J Bone Jt Infect 2018; 3(1):27-36. doi:10.7150/jbji.22799

Research Paper

Combined Staining Techniques for Demonstration of Staphylococcus aureus Biofilm in Routine Histopathology

Louise Kruse Jensen1✉, Nicole Lind Henriksen1, Thomas Bjarnsholt2,3, Kasper Nørskov Kragh2, Henrik Elvang Jensen1

1. Department of Veterinary Clinical and Animal Sciences, Ridebanevej 3, 1870 Frederiksberg C, University of Copenhagen, Denmark
2. Costerton Biofilm Center, Department of Immunology and Microbiology, Blegdamsvej 3B, 2200 Copenhagen N, University of Copenhagen, Denmark
3. Department of Clinical Microbiology, Juliane Maries Vej 22, 2100 Copenhagen Ø, Copenhagen University Hospital, Denmark


Aim: Visualization of Staphylococcus aureus biofilm using histochemical staining and combined histochemistry (HC) and immunohistochemistry (IHC).

Methods: The ability of S. aureus S54F9 to form biofilm was tested in vitro. Hereafter, infected bone tissue was collected from two different porcine models of osteomyelitis inoculated with S. aureus 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 S. aureus. 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.

Results: S. aureus S54F9 showed an ability to form biofilm in vitro. Visualization of biofilm, i.e. 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.

Conclusions: A combination of HC and IHC can be used to diagnose and characterise biofilm infections on a routine basis.

Keywords: Biofilm, Staphylococcus aureus, bone infection, histology, light microscopy

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How to cite this article:
Jensen LK, Henriksen NL, Bjarnsholt T, Kragh KN, Jensen HE. Combined Staining Techniques for Demonstration of Staphylococcus aureus Biofilm in Routine Histopathology. J Bone Jt Infect 2018; 3(1):27-36. doi:10.7150/jbji.22799. Available from http://www.jbji.net/v03p0027.htm