To investigate the dynamics of a disinfection regimen using 1% trypsin and 1% proteinase K in combination with 2% chlorhexidine (with or without ultrasonics) using a nutrient-stressed endodontic multispecies model biofilm.MethodologyNutrient-stressed biofilms (Propionibacterium acnes, Staphylococcus epidermidis, Actinomyces radicidentis, Streptococcus mitis and Enterococcus faecalis OMGS 3202) were grown in prepared root canals of single-rooted teeth. The treatment groups included 1% trypsin and 2% chlorhexidine (CHX), 1% proteinase K and 2% CHX (with and without ultrasonics). 2% CHX was the positive control and untreated group, and sterile saline (with and without ultrasonics) was the negative control. The biofilms were investigated using confocal laser scanning microscopy (CLSM) with live/dead staining and quantitative microbial culture.ResultsThe trypsin and CHX group with ultrasonics was significantly more effective in reducing viable counts and the substratum coverage than those of all other groups (P