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Diagnosis of prosthetic joint infections (PJIs) remains a challenge for microbiologists, despite new techniques for bacteria isolation have been developed in recent years. A widely recognized standard method has not yet been indicated mainly because of limitations due difficult procedures and need of dedicated instrumentation. We evaluated the ability of a sulfhydryl compound routinely used in microbiology laboratories, dithiothreitol (DTT), to dislodge bacteria from biofilm, keeping them alive and cultivable for identification and antibiotic susceptibility testing. We compared DTT treatment against sonication of prosthesis and culture of periprosthetic tissues, in order to establish if it could be introduced in routine microbiological diagnosis of PJIs. The study was conducted on 76 patients, 34 with aseptic loosening of their prosthesis and 42 who were diagnosed for PJI. DTT treatment gave results similar to sonication in terms of bacterial yielding. Sonication provided higher sensitivity (71.4%) and specificity (94.1%) respect to periprosthetic tissue culture, while DTT showed the same specificity of sonication but a better sensitivity (85.7%), especially when the causative microorganism was Staphylococcus epidermidis. In conclusion, we demonstrated that DTT could be used for PJIs diagnosis, thanks to its ease of use and its high sensitivity and specificity.