1a and ATCC in the test group of modified capsular swelling group were (0.541 ± 0.038), (0.510 ± 0.060), (0.487 ± 0.041) and (0.595 ± 0.027) (0.215 ± 0.022), (0.247 ± 0.018), (0.213 ± 0.033) and (0.220 ± 0.016) μm respectively in the control group (P <0.01). (3) The capsule thickness of control group 2 was smaller than that of control group 1 (P <0.01). The capsule thickness of experimental group 2 was smaller than that of experimental group 1 (P <0.01). Conclusion: As a quantitative test, the modified capsule swelling test is more stable and accurate than the traditional capsule swelling test.