Comparison of 2 fixatives in the porcine colon for in situ microbiota studies

Fixation is the first step towards preservation of tissues and can impact downstream histological applications. Historically, formalin has been the fixative of choice in both research and clinical settings due to cost, accessibility, and broad applicability. Here, we describe a method for collection of porcine colon, and compare the usage of Carnoy's solution (CS) to a 10% neutral buffered formalin (NBF) in tissue fixation. Consecutive colon samples were collected from 24 four-wk-old piglets and fixed in CS for 45 min or NBF for 24 h. We measured the thickness of the inner mucus layer using Alcian Blue stain and found thicker inner mucus layers in porcine colons fixed with CS as compared to NBF (P < 0.0001). Carnoy's solution-fixed colon exhibited greater bacterial cell counts than NBF-fixed colon (P < 0.0022) after labeling with an eubacterial probe in fluorescent in situ hybridization (FISH). No difference was observed between the mucosal height (P = 0.42) and number of goblet cells (P = 0.66) between the 2 fixatives. From this, we concluded CS is more suitable than NBF for the preservation of the mucus layer and the associated mucosal bacteria in the porcine colon without compromising on overall tissue morphology. This study provides a useful sampling and fixation methodology for histology studies in the porcine gastrointestinal tract, and may be beneficial to microbiota, pathology, and nutrition studies.