Fluorescence-Based Viability Assessment of Oral Anaerobic Bacteria

Résumé

Source: Calatayud Arroyo, M., et al. Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface. J. Vis. Exp. (2018)

The video demonstrates a method to evaluate the viability of anaerobic oral bacteria using fluorescent dyes and flow cytometry. It begins with culturing bacteria in oxygen-free broth, followed by resuspending a single colony from a blood agar plate. After incubation and dilution, the culture is stained with membrane-permeable and impermeable dyes. Flow cytometry is then used to distinguish live cells, which fluoresce green, from dead cells, which fluoresce red.

Protocole

1. Strains and Culture Conditions

NOTE: The synthetic oral community was composed of strains commonly present in the oral microbiome.

1. Obtain the following strains from the American Type Culture Collection (ATCC): Aggregatibacter actinomycetemcomitans (ATCC 43718), Fusobacterium nucleatum (ATCC 10953), Porphyromonas gingivalis (ATCC 33277), Prevotella intermedia (ATCC 25611), Streptococcus mutans (ATCC 25175), Streptococcus sobrinus (ATCC 33478), Actinomyces naeslundii (ATCC 51655), Streptococcus gordonii (ATCC 49818), Actinomyces viscosus (ATCC 15987), and Streptococcus mitis (ATCC 49456).
2. Acquire Veillonella parvula from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (DSM 2007), Streptococcus sanguinis from the BCCM/LMG Bacteria Collection (LMG 14657), and use Streptococcus salivarius strain TOVE-R9 and Streptococcus oralis.

2. Development of a Multispecies Community Representative of the Oral Microbiome

NOTE: Generate a synthetic community to simulate the potential adhesion capacity of oral bacteria to the in vitro gut epithelium. Grow bacteria on blood agar plates supplemented with 5 μg/mL hemin, 1 μg/mL menadione, and 5% sterile defibrinated horse blood. In brief:

1. Dissolve 100 mg of hemin in 2 mL of 1 M NaOH, add 100 mL of distilled autoclaved water. Store in a dark container. Sterilize through a 0.22 µm filter before adding to medium.
2. Dissolve 100 mg of menadione in 20 mL of 96% ethanol. Sterilize through a 0.22 µm filter before adding to the medium.
3. Prepare 1 L of blood agar medium (see Table of Materials) according to the manufacturer’s instructions and autoclave. Let it cool down before adding the horse blood and the supplements. Mix and pour the plates.
4. Prepare modified Brain Heart Infusion (BHI) broth. Add 5 μg/mL of hemin and 1 μg/mL of menadione after autoclaving.
5. Dispense 9 mL of the medium in Hungate tubes, and close with a rubber stopper and an aluminium cap. Flush the tubes with N₂/CO₂ (90%/10%).
6. Retrieve 1 mL of anaerobic medium with a syringe and place it in a 1.5 mL microcentrifuge tube. Pick single colonies of each bacterial species, resuspend in the medium, and transfer back into the anaerobic modified BHI. Incubate for 48 h at 37 °C, except for S. salivarius, which must be incubated for 24 h.
7. Measure the cell number at the end of the incubation, using flow cytometry and SYBR Green/Propidium Iodide stain. Dilute the cultures to 10⁵ cells mL^-1, using modified BHI.

Matériaux

Liste des matériaux utilisés dans cet article

Nom	Entreprise	Numéro de catalogue	Commentaires
STRAINS
Aggregatibacter actinomycetemcomitans	American Type Culture Collection	ATCC 43718
Fusobacterium nucleatum	American Type Culture Collection	ATCC 10953
Porphyromonas gingivalis	American Type Culture Collection	ATCC 33277
Prevotella intermedia	American Type Culture Collection	ATCC 25611
Streptococcus mutans	American Type Culture Collection	ATCC 25175
Streptococcus sobrinus	American Type Culture Collection	ATCC 33478
Actinomyces viscosus	American Type Culture Collection	ATCC 15987
Streptococcus salivarius TOVE-R
Streptococcus mitis	American Type Culture Collection	ATCC 49456
Streptococcus sanguinis	BCCM/LMG Bacteria Collection	LMG 14657
Veillonella parvula	Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures	DSM 2007
Streptococcus gordonii	American Type Culture Collection	ATCC 49818
CELL LINES
Caco-2 cells	European Collection of Authenticated Cell Cultures	86010202
HT29-MTX cells	European Collection of Authenticated Cell Cultures	12040401
REAGENTS AND CONSUMABLES
Brain Heart Infusion (BHI) broth	Oxoid	CM1135
Blood Agar 2	Oxoid	CM0055	Blood Agar medium
Menadione	Sigma	M9429
Hemin	Sigma	H9039
5% sterile defibrinated horse blood	E&O Laboratories Ltd,	P030
SYBR Green I	Invitrogen	S7585
Propidium Iodide	Invitrogen	P1304MP
PBS	Gibco	14190250
EQUIPMENT
BD Accuri C6 Flow cytometer	BD Biosciences	653118
Memmert CO₂ incubator	Memmert GmbH & Co.	ICO150med
Millipore Milli-Q academic, ultra pure water system	Millipore, Merck KGaA	-