Kania Romain E, Lamers Gerda E M, Vonk Marcel J, Huy Patrice Tran Ba, Hiemstra Pieter S, Bloemberg Guido V, Grote Jan J
Department of Otorhinolaryngology-Head and Neck Surgery, Leiden University Medical Center, Leiden University, Leiden, the Netherlands.
Arch Otolaryngol Head Neck Surg. 2007 Feb;133(2):115-21. doi: 10.1001/archotol.133.2.115.
To demonstrate mucosal biofilms in human tissue by direct visualization of bacteria and glycocalyx using confocal laser scanning microscopy with double fluorescent staining on tonsils and to compare the findings with the results of scanning electron microscopy analysis.
Prospective study.
Tertiary university-based referral center.
Twenty-four tonsils were obtained from children with chronic or recurrent tonsillitis.
Tonsils were prepared for analysis by scanning electronic microscopy and by confocal laser scanning microscopy.
Double fluorescent staining for confocal laser scanning microscopy consisted of propidium iodide staining to detect bacterial cells and fluorescein isothiocyanate concanavalin A staining to detect the glycocalyx matrix. Images were analyzed for characteristic biofilm morphologic features by 3 investigators who evaluated the images independently in a blinded retrospective manner. Consensus of all observers was required to demonstrate the presence of a biofilm in a specimen.
Findings from analyses using scanning electronic microscopy suggested the presence of biofilm formations on tonsils by showing bacterial cells in microcolonies. Double-staining technique using confocal laser scanning microscopy showed bacterial cells and the glycocalyx matrix, providing visual evidence for the presence of biofilms on tonsils.
Using a novel visualization approach in single sections of human mucosal tissue, the presence of biofilms was demonstrated on tonsils in most (17/24 [70.8%]) patients with tonsillitis.
通过共聚焦激光扫描显微镜对扁桃体进行双重荧光染色直接观察细菌和糖萼，以展示人体组织中的黏膜生物膜，并将结果与扫描电子显微镜分析结果进行比较。
前瞻性研究。
大学三级转诊中心。
从患有慢性或复发性扁桃体炎的儿童中获取24个扁桃体。
对扁桃体进行处理，以便通过扫描电子显微镜和共聚焦激光扫描显微镜进行分析。
共聚焦激光扫描显微镜的双重荧光染色包括用碘化丙啶染色检测细菌细胞，用异硫氰酸荧光素伴刀豆球蛋白A染色检测糖萼基质。由3名研究人员对图像进行分析，以确定生物膜的特征形态学特征，他们以盲法回顾性方式独立评估图像。标本中生物膜的存在需要所有观察者达成共识。
扫描电子显微镜分析结果表明扁桃体上存在生物膜形成，表现为微菌落中的细菌细胞。共聚焦激光扫描显微镜的双重染色技术显示了细菌细胞和糖萼基质，为扁桃体上生物膜的存在提供了视觉证据。
通过在人体黏膜组织的单个切片中采用一种新颖的可视化方法，在大多数（17/24 [70.8%]）扁桃体炎患者的扁桃体上证实了生物膜的存在。
