Enhancing Histological Techniques for Small Crustaceans: Evaluation of Fixation, Decalcification, and Enzymatic Digestion in Neocaridina Shrimp

Abstract

Histological techniques are essential for studying small crustaceans', such as Neocaridina shrimp, anatomy and physiology. However, their small size and rapid tissue autolysis present challenges for fixation and processing. This study aimed to optimize histological methods for Neocaridina shrimp by evaluating different protocols for fixation, decalcification, and enzymatic digestion. Shrimp were fixed using 10% neutral-buffered formalin (NBF) and Bouin's or Davidson's fluid with or without modifications such as trypsin digestion, decalcification, or abdomen removal. Tissue preservation, section quality, and staining properties were assessed. Davidson's fluid consistently gave generally acceptable fixation results, with minimal autolysis and good tissue preservation. Trypsin digestion increased tissue damage and autolysis, particularly in the liver and pancreas. Decalcification improved the quality of the sections; however, it increased autolysis and resulted in less specific staining. The optimal protocol involved the removal of the abdomen, followed by fixation in Davidson's fluid and decalcification, which resulted in rapid penetration of the fixative, minimal autolysis, and a beneficial effect on staining. This study highlights the importance of adapting histological methods to the specific characteristics of small crustaceans and provides a basis for future research on Neocaridina shrimp. Implementing these optimized techniques will improve the quality and reliability of histological analyses in crustacean research, deepening the understanding of their biology and facilitating their use as model organisms in various scientific fields.

Keywords: crustacean histology; decalcification; fixation; protocol optimization.

Figure 1

Shrimp tissue comparison for unmodified fixation variants. Green frame: microscopic images of: (A1–C1)—hepatopancreas, (A2–C2)—nervous tissue, (A3–C3)—muscle tissue. HE stain, scale 100 μm. Red frame: digital image analysis of stainability of tissue (raw image, image with mask applied, color histogram of image, and mean RGB value of image as color).

Figure 2

Shrimp tissue comparison for enzymatic digestion fixation variants. Green frame: microscopic images of: (A1–C1)—hepatopancreas, (A2–C2)—nervous tissue, (A3–C3)—muscle tissue. HE stain, scale 100 μm. Red frame: digital image analysis of stainability of tissue (raw image, image with mask applied, color histogram of image, and mean RGB value of image as color).

Figure 3

Shrimp tissue comparison for decalcification fixation variants. Green frame: microscopic images of: (A1–C1)—hepatopancreas, (A2–C2)—nervous tissue, (A3–C3)—muscle tissue. HE stain, scale 100 μm. Red frame: digital image analysis of stainability of tissue (raw image, image with mask applied, color histogram of image, and mean RGB value of image as color).

Figure 4

Shrimp tissue comparison for abdomen removal fixation variants. Green frame: microscopic images of: (A1–C1)—hepatopancreas, (A2–C2)—nervous tissue, (A3–C3)—muscle tissue. HE stain, scale 100 μm. Red frame: digital image analysis of stainability of tissue (raw image, image with mask applied, color histogram of image, and mean RGB value of image as color).