Combined Staining Techniques for Demonstration of Biofilm in Routine Histopathology.

Visualization of biofilm using histochemical staining and combined histochemistry (HC) and immunohistochemistry (IHC). The ability of S54F9 to form biofilm was tested . Hereafter, infected bone tissue was collected from two different porcine models of osteomyelitis inoculated with strain S54F9. The infection time was five and fifteen days, respectively. Twenty-five different histochemical staining protocols were tested in order to find the stains that could identify extracellular biofilm matrix. Protocols with an optimal visualization of biofilm extracellular matrix were combined with an immunohistochemical protocol based on a specific antibody against The combined protocols were applied to the tissue from the porcine models and to infected bone tissue from a child suffering from chronic staphylococcal osteomyelitis for more than a year. S54F9 showed an ability to form biofilm . Visualization of biofilm, . bacterial cells and extracellular matrix in different colours, was seen when the immunohistochemical protocol was combined with Alcian Blue pH3, Luna and Methyl-pyronin green. The bacterial cells were red to light brown and the extracellular matrix either light blue, blue or orange depending on the histochemical stain. In the porcine models and the human case 10 and 90 percent, respectively, of the bacterial aggregates in a 100x magnification field displayed both the extracellular matrix and the bacterial cells simultaneously in two different colours. A combination of HC and IHC can be used to diagnose and characterise biofilm infections on a routine basis.