Establishing Bacterial–Fungal Cross-Kingdom Biofilms for Studying Oral Pathogenesis

Abstract

Source: Schlafer, S, and Frost Kristensen, M. Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy. J. Vis. Exp. (2020)

This video demonstrates the cultivation of Streptococcus mutans and Candida albicans cross-kingdom biofilms under oral-like conditions to model bacterial–fungal interactions, enabling the study of microbial synergy, glucan-mediated adhesion, and acidogenic virulence in oral pathogenesis.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Cultivation of Cross-kingdom Biofilms

1. Grow Streptococcus mutans (S. mutans) DSM 20523 and Candida albicans (C. albicans) NCPF 3179 on blood agar plates at 37 °C under aerobic conditions.
2. Transfer single colonies of each organism to test tubes filled with 5 mL of brain heart infusion (BHI). Grow for 18 h under aerobic conditions at 37 °C.
3. Centrifuge the overnight cultures at 1,200 x g for 5 min. Discard the supernatant, resuspend the cells in physiological saline and adjust the OD_{550 nm} to 0.5 for C. albicans (~10⁷ cells/mL) and S. mutans (~10⁸ cells/mL). Dilute the S. mutans suspension 1:10 with sterile physiological saline (~10⁷ cells/mL).
4. Pipette 50 µL of sterile salivary solution, into the wells of an optical bottom 96-well plate for microscopy. Incubate for 30 min at 37 °C. Wash the wells 3x with 100 µL of sterile physiological saline. Empty the wells.
5. Add 100 µL of C. albicans suspension to each well. Incubate at 37 °C for 90 min. Wash 3x with sterile physiological saline.
   NOTE: Do not empty the wells completely during washing. Leave a reservoir of 20 µL to avoid excessive shear forces.
6. Add 100 µL of heat-inactivated fetal bovine serum (inactivated at 56 °C for 30 min) to each well. Incubate at 37 °C for 2 h. Wash 3x with sterile physiological saline. Empty the wells, leaving a reservoir of 20 µL.
7. Add 100 µL of S. mutans suspension (prepared in step 1.3) to each well. Add 150 µL of BHI containing 5% sucrose. Incubate at 37 °C for 24 h or longer. When cultivating older biofilms, change the medium daily to fresh BHI. At the end of the cross-kingdom biofilm growth phase, wash 5x with sterile physiological saline.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Blood agar plates	Statens Serum Institut	677
Brain heart infusion	Oxoid	CM1135
Brain heart infusion + 5 % sucrose	BDH laboratory supplies	10274
Candida albicans	National Collection of Pathogenic Fungi	NCPF 3179
Fetal bovine serum	Gibco Life technologies	10270
GS-6R refrigerated centrifuge	Beckman	N/A
µ-Plate 96 Well Black	Ibidi	89626
Sterile physiological saline	VWR	6404
Streptococcus mutans	Deutsche Sammlung von Mikroorganismen und Zellkulturen	DSM 20523
Vis-spectrophotometer V-3000PC	VWR	N/A