Fluorescence-Based Viability Assessment of Oral Anaerobic Bacteria

Begin with a sealed tube containing a nutrient-rich broth maintained under anaerobic conditions.

Using a syringe, transfer a small amount of the broth into another tube.

Take a blood agar plate containing colonies of an anaerobic oral bacterial strain. Pick a single colony and resuspend it in the broth.

Return the suspension to the sealed tube and incubate to promote bacterial growth.

Dilute the culture in a buffer to achieve an optimal cell concentration for cell viability assessment.

Add a solution containing a membrane-permeable dye and a membrane-impermeable dye, then incubate.

The permeable dye enters all cells and binds to intracellular nucleic acids.

The impermeable dye penetrates only dead cells with damaged cell envelopes and binds to their nucleic acids.

Analyze the sample using flow cytometry.

Dead cells exhibit dominant red fluorescence, whereas live cells show only green fluorescence, allowing assessment of bacterial viability.

To begin, autoclave the medium, and then add 5 microgram per milliliter of hemin and 1 microgram per milliliter of menadione. Dispense 9 milliliters of the medium into Hungate tubes, and close the tubes with a rubber stopper and an aluminum cap. Then flus