Comparison of cell viability assessment and visualization of Aspergillus niger biofilm with two fluorescent probe staining methods
Filamentous fungi are ubiquitous and frequent components of biofilms. A means to visualize them and quantify their viability is essential for understanding their development and disruption. However, quantifying filamentous fungal biofilms poses challenges because, unlike yeasts and bacteria, they ar...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2022-12-01
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| Series: | Biofilm |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2590207522000247 |
| _version_ | 1811178656103399424 |
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| author | Aswathy Shailaja Terri F. Bruce Patrick Gerard Rhonda R. Powell Charles A. Pettigrew Julia L. Kerrigan |
| author_facet | Aswathy Shailaja Terri F. Bruce Patrick Gerard Rhonda R. Powell Charles A. Pettigrew Julia L. Kerrigan |
| author_sort | Aswathy Shailaja |
| collection | DOAJ |
| description | Filamentous fungi are ubiquitous and frequent components of biofilms. A means to visualize them and quantify their viability is essential for understanding their development and disruption. However, quantifying filamentous fungal biofilms poses challenges because, unlike yeasts and bacteria, they are not composed of discrete cells of similar size. This research focused on filamentous fungal biofilms that are representative of those in the built environment. The objective of this study was to develop a rapid method to examine biofilm structure and quantify live (metabolically active/ membrane undamaged) and dead (inactive/ membrane damaged) cells in Aspergillus niger biofilms utilizing a fluorescent probe staining method and confocal laser scanning microscopy (CLSM). For this, we compared two commercially available probe staining kits that have been developed for bacterial and yeast systems. One method utilized the classic cell stain FUN 1 that exhibits orange-red fluorescent intravacuolar structures in metabolically active cells, while dead cells are fluoresced green. The second method utilized a combination of SYTO9 and propidium iodide (PI), and stains cells based on their membrane morphology. SYTO9 is a green fluorescent stain with the capacity to penetrate the living cell walls, and PI is a red fluorescent stain that can only penetrate dead or dying cells with damaged cell membranes. Following staining, the biofilms were imaged using CLSM and biofilm volumes and thickness were quantified using COMSTAT, a computer program that measures biofilm accumulation from digital image stacks. The results were compared to independent measurements of live-dead cell density, as well as a classic cell viability assay-XTT. The data showed that the combination of SYTO9 and PI is optimal for staining filamentous fungal biofilms. |
| first_indexed | 2024-04-11T06:22:04Z |
| format | Article |
| id | doaj.art-ae44e498d6ab4c03b1da279e5220b1a1 |
| institution | Directory Open Access Journal |
| issn | 2590-2075 |
| language | English |
| last_indexed | 2024-04-11T06:22:04Z |
| publishDate | 2022-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Biofilm |
| spelling | doaj.art-ae44e498d6ab4c03b1da279e5220b1a12022-12-22T04:40:32ZengElsevierBiofilm2590-20752022-12-014100090Comparison of cell viability assessment and visualization of Aspergillus niger biofilm with two fluorescent probe staining methodsAswathy Shailaja0Terri F. Bruce1Patrick Gerard2Rhonda R. Powell3Charles A. Pettigrew4Julia L. Kerrigan5Department of Plant and Environmental Sciences, Clemson University, Clemson, SC, USA; Corresponding author.Clemson Light Imaging Facility, Clemson University, Clemson, SC, USADepartment of Mathematical Sciences, Clemson University, Clemson, SC, USAClemson Light Imaging Facility, Clemson University, Clemson, SC, USAProcter & Gamble, Global Microbiology, Mason, OH, USADepartment of Plant and Environmental Sciences, Clemson University, Clemson, SC, USAFilamentous fungi are ubiquitous and frequent components of biofilms. A means to visualize them and quantify their viability is essential for understanding their development and disruption. However, quantifying filamentous fungal biofilms poses challenges because, unlike yeasts and bacteria, they are not composed of discrete cells of similar size. This research focused on filamentous fungal biofilms that are representative of those in the built environment. The objective of this study was to develop a rapid method to examine biofilm structure and quantify live (metabolically active/ membrane undamaged) and dead (inactive/ membrane damaged) cells in Aspergillus niger biofilms utilizing a fluorescent probe staining method and confocal laser scanning microscopy (CLSM). For this, we compared two commercially available probe staining kits that have been developed for bacterial and yeast systems. One method utilized the classic cell stain FUN 1 that exhibits orange-red fluorescent intravacuolar structures in metabolically active cells, while dead cells are fluoresced green. The second method utilized a combination of SYTO9 and propidium iodide (PI), and stains cells based on their membrane morphology. SYTO9 is a green fluorescent stain with the capacity to penetrate the living cell walls, and PI is a red fluorescent stain that can only penetrate dead or dying cells with damaged cell membranes. Following staining, the biofilms were imaged using CLSM and biofilm volumes and thickness were quantified using COMSTAT, a computer program that measures biofilm accumulation from digital image stacks. The results were compared to independent measurements of live-dead cell density, as well as a classic cell viability assay-XTT. The data showed that the combination of SYTO9 and PI is optimal for staining filamentous fungal biofilms.http://www.sciencedirect.com/science/article/pii/S2590207522000247Aspergillus nigerCell viabilityCOMSTATConfocal laser scanning microscopyFilamentous fungal biofilmsFluorescent probes |
| spellingShingle | Aswathy Shailaja Terri F. Bruce Patrick Gerard Rhonda R. Powell Charles A. Pettigrew Julia L. Kerrigan Comparison of cell viability assessment and visualization of Aspergillus niger biofilm with two fluorescent probe staining methods Biofilm Aspergillus niger Cell viability COMSTAT Confocal laser scanning microscopy Filamentous fungal biofilms Fluorescent probes |
| title | Comparison of cell viability assessment and visualization of Aspergillus niger biofilm with two fluorescent probe staining methods |
| title_full | Comparison of cell viability assessment and visualization of Aspergillus niger biofilm with two fluorescent probe staining methods |
| title_fullStr | Comparison of cell viability assessment and visualization of Aspergillus niger biofilm with two fluorescent probe staining methods |
| title_full_unstemmed | Comparison of cell viability assessment and visualization of Aspergillus niger biofilm with two fluorescent probe staining methods |
| title_short | Comparison of cell viability assessment and visualization of Aspergillus niger biofilm with two fluorescent probe staining methods |
| title_sort | comparison of cell viability assessment and visualization of aspergillus niger biofilm with two fluorescent probe staining methods |
| topic | Aspergillus niger Cell viability COMSTAT Confocal laser scanning microscopy Filamentous fungal biofilms Fluorescent probes |
| url | http://www.sciencedirect.com/science/article/pii/S2590207522000247 |
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