1 Cell surface structures of rugose colony-forming bacteria on Congo red agar plates isolated from human whole saliva under aerobic conditions

Abstract

It is widely known that most bacteria produce exopolysaccaride and exist in the form of communities, commonly referred to as biofilm. Since the ability of bacteria to form biofilm is considered an important factor in persistent infections, it is important to isolate these bacteria from lesions and study their mechanism of biofilm formation. The isolation of biofilm-forming bacteria is difficult because it requires the measurement of viscosity of culture supernatants and scanning electron microscopic (SEM) observation of cell surface structures. We used Congo red agar plates (CRA) to isolate biofilm-forming bacteria from the oral cavity. Whole saliva from five healthy volunteers was diluted to 10^<-4> and used to inoculate CRA plates. The bacterial cultures were incubated aerobically for 24 hours at 37℃. Using a stereoscopic microscope, the colonies were divided into two groups, the smooth and rugose periphery types. The cell surface structures of the bacterial strains obtained from 30 arbitrarily selected colonies in each group were further studied by SEM and 16S rRNA sequencing. Bacterial strains from the smooth colony group showed biofilm-negative cell surfaces regardless of colony color. On the other hand, bacterial strains that formed rugose colonies on CRA under aerobic conditions expressed dense fibrillar structures, which is a typical phenotype for biofilm-forming bacteria. These results suggest that oral bacteria form rugose colonies cultured under aerobic condition on CRA have the capacity to form biofilm. We also found that CRA can be applied as a simple method for screening oral biofilm-forming bacteria.