Korea Food Research Institute; Department of Food Biotechnology, Korea University of Science and Technology;
Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute.
J Vis Exp. 2023 Aug 4(198). doi: 10.3791/65517.
Acinetobacter causes nosocomial infections and its biofilm formation can contribute to the survival on dry surfaces such as hospital environments. Thus, biofilm quantification and visualization are important methods to assess the potential of Acinetobacter strains to cause nosocomial infections. The biofilms forming on the surface of the microplate can be quantified in terms of volume and cell numbers. Biofilm volumes can be quantified by staining using crystal violet, washing, destaining using ethanol, then measuring the solubilized dye using a microplate reader. To quantify the number of cells embedded in the biofilms, the biofilms are scrapped off using cell scrapers, harvested in the saline, vigorously agitated in the presence of glass beads, and spread on Acinetobacter agar. Then, the plates are incubated at 30 °C for 24-42 h. After incubation, the red colonies are enumerated to estimate the number of cells in biofilms. This viable count method can also be useful for counting Acinetobacter cells in mixed-species biofilms. Acinetobacter biofilms can be visualized using fluorescent dyes. A commercially available microplate designed for microscopic analysis is employed to form biofilms. Then, the bottom-surface attached biofilms are stained with SYTO9 and propidium iodide dyes, washed, then visualized with confocal laser scanning microscopy.
不动杆菌会引起医院感染，其生物膜的形成有助于其在医院环境等干燥表面存活。因此，生物膜的定量和可视化是评估不动杆菌菌株引起医院感染潜力的重要方法。可以通过结晶紫染色、清洗、乙醇脱色，然后使用微孔板读数仪测量溶解染料的方法来定量平板表面形成的生物膜体积和细胞数量。为了定量嵌入生物膜中的细胞数量，可以使用细胞刮刀刮取生物膜，在盐水中收集，在玻璃珠存在下剧烈搅拌，并在不动杆菌琼脂上展开。然后，将平板在 30°C 下孵育 24-42 小时。孵育后，通过计数红色菌落来估计生物膜中的细胞数量。这种活菌计数方法也可用于计数混合物种生物膜中的不动杆菌细胞。可以使用荧光染料对不动杆菌生物膜进行可视化。使用专为显微镜分析设计的商用微孔板形成生物膜。然后，用 SYTO9 和碘化丙啶染料对附着在底部表面的生物膜进行染色、清洗，然后使用共聚焦激光扫描显微镜进行可视化。
