Journal cover Journal topic
Journal of Bone and Joint Infection An open-access journal of the European Bone and Joint Infection Society and the MusculoSkeletal Infection Society
Journal topic

Journal metrics

h5-index value: 15
Volume 3, issue 1
J. Bone Joint Infect., 3, 27–36, 2018
© Author(s) 2018. This work is distributed under
the Creative Commons Attribution 4.0 License.
J. Bone Joint Infect., 3, 27–36, 2018
© Author(s) 2018. This work is distributed under
the Creative Commons Attribution 4.0 License.

Original full-length article 20 Feb 2018

Original full-length article | 20 Feb 2018

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

Louise Kruse Jensen1, Nicole Lind Henriksen1, Thomas Bjarnsholt2,3, Kasper Nørskov Kragh2, and Henrik Elvang Jensen1 Louise Kruse Jensen et al.
  • 1Department of Veterinary Clinical and Animal Sciences, Ridebanevej 3, 1870 Frederiksberg C, University of Copenhagen, Denmark
  • 2Costerton Biofilm Center, Department of Immunology and Microbiology, Blegdamsvej 3B, 2200 Copenhagen N, University of Copenhagen, Denmark
  • 3Department of Clinical Microbiology, Juliane Maries Vej 22, 2100 Copenhagen Ø, Copenhagen University Hospital, Denmark

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

Abstract. 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.

Publications Copernicus