Microbiota from IBD patients form larger biofilms than microbiota from healthy controls and contains higher intracellular iron. A, Human microbiota biofilms (healthy; Crohn’s Disease, CD: ulcerative colitis UC) were grown on the Calgary Biofilm Device. Confocal laser scanning microscopy of biofilms was performed, the extracellular matrix of biofilms was stained by acridine orange (green) and bacteria DNA with TO-PRO-3 (red). Scale bars correspond to 70 μm. B, Biofilms from IBD patients had a higher relative biomass compared to healthy biofilms (Crystal violet staining, n = 145 to 313 biofilms per group). C, Biofilms from IBD patients contained higher numbers of colony forming units (CFU per biofilm) compared to biofilms from healthy biofilms (n = 21 to 24 biofilms per group). D, Equivalent numbers of bacteria from IBD biofilms contained higher concentration of intracellular iron compared to healthy biofilms (n = 3 3 biofilms per group). B, 1-way ANOVA followed by Dunnett’s tests. C, D, Kruskal-Wallis followed by Dunn’s tests versus healthy group _aP < 0.01, _bP < 0.001