1.Streak F. nucleatum ATCC 23726 from a frozen stock on a TSPC agar plate and incubate 3 days in an anaerobic chamber at 37 °C to obtain single colonies.

2.Inoculate a colony of F. nucleatum into an 8 mL of TSPC and incubate statically overnight at 37 °C 5% CO2 incubator. The next day, transfer all 8 mL of the overnight culture into 42 mL of fresh TSPC in a 50 mL centrifuge tube. Grow cells at 37 °C until the OD600 reaches 1.2

3.Chill the culture on ice for at least 10 min. Harvest the cell pellet by centrifugation at 4°C and discard the supernatant. Wash the cell pellet twice with 25 mL of prechilled sterile water. Resuspend the bacteria in 1 mL of 10% cold glycerol, and then aliquot into 1.5 mL centrifuge tubes (~200 μL each).

4.Snap-freeze the samples with a dry-ice-ethanol bath, and store at −80°C.

ATCC 23726 培养基配置：

TSPC Agar plates Tryptic soy broth powder: 15 g BactoTM peptone: 5 g Agar: 7.5 g L-Cysteine stock (see above) Water: up to 0.5 L Thoroughly mix all components except cysteine, autoclave, cool in 56°C water bath for ~20 minutes, add 5 mL L-cysteine stock, and pour ~25 mL into 100 mm petri dishes under sterile conditions.