Triacid staining solution (neutral mixture) contains dye complex. Here, we speculate on the structure and characteristics of the dye complex in various triacid staining solutions. It was inferred that the dye complexes in the Ehrlich (methyl green + 2 acid fuchsin, methyl green + 2 orange G, methyl green + acid fuchsin + orange G), Romanowsky (eosin + methylene blue), and Pappenheim (eosin + methylene azure) triacid stains behave as acidic dyes, and that the dye complexes do not dissociate against weakly acidophilic objects, resulting in staining with a complex color (purple). Therefore, it can be said that the objects to which the dye complexes bind without dissociation do not need to be basophilic. When the stained material is highly acidophilic or basic, the dye complex dissociates, and the acidophilic object is stained by the acidic dye and the basiophilic object by the basic dye. The dissociation of dye complexes depends on the degree of acidophilicity and basophilicity of the stained material, and the dissociation depends on the types of dye composing the dye complexes. The dye complex in a triacid stain can be defined as a purple acidic dye in which the bonds between the acidic and basic dyes in the complex can be broken, depending on the acidophilic and basophilic strength of the object.

Previously, we speculated on the staining principle of triacid staining solution (neutral mixture) containing a dye complex. The principle could be explained solely by the relationship between the stained object and the dye complex. We propose here to extend the staining principles of triacid stain to explain the staining principles of other dye mixtures such as May-Grünwald (MG), Giemsa and MG-Giemsa. In a mixture of acidic and basic dyes, the staining characteristics of the object were presumably determined by the degree of stainability (degree of acidophilicity or basophilicity) of the object, the degree of stainability of both dyes (degree of basophilicity of the acidic dye and degree of acidophilicity of the basic dye), and the ratio and concentration of each dye.

‘Hikoshima-haruna’ (Brassica rapa var. narinosa) is a local traditional vegetable in Shimonoseki (Yamaguchi, Japan). The mature leaves are edible and much of the harvest is processed into pickles for consumption in the Shimonoseki area. To expand the consumption of ‘Hikoshima-haruna’, we verified the use of ‘Hikoshima-haruna’ powder in fish paste products as a part of the utilization research. We prepared heated gels (Kamaboko) mixed with 0-10% ‘Hikoshima-haruna’ powder and measured their breaking strength. The Kamaboko containing 1.0-4.0% powder exhibited a bright green color, preserving the characteristics of ‘Hikoshima-haruna’ as a plant with bright green leaves. Although the breaking strength of the two-step heating (30℃ for 30 min followed by 85℃ for 20 min) gels tended to decrease with increasing powder concentration, the 1.0% powder concentration exhibited a slightly greater breaking strength than the other concentrations. Therefore, when applying ‘Hikoshima-haruna’ powder to fish paste products, its use as Kamaboko at a powder concentration of 1.0% was considered appropriate.