Figure 3 Mucosal barrier in suppurative appendicitis. (A) Alcian blue/periodic acid Schiff stain. The appendiceal lumen is nearly completely filled with leucocytes in suppurative appendicitis. The mucus is absent even in cases where the mucosal integrity is preserved. Leucocytes are observed in direct contact with the epithelial surface without any visible mucus gap in between (white arrows). (B,C) Simultaneous photomicrograph of DAPI fluorescence (blue colour represents mainly nuclei of human cells) and orange fluorescence of the universal bacterial Eub 338 Cy3 probe are presented. The lumen of the intestine is filled with leucocytes, and scattered bacteria. Single bacteria and bacterial islands orange fluorescence are seen between leucocytes filling the lumen of the appendix. The inset (a) in B shows an aphthoid epithelial defect with bacteria attached to the submucosa (Eub 338 Cy3 orange fluorescence). The arrows in the C point towards a more extended epithelial defect. (D) Simultaneous multi‐colour hybridisation. The bacteria hybridised with the Bac 303 Cy3 probe (Bacteroides) are orange and the bacteria hybridised with the Fprau Cy5 probe (Fusobacterium prausnitzii) are red, all other bacteria are stained with FITC and appear green. The insertion (b) shows a photomicrograph of a microabscess within the same section. The infiltrating bacteria and the bacteria within abscess are of various microbial species and the numbers of single bacterial species vary significantly.