A Method Using CRA for Detecting Bacteria with Biofilm-Forming Capabilities

It is well known that biofilm-forming bacteria produce large amounts of extracellular polysaccharides (EPS), which are commonly present as cell surface-associated meshwork structures that could cause persistent infections. However, the isolation of biofilm-forming bacteria could be time consuming which requires processing such as morphological characterization using scanning electron microscopy (SEM) and/or other approaches. Objective: To develop a rapid and easy-to-use method for isolating biofilm-forming bacteria by the use of a Congo red agar (CRA) plate assay for screening of bacteria isolated from human saliva. Methods: Whole saliva samples were collected from 5 healthy volunteers. Samples were diluted by ten thousand fold, inoculated onto the CRA plates, and incubated at 37 °C for 24 hrs aerobically or for 48 hrs anaerobically. Each isolated colony was grouped under a stereomicroscope into two morphological types: either rugose or smooth. Thirty colonies were randomly selected from each category and subjected to SEM observation of cell surface structures and 16S rRNA sequencing for identification. Results: Under aerobic condition, the rugose types of colonies on the CRA plate were found to be bacterial strains possessing dense meshwork structures, which included Streptococcus spp., Neisseria spp. and Rothia spp.. The smooth types of colonies were not biofilm-forming bacteria. In contrast, a similar correlation was not observed when cells were grown anaerobically. Conclusion: Under aerobic condition, biofilm-forming bacteria were easily detected by changes in colonial appearance on the CRA plate, and this approach did not work under anaerobic condition. Therefore, a CRA screening method is useful for isolating oral biofilm-forming bacteria under aerobic condition.