Kulišová Markéta, Maťátková Olga, Brányik Tomáš, Zelenka Jaroslav, Drábová Lucie, Kolouchová Irena Jarošová
Department of Biotechnology, University of Chemistry and Technology, Prague, Technická 5, Prague 166 28, Czech Republic.
Research Institute of Brewing and Malting, Lipová 511/15, Prague 120 44, Czech Republic.
J Microbiol Methods. 2023 Feb;205:106676. doi: 10.1016/j.mimet.2023.106676. Epub 2023 Jan 21.
Microscopic filamentous fungi are ubiquitous microorganisms that adapt very easily to a variety of environmental conditions. Due to this adaptability, they can colonize a number of various surfaces where they are able to start forming biofilms. Life in the form of biofilms provides them with many benefits (increased resistance to desiccation, UV radiation, antimicrobial compounds, and host immune response). The aim of this study is to find a reliable and reproducible methodology to determine biofilm growth of selected microscopic filamentous fungi strains. Several methods (crystal violet staining, MTT assay, XTT assay, resazurin assay) for the determination of total biofilm biomass and its metabolic activity were tested on four fungi - Alternaria alternata, Aspergillus niger, Fusarium culmorum and Fusarium graminearum, and their biofilm was also imaged by spinning disc confocal microscopy using fluorescent dyes. A reproducible biofilm quantification method is essential for the subsequent testing of the biofilm growth suppression using antifungal agents or physical methods. Crystal violet staining was found to be a suitable method for the determination of total biofilm biomass of selected strains, and the MTT assay for the determination of metabolic activity of the biofilms. Calcofluor white and Nile red fluorescent stains successfully dyed the hyphae of microscopic fungi.
微小丝状真菌是普遍存在的微生物，能非常容易地适应各种环境条件。由于这种适应性，它们能够在许多不同表面定殖，并在这些表面开始形成生物膜。以生物膜形式存在的生活方式为它们带来了许多益处（增强对干燥、紫外线辐射、抗菌化合物和宿主免疫反应的抵抗力）。本研究的目的是找到一种可靠且可重复的方法来测定所选微小丝状真菌菌株的生物膜生长情况。在四种真菌——链格孢、黑曲霉、禾谷镰刀菌和黄色镰刀菌上测试了几种用于测定生物膜总生物量及其代谢活性的方法（结晶紫染色、MTT 法、XTT 法、刃天青法），并且还使用荧光染料通过转盘共聚焦显微镜对它们的生物膜进行了成像。一种可重复的生物膜定量方法对于随后使用抗真菌剂或物理方法测试生物膜生长抑制至关重要。发现结晶紫染色是测定所选菌株生物膜总生物量的合适方法，而 MTT 法是测定生物膜代谢活性的合适方法。荧光增白剂和尼罗红荧光染料成功地对微小真菌的菌丝进行了染色。
