Kondo Masakazu

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.

Identification of leukocytes on tissue sections is important to elucidate the mechanism of swim bladder lesions. To determine the best fixative solution for a koi carp swim bladder, the swim bladders were fixed in 10% formalin, Bouin's, MFAA and Davidson's solution. The swim bladders fixed in MFAA or Davidson's solution were severely detached and twisted, whereas those fixed in 10% formalin and Bouin's solution kept their external shape. However, the majority of the 10% formalin-fixed specimens showed detachment of the tunica interna from the tunica externa under the light microscope. Therefore, Bouin’s solution was determined to be the most suitable fixing solution for the swim bladder. Imprints (head kidney-touched slides glass were fixed with Bouin's solution) and tissue sections of head kidney fixed in Bouin’s solution were stained with Mayer’s hematoxylin and eosin (HE) and May-Grünwald·Giemsa (MGG), and the best staining method for leukocytes identification was investigated. In the HE-stained specimens, identification of leukocytes by staining was difficult. On the other hand, MGG-stained specimens could be identified by staining. Fixation with Bouin's solution and MGG staining was determined to be the most suitable method for leukocytes identification in the swim bladder.

Artur Pappenheim reported a poor description of the morphology of blood neutrophils from lamprey (Lampetra planeri； adult and ammocoetes) and hagfish (Myxine glutinosa) in Folia Haematologica (volume 8, 1909). Here, we inferred on the morphological characteristics of cyclostome neutrophils observed by Pappenheim based on his descriptions and our previous reports. He recognized neutrophilic leukocytes (mature neutrophils) in lamprey (adult and ammocoetes) and hagfish, and neutrophilic myelocytes (immature neutrophils) in ammocoetes and hagfish. This means the existence of specific granules and azure granules. However, the specific granules were considered as an inner layer (L0) of the striated granule (NG2), and the azure granules outer (L1) layer of the striated granule (NG2). The specific granules (=L0 of NG2) would probably have been stained with triacid (purple), acid dyes, and May-Grünwald·Giemsa (MGG； purple), but not with methylgreen-pyronine (MP). The azure granules (=L1 of NG1a) would also have stained purple with MGG, but negative with triacid, acidic dyes and MP.

The monocytes of lampreys [Lethenteron camtschaticum (adult), L. reissneri (adult), L. hattai (adult, ammocoetes)] were round or oval, low nucleus/cytoplasm ratio, and have striated (two-layered) granules (MoG). The MoG consisted of a basophilic inner layer (L0) and a chromophobic outer layer (L1). The L0 were positive for acid phosphatase, α-naphthyl acetate esterase, naphthol AS-D chloroacetate esterase and Sudan black B. Also, the L0 showed orthochromatic (blue) with toluidine blue. However, ꞵ-glucuronidase, α-naphthyl butyrate esterase and peroxidase were not detected in the monocytes, which were negative for periodic acid Schiff reaction, alcian blue, Sudan III, and oil red O. Kenji Kiyono reported the two types of monocytes (as blood histocytes), real and dubious monocytes, from the blood of hagfish Eptatretus burgeri. He observed both monocyte types in grown-up hagfish, but only real monocytes in undeveloped (details not stated) hagfish. His real monocytes in undeveloped hagfish were speculated as the monocytes of lamprey (mistaking the specimen), and the real monocytes in grown-up hagfish as poorly stained neutrophils. The dubious monocytes of Kiyono were considered as the real monocytes of hagfish.