Background: Gastrointestinal (GI) mucus protects the epithelium and provides a niche for the mucosal microbiota. Tissue fixation methods may influence mucus integrity and the preservation of associated bacteria, which are critical for mucosal health studies.
Objective: To compare Carnoy’s solution (CS) and 10% neutral buffered formalin (NBF) for their ability to preserve GI mucus and mucus-associated bacteria in canine GI endoscopic biopsies. Animals: Six healthy laboratory-reared adult dogs.
Methods: Endoscopic biopsies from the stomach, ileum, and colon were fixed in CS or NBF. Tissue sections were stained with hematoxylin and eosin (H&E) and Alcian blue (AB) to quantify mucus (% AB staining via HALO). Fluorescence in situ hybridization (FISH) used the universal EUB338-Cy5 probe to identify and visualize mucosal bacteria. Labeled bacteria were counted across 5 mucosal fields at 400x using MetaMorph® software. Statistical analysis was performed using GraphPad Prism.
Results: Mucosal AB staining did not differ significantly between fixatives at any GI site or for all GI sites combined (P > 0.05). Total bacterial counts were similar in stomach and colon biopsies; however, CS-fixed ileum samples exhibited higher bacterial abundance (mean total bacteria/field = 775) vs. NBF-fixed ileum (mean total bacteria/field = 261, P < 0.05). Conclusions and Clinical Importance: Both CS and NBF adequately preserve GI mucus, but NBF may reduce bacterial abundance in the ileum, potentially affecting microbiota studies.
