Sommerfeld Ross Stacy, Tu Mai Han, Falsetta Megan L, Ketterer Margaret R, Kiedrowski Megan R, Horswill Alexander R, Apicella Michael A, Reinhardt Joseph M, Fiegel Jennifer
The University of Iowa, Department of Pharmaceutical Sciences and Experimental Therapeutics, S215 PHAR, 115 S. Grand Ave, Iowa City, IA 52242, United States.
The University of Iowa, Department of Microbiology, Iowa City, IA 52242, United States.
J Microbiol Methods. 2014 May;100:111-20. doi: 10.1016/j.mimet.2014.02.020. Epub 2014 Mar 13.
Bacterial biofilms grow on many types of surfaces, including flat surfaces such as glass and metal and irregular surfaces such as rocks, biological tissues and polymers. While laser scanning confocal microscopy can provide high-resolution images of biofilms grown on any surface, quantification of biofilm-associated bacteria is currently limited to bacteria grown on flat surfaces. This can limit researchers studying irregular surfaces to qualitative analysis or quantification of only the total bacteria in an image. In this work, we introduce a new algorithm called modified connected volume filtration (MCVF) to quantify bacteria grown on top of an irregular surface that is fluorescently labeled or reflective. Using the MCVF algorithm, two new quantification parameters are introduced. The modified substratum coverage parameter enables quantification of the connected-biofilm bacteria on top of the surface and on the imaging substratum. The utility of MCVF and the modified substratum coverage parameter were shown with Pseudomonas aeruginosa and Staphylococcus aureus biofilms grown on human airway epithelial cells. A second parameter, the percent association, provides quantified data on the colocalization of the bacteria with a labeled component, including bacteria within a labeled tissue. The utility of quantifying the bacteria associated with the cell cytoplasm was demonstrated with Neisseria gonorrhoeae biofilms grown on cervical epithelial cells. This algorithm provides more flexibility and quantitative ability to researchers studying biofilms grown on a variety of irregular substrata.
细菌生物膜生长在多种类型的表面上，包括玻璃和金属等平面以及岩石、生物组织和聚合物等不规则表面。虽然激光扫描共聚焦显微镜可以提供在任何表面上生长的生物膜的高分辨率图像，但目前生物膜相关细菌的定量仅限于在平面上生长的细菌。这可能会限制研究不规则表面的研究人员只能进行定性分析或仅对图像中的总细菌进行定量。在这项工作中，我们引入了一种名为改进连通体积过滤（MCVF）的新算法，用于对生长在荧光标记或反射的不规则表面上的细菌进行定量。使用MCVF算法，引入了两个新的定量参数。改进的基质覆盖参数能够对表面顶部和成像基质上的连通生物膜细菌进行定量。在生长于人呼吸道上皮细胞上的铜绿假单胞菌和金黄色葡萄球菌生物膜上展示了MCVF和改进的基质覆盖参数的实用性。第二个参数，关联百分比，提供了关于细菌与标记成分共定位的定量数据，包括标记组织内的细菌。在生长于宫颈上皮细胞上的淋病奈瑟菌生物膜上证明了对与细胞质相关细菌进行定量的实用性。该算法为研究在各种不规则基质上生长的生物膜的研究人员提供了更大的灵活性和定量能力。
