title: Demonstration of bacterial cells and glycocalyx in biofilms on human tonsils.
abstract: OBJECTIVE
  To demonstrate mucosal biofilms in human tissue by direct visualization of bacteria and glycocalyx using confocal laser scanning microscopy with double fluorescent staining on tonsils and to compare the findings with the results of scanning electron microscopy analysis.
  
  
  METHODS
  Prospective study.
  
  
  METHODS
  Tertiary university-based referral center.
  
  
  METHODS
  Twenty-four tonsils were obtained from children with chronic or recurrent tonsillitis.
  
  
  METHODS
  Tonsils were prepared for analysis by scanning electronic microscopy and by confocal laser scanning microscopy.
  
  
  METHODS
  Double fluorescent staining for confocal laser scanning microscopy consisted of propidium iodide staining to detect bacterial cells and fluorescein isothiocyanate concanavalin A staining to detect the glycocalyx matrix. Images were analyzed for characteristic biofilm morphologic features by 3 investigators who evaluated the images independently in a blinded retrospective manner. Consensus of all observers was required to demonstrate the presence of a biofilm in a specimen.
  
  
  RESULTS
  Findings from analyses using scanning electronic microscopy suggested the presence of biofilm formations on tonsils by showing bacterial cells in microcolonies. Double-staining technique using confocal laser scanning microscopy showed bacterial cells and the glycocalyx matrix, providing visual evidence for the presence of biofilms on tonsils.
  
  
  CONCLUSIONS
  Using a novel visualization approach in single sections of human mucosal tissue, the presence of biofilms was demonstrated on tonsils in most (17/24 [70.8%]) patients with tonsillitis.
authors.affiliation: Department of Otorhinolaryngology-Head and Neck Surgery, Leiden University Medical Center, Leiden University, Leiden, the Netherlands. romain.kania@lrb.ap-hop-paris.fr
authors.forename: Romain E
authors.initials: RE
authors.lastname: Kania
authors.forename: Gerda E M
authors.initials: GE
authors.lastname: Lamers
authors.forename: Marcel J
authors.initials: MJ
authors.lastname: Vonk
authors.forename: Patrice Tran Ba
authors.initials: PT
authors.lastname: Huy
authors.forename: Pieter S
authors.initials: PS
authors.lastname: Hiemstra
authors.forename: Guido V
authors.initials: GV
authors.lastname: Bloemberg
authors.forename: Jan J
authors.initials: JJ
authors.lastname: Grote
mesh_terms: D001419:Bacteria / Q000166:cytology / Q000302:isolation & purification* / Q000648:ultrastructure; D018441:Biofilms; D002648:Child; D003208:Concanavalin A; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives; D005456:Fluorescent Dyes; D019276:Glycocalyx / Q000382:microbiology* / Q000648:ultrastructure; D006801:Humans; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D014066:Palatine Tonsil / Q000382:microbiology* / Q000601:surgery; D011446:Prospective Studies; D014069:Tonsillitis / Q000382:microbiology*
chemical_list: D005456:Fluorescent Dyes; C042784:fluorescein isothiocyanate-concanavalin A; D003208:Concanavalin A; D016650:Fluorescein-5-isothiocyanate