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

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

1. 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.
2. Fill a 6-well microplate with 5 ml of the bacterial suspension (or sterile 0.1 M potassium phosphate buffer for the negative control).
3. Cut the cellophane membrane of the fungal culture with a sterile razor blad....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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