We studied 126 blood samples of wild horseshoe crabs (Tachypleus tridentatus)that have been kept in captivity for 4-7 years. The blood was tested with two kinds of anticoagulant (0.02 M theophylline or 0.01 M NEM) and three kinds of fixatives (methanol, 95% ethanol and 10% formalin). The number of hemocyte was calculated by disposable hemocytometer, and stained hemocyte blood smear were analyzed. The best results of treatments were with 0.02 M theophylline anticoagulant and 20X formalin to fix hemocyte. The types of hemocyte were discriminated by the staining property of hemocyte intracellular particles through DIFF Quik Stain. The hemocytes cuonts were 24,955 ± 15,968/uL for male and 19,004 ± 13,755/uL for female. Stained hemocyte was classed into granule flattened cells, degranulated flattened cells, and contract flattened cells with a ratio of 95:3:2.
In the past staining blood cell smears of horseshoe crab was difficult. Hemocyte easily explodes in vitro, which in turn releases agglutinating particles. Our method makes staining possible and provides a new way to classify horseshoe crab blood cells.