Improved fixation of the whole bodies of fish by a double-fixation method with formalin solution and Bouin's fluid or Davidson's fluid

Abstract

To prevent fixation defects or artifacts in the whole bodies of fish caused by conventional fixatives, such as formalin solution, Bouin's fluid (BF), and Davidson's fluid (DF), the optimal fixatives and fixing method were examined. An improved method of fixing the whole bodies of fish was examined that makes use of a combination of 20% formalin and BF or DF. The fixatives were examined with four representative tissues, i.e., the gill, liver, intestinal tract, and kidney, to evaluate end points including the appearance of degraded tissues and artifacts caused by each fixative, overall morphological clarity of nuclei, staining intensity, and integrity of the other tissues. The best results were obtained when the fresh whole bodies were initially fixed in 20% formalin (primary fixation) at 4°C for 1 h and subsequently fixed in BF for 5 h at 4°C (secondary fixation). Therefore, the current findings led the authors to conclude that the combination of primary fixation with 20% formalin at 4°C for 1 h and secondary fixation with BF at 4°C for 5 h was suitable for fixation of the whole bodies of fish.

Keywords: 20% formalin; Bouin’s fluid; double fixation; fish; fixative; histopathology.

Fig. 1.

The histopathological findings observed in the gills of fish fixed with conventional fixatives and improved fixatives. 1a: Fixed with 20% formalin (FS) only for 6 h at 4°C. Well-preserved gill filaments are observed. 1b: Fixed with Bouin’s fluid (BF) only for 6 h at 4°C. The epithelium detached artifactually from the capillary, resulting in formation of a space in the gill lamella. 1c: Fixed with BF for 5 h after 20% FS fixation for 1 h at 4°C. Almost well-preserved gill lamellae are observed.

Fig. 2.

The histopathological findings observed in the livers of fish fixed with conventional fixatives and improved fixatives. 2a: Fixed with 20% FS only for 1 h at 4°C. Though well-fixed liver is observed, a portion with an artifactual defect is apparent around the gallbladder. The area with the defect is rather wide in this sample. 2b: Fixed with BF only for 6 h at 4°C. A structure similar to that in 2a is observed. However, the area with the defect is smaller than that with 20% FS only fixation. 2c: Fixed with BF for 5 h after 20% FS fixation for 1 h at 4°C. An almost well-fixed liver is observed, and a portion with an artifactual defect is apparent around the gallbladder. However, the area with the defect is smaller than those with 20% FS only fixation.

Fig. 3.

The histopathological findings observed in the intestinal tracts of fish fixed with conventional fixatives and improved fixatives. 3a: Fixed with 20% FS only for 1 h at 4°C. A defectively fixed region caused by autolysis is observed. 3b: Fixed with BF only for 6 h at 4°C. Though a well-fixed intestinal tract is observed, space formation is prominent as a result of lifting of the mucosal epithelium. 3c: Fixed with BF for 5 h after 20% FS fixation for 1 h at 4°C. A well-fixed intestinal tract is observed.

Fig. 4.

The histopathological findings observed in the kidney of fish fixed with conventional fixatives and improved fixatives. 4a: Fixed with 20% FS only for 1 h at 4°C. A well-fixed structure is observed. 4b: Fixed with BF for 6 h at 4°C. Shrunken epithelia of urinary tubules and space formation around the renal tubules are observed. 4c: Fixed with BF for 5 h after 20% FS fixation for 1 h at 4°C. The similar figure is also observed; however, it looks rather slight.

Fig. 5.

The histopathological findings observed in the other organs or tissue of fish fixed with conventional fixatives and improved fixatives. 5a: Skin fixed with 20% FS only for 1 h at 4°C. A well-fixed structure of the skin is observed. 5b: Skin fixed with DF for 6 h at 4°C. Lifting of the epithelium is observed, resulting in space formation under the epidermis or dermis. 5c: Skin fixed with BF for 5 h after 20% FS fixation for 1 h at 4°C. Artifactual space formation similar to that in 5b is also observed. 5d: Vessels fixed with 20% FS only for 1 h at 4°C. Though hemolysis is observed, shrunken erythrocytes are not prominent. 5e: Vessels fixed with BF only for 6 h at 4°C. Hemolytic and shrunken erythrocytes are prominent. 5f: Vessels fixed with BF for 5 h after 20% FS fixation for 1 h at 4°C. Shrunken erythrocytes are prominent.