Monocytes were observed in the blood of inshore hagfish Eptatretus burgei. The monocytes were round or oval, high nucleus/cytoplasm ratio, and have several round or oval eosinophilic granules, which show dark red or blackish red color when stained with May-Grünwald (MG), Giemsa, and MG-Giemsa stain. The granules were positive for alkaline phosphatase, acid phosphatase, and α-naphthyl butyrate esterase. Also, the granules show metachromatic (reddish purple) with toluidine blue. However, ꞵ-glucuronidase, α-naphthyl acetate esterase, naphthol AS-D chloroacetate esterase and peroxidase were not detected in the monocytes, and negative for periodic acid Schiff reaction, alcian blue, Sudan black B, Sudan III, and oil red O. The monocytes engulfed many yeast particles (zymosan).

Structure and development of eosinophilic granules (EG1) in eosinophils from larva (ammocoetes) of far eastern brook lamprey Lethenteron reissneri were speculated. EG1 is stratified granules (two-layer) and consists of inner eosinophilic layer (L0) and chromophobic outer layer (L1). Three subtypes of EG1 are identified based on the optical artificial image (OAI) of inclusion structure (IS) in L0: EG1a, EG1b, and EG1c. The EG1a had no OAI (probably no IS). The EG1b and EG1c contain IS in L0. The OAI of both EG1b and EG1c were larger than IS. The former was round or oval chromophobic area (OAI-1), and the latter was expanded and rugged (three-dimensional) image (OAI-2) surrounded with OAI-1. EG1a was thought to be a prototype of EG1, which would develop into EG1b and then EG1c.

Neutrophil granules (NG) of West African lungfish Protopterus annectens were classified into two types of stratified [two-layer; inner layer (L0) and outer layer (L1)] granules (type A, NG-A; type B, NG-B). The L1 of NG-A and both layers of NG-B were chromophobic, and L0 of NG-A showed polychromatophilic [eosinophilic or basophilic (orthomethylenophilic or metaazurophilic)]. The L0 of NG-A showed metachromatic (reddish purple) with toluidine blue. On the other hand, L0 of NG-B were positive for acid phosphatase, α-naphthyl acetate esterase, α-naphthyl butyrate esterase and naphthol AS-D chloroacetate esterase. Both types of granules were negative in periodic acid Schiff reaction, alcian blue, Sudan black B, Sudan III, and oil red O. Alkaline phosphatase, β-glucuronidase, and peroxidase were not detected in either granule.

The term “neutrophilic” and “neutrophilic granule” was first introduced by Paul Ehrlich to identify specific granules of major polymorphonuclear leukocytes (neutrophils). He used an original staining method, triacid stain, for this purpose. The staining solution, called “neutral mixture”, which was mixture of acidic dye solution and basic dye solution, with soluble form in water (not neutral dye). In this paper, we speculate the structure and characteristics of the dye complex in the neutral mixture. The dye complex has free acidic groups and is expected to behave as an acidic dye. The dye complex bound to the neutrophilic granules stains the granules with the color tone of the dye complex (purple) because the acid and basic dyes do not dissociate. On the other hand, in the vicinity of the eosinophilic granules and nucleus, the dye complex dissociates into acid and basic dyes, and the former bind to the acid dye and the latter to the basic dye. It was inferred that this dye complex is not formed during staining with stains containing neutral dyes, such as May-Grünwald (methylene blue eosinate), Wright (polychromtic methylene blue eosinate), and Giamsa (containing azure II eosinate).