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Shailaja, Aswathy [1], Kerrigan, Julia [1], Bruce, Terri [2], Gerard, Patrick [3], Powell, Rhonda [2], Pettigrew, Charles [4].

A new approach to assess the cell viability and three-dimensional structural analysis of Aspergillus niger biofilm using fluorescent probes staining and COMSTAT program.

Aspergillus niger is a filamentous fungus that adheres to different substrate surfaces and forms biofilms consisting of dense hyphal networks encased within and bound by a matrix composed of extracellular polymeric substances (EPS). Both yeast and bacterial biofilms have been widely studied by different microscopic methods; however, microstructures of biofilms containing filamentous fungi is less well known. Previously, we developed a protocol to grow A. niger biofilms in a controlled reactor under low-shearing force to reflect those that form in the built environment. To evaluate the cell viability and quantification of A. niger biofilm structure, we developed molecular probes staining methods and utilized a computer program, COMSTAT, a computer program which quantitatively measure biofilm accumulation from the digital image stacks. The results were then compared to independent measurements of live-dead cell density. For this study, two different types of fluorescent probes kit methods that have been developed for bacterial and yeast system are being used. One method utilizes the classical cell stain FUN-1 that exhibits orange-red fluorescent intravacuolar structures in metabolically active cells, while dead cells exhibit green-yellow fluorescence. The second method used a combination of SYTO9 and Propidium iodide (PI). SYTO9 is a green fluorescent stain with a capacity to penetrate the active cell walls, and PI, is a red fluorescent stain that can penetrates the damaged cell membrane. Biofilms stained with both kit methods were compared for reliability using confocal laser scanning microscopy (CLSM). The data showed that the combination of SYTO9 and PI are reliable vital stain. To compare the accuracy of fluorescent probe staining method of cell viability assessment, a classical cell viability XTT assay method was used, and the statistical data was compared with the fluorescent probe staining method.  

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1 - Clemson University, Plant and Environmental Sciences, Clemson, SC, USA
2 - Clemson University, Clemson Light Imaging Facility, Clemson, SC, USA
3 - Clemson University, Mathematical and Statistical Sciences, Clemson, SC, USA
4 - Procter & Gamble, Global Microbiology, Mason, OH, USA

Aspergillus niger
Cell viability
Confocal laser scanning microscopy
Fluorescent probes

Presentation Type: Oral Paper
Session: MY2, Mycology: Human and Animal Pathogens
Location: /
Date: Tuesday, July 20th, 2021
Time: 2:00 PM(EDT)
Number: MY2007
Abstract ID:1019
Candidate for Awards:None

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