Jensen Louise Kruse, Henriksen Nicole Lind, Bjarnsholt Thomas, Kragh Kasper Nørskov, Jensen Henrik Elvang
Department of Veterinary Clinical and Animal Sciences, Ridebanevej 3, 1870 Frederiksberg C, University of Copenhagen, Denmark.
Costerton Biofilm Center, Department of Immunology and Microbiology, Blegdamsvej 3B, 2200 Copenhagen N, University of Copenhagen, Denmark.
J Bone Jt Infect. 2018 Feb 20;3(1):27-36. doi: 10.7150/jbji.22799. eCollection 2018.
Visualization of biofilm using histochemical staining and combined histochemistry (HC) and immunohistochemistry (IHC). The ability of S54F9 to form biofilm was tested . Hereafter, infected bone tissue was collected from two different porcine models of osteomyelitis inoculated with strain S54F9. The infection time was five and fifteen days, respectively. Twenty-five different histochemical staining protocols were tested in order to find the stains that could identify extracellular biofilm matrix. Protocols with an optimal visualization of biofilm extracellular matrix were combined with an immunohistochemical protocol based on a specific antibody against The combined protocols were applied to the tissue from the porcine models and to infected bone tissue from a child suffering from chronic staphylococcal osteomyelitis for more than a year. S54F9 showed an ability to form biofilm . Visualization of biofilm, . bacterial cells and extracellular matrix in different colours, was seen when the immunohistochemical protocol was combined with Alcian Blue pH3, Luna and Methyl-pyronin green. The bacterial cells were red to light brown and the extracellular matrix either light blue, blue or orange depending on the histochemical stain. In the porcine models and the human case 10 and 90 percent, respectively, of the bacterial aggregates in a 100x magnification field displayed both the extracellular matrix and the bacterial cells simultaneously in two different colours. A combination of HC and IHC can be used to diagnose and characterise biofilm infections on a routine basis.
使用组织化学染色以及联合组织化学（HC）和免疫组织化学（IHC）对生物膜进行可视化。测试了S54F9形成生物膜的能力。此后，从接种菌株S54F9的两种不同猪骨髓炎模型中收集感染的骨组织。感染时间分别为5天和15天。测试了25种不同的组织化学染色方案，以找到能够识别细胞外生物膜基质的染色剂。将生物膜细胞外基质可视化效果最佳的方案与基于针对……的特异性抗体的免疫组织化学方案相结合。将联合方案应用于猪模型的组织以及一名患有慢性葡萄球菌骨髓炎一年多的儿童的感染骨组织。S54F9显示出形成生物膜的能力。当免疫组织化学方案与阿尔辛蓝pH3、卢娜染色法和甲基派洛宁绿相结合时，可以看到生物膜、细菌细胞和细胞外基质呈现不同颜色。细菌细胞呈红色至浅棕色，细胞外基质根据组织化学染色呈浅蓝色、蓝色或橙色。在猪模型和人类病例中，在100倍放大视野下，分别有10%和90%的细菌聚集体同时呈现出两种不同颜色的细胞外基质和细菌细胞。HC和IHC的联合可用于常规诊断和表征生物膜感染。
