A Method for Sample Preparation to Visualize Biofilm Forming Bacteria on Fungal Hyphae

Published: March 29, 2024

Abstract

Source: Guennoc, Cora Miquel, et al, A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy. J. Vis. Exp. (2017).

This video describes a method to grow and qualitatively analyze bacterial biofilms on fungal hyphae using microscopy. Bacterial biofilms frequently form on fungal surfaces and involve numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation.

Protocol

1. Preparation of an In Vitro Biofilm of Bacteria on the Fungal Colony Centrifuge the bacterial culture at 5,000 x g for 3 min and suspend the pellet in 25 ml of sterile 0.1 M potassium phosphate buffer (25 g/L KH2PO4 and 2.78 g/L K2HPO4, pH 5.8). Repeat this step once and adjust the final bacterial concentration to 109 cells/ml with the same buffer. Fill a 6-well microplate with 5 ml of the bacterial suspension (or ste…

Divulgazioni

The authors have nothing to disclose.

Materials

6 well Falcon Tissue Culture Plates  Fisher Scientific 08-772-33 Used in 2.2 & 3.1
Congo Red Fisher Scientific C580-25 Used in 3.1.4.1
FUN 1 Cell Stain Thermo Fisher Scientific F7030 Used in 3.1.4.1
Wheat Germ Agglutinin, Alexa Fluor 633 Conjugate Thermo Fisher Scientific W21404 Used in 3.1.4.1
DAPI solution Thermo Fisher Scientific 62248 Used in 3.1.4.2
Propidium iodide Thermo Fisher Scientific P3566 Used in 3.1.4.3
FilmTracer SYPRO Ruby Biofilm Matrix protein Stain Thermo Fisher Scientific F10318 Used in 3.1.4.4
Fluoromount-G Slide Mounting Medium Fisher Scientific OB100-01 Used in 3.1.7
LSM780 Axio Observer Z1 Zeiss Used in 3.2.1
ZEN 2.1 lite black software Zeiss Used in 3.2.1
High Vacuum Coater Leica EM ACE600 Leica Used in 4
GeminiSEM-FEG Zeiss Used in 4

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Citazione di questo articolo
A Method for Sample Preparation to Visualize Biofilm Forming Bacteria on Fungal Hyphae. J. Vis. Exp. (Pending Publication), e22067, doi: (2024).

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