title: How to Study Biofilms after Microbial Colonization of Materials Used in Orthopaedic Implants.
abstract: Over the years, various techniques have been proposed for the quantitative evaluation of microbial biofilms. Spectrophotometry after crystal violet staining is a widespread method for biofilm evaluation, but several data indicate that it does not guarantee a good specificity, although it is rather easy to use and cost saving. Confocal laser microscopy is one of the most sensitive and specific tools to study biofilms, and it is largely used for research. However, in some cases, no quantitative measurement of the matrix thickness or of the amount of embedded microorganisms has been performed, due to limitation in availability of dedicated software. For this reason, we have developed a protocol to evaluate the microbial biofilm formed on sandblasted titanium used for orthopaedic implants, that allows measurement of biomass volume and the amount of included cells. Results indicate good reproducibility in terms of measurement of biomass and microbial cells. Moreover, this protocol has proved to be applicable for evaluation of the efficacy of different anti-biofilm treatments used in the orthopaedic setting. Summing up, the protocol here described is a valid and inexpensive method for the study of microbial biofilm on prosthetic implant materials.
authors.affiliation: Laboratory of Clinical Chemistry and Microbiology, IRCCS Galeazzi Orthopaedic Institute, via R. Galeazzi 4, 20161 Milan, Italy. lorenzo.drago@unimi.it.|Laboratory of Clinical Microbiology, Department of Biomedical Sciences for Health, University of Milan, via L. Mangiagalli 31, 20133 Milan, Italy. lorenzo.drago@unimi.it.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Drago
authors.affiliation: Laboratory of Clinical Chemistry and Microbiology, IRCCS Galeazzi Orthopaedic Institute, via R. Galeazzi 4, 20161 Milan, Italy. serse.agrappi@gmail.com.
authors.forename: Serse
authors.initials: S
authors.lastname: Agrappi
authors.affiliation: Laboratory of Clinical Chemistry and Microbiology, IRCCS Galeazzi Orthopaedic Institute, via R. Galeazzi 4, 20161 Milan, Italy. monica.bortolin85@gmail.com.
authors.forename: Monica
authors.initials: M
authors.lastname: Bortolin
authors.affiliation: Laboratory of Clinical Microbiology, Department of Biomedical Sciences for Health, University of Milan, via L. Mangiagalli 31, 20133 Milan, Italy. toscano.marco1@gmail.com.
authors.forename: Marco
authors.initials: M
authors.lastname: Toscano
authors.affiliation: Department of Bone and Joint Infections and Reconstructive Surgery, IRCCS Galeazzi Orthopaedic Institute, via R. Galeazzi 4, 20161 Milan, Italy. carlo.romano@grupposandonato.it.
authors.forename: Carlo Luca
authors.initials: CL
authors.lastname: Romanò
authors.affiliation: Laboratory of Clinical Chemistry and Microbiology, IRCCS Galeazzi Orthopaedic Institute, via R. Galeazzi 4, 20161 Milan, Italy. devecchi.elena@gmail.com.
authors.forename: Elena
authors.initials: E
authors.lastname: De Vecchi
mesh_terms: D018441:Biofilms / Q000254:growth & development*; D018533:Biomass; D002175:Candida / Q000502:physiology; D007595:Joint Prosthesis / Q000382:microbiology*; D055624:Methicillin-Resistant Staphylococcus aureus / Q000502:physiology; D018613:Microscopy, Confocal / Q000379:methods; D011550:Pseudomonas aeruginosa / Q000502:physiology; D014025:Titanium / Q000737:chemistry
chemical_list: D014025:Titanium
keywords: biofilm; confocal laser scanning microscopy; fluorescent stains; images analysis; prosthetic implants