The plaque samples were collected using the tips of sterile wooden toothpicks from carious lesions. The toothpicks were cut off and instantly dipped into 1 ml sterile phosphate-buffered saline (HiMedia, India) and stored at 4°C. Plaque samples were vortexed for a minute to disperse the plaque and obtain a homogeneous suspension. The samples were diluted by 100-fold in 1x sterile phosphate-buffered saline and plated on Mitis Salivarius bacitracin (MSB) agar. The MSB agar composed of mitis salivarius agar (HiMedia, India) and supplemented with 15% of sucrose, 1% of agar, 0.0001% potassium tellurite solution and 0.2 units/ml of bacitracin (HiMedia, India). The plates were incubated anaerobically at 37°C for 48 h.[19 (link)]
After the incubation period, the colonies were identified on the basis of colony morphology.[20 (link)] The typical colonies from each sample plate were transferred to brain–heart infusion (BHI) broth (HiMedia, India) and incubated at 37°C for 18 h. After the incubation period, the broth cultures were streaked on MSB agar and anaerobically incubated at 37°C for 48 h. The overnight bacterial cultures were stored in 80% glycerol stock at −20°C.
After the incubation period, the colonies were identified on the basis of colony morphology.[20 (link)] The typical colonies from each sample plate were transferred to brain–heart infusion (BHI) broth (HiMedia, India) and incubated at 37°C for 18 h. After the incubation period, the broth cultures were streaked on MSB agar and anaerobically incubated at 37°C for 48 h. The overnight bacterial cultures were stored in 80% glycerol stock at −20°C.