Periprosthetic joint infection (PJI) is one of the most challenging complications after joint replacement. However, when treated correctly, chances of recovery are good. The most important step in correct diagnosis and management of PJI is the detection of the causative germ. In the last years, the use of sonication in the diagnostic process has become more important. However, this diagnostic methodology has been controversially discussed when used in combination with antibiotic loaded bone cement (PMMA), which is frequently used in joint replacement surgeries. The aim of this study was thus to analyse in vitro bacterial growth in sonication fluid cultures obtained from antibiotic loaded PMMA which were contaminated with various bacterial biofilms.Sonication fluid obtained from antibiotic loaded PMMA (Copal G+V and Copal G+C) and plain Palacos R (control) contaminated either with S. aureus, E. faecalis, S. sanguinis or P.acnes, were analysed for bacterial re-growth in a standardised in-vitro setting.In vitro bacterial growth was not interfered by released antibiotics from sonication of antibiotic loaded PMMA for S. aureus, E. faecalis and S. sanguinis. However, for P. acnes bacterial counts were affected by the released antibiotics as well as by the time delay between sonication and analysis. The in-vitro data suggest sonication to be an easy and sensitive diagnostic modality to detect easy-to-detect bacteria, however, results are alarming for the difficult-to-detect bacteria P. acnes, indicating that further attention and research is necessary to improve the detection of difficult-to-detect bacteria.