231
J. Anat. (1978), 125, 2, pp. 231-235 With 3 figures Printed in Great Britain
Cytochemistry of blood cells in two West African amphibians A. E. CAXTON-MARTINS
Division of Human Biology and Behaviour, Faculty of Health Sciences, University of Ife, Ile-Ife, Nigeria
(Accepted 28 December 1976) INTRODUCTION
A great deal of information is available concerning the cytochemistry of blood cells in mammals (see Bainton & Farquhar, 1968), birds (Merkal & Mora, 1962) and reptiles (Efrati, Nir & Yaari, 1970). To the best knowledge of the author, however, there is no such information available for amphibia and fish, and so a study was undertaken of the cell populations in blood smears of two West African amphibians - the toad (Bufo regularis) and the frog (Rana temporaris) - with particular reference to lipid, glycogen and peroxidase, acid phosphatase and alkaline phosphatase activities. MATERIALS AND METHODS
Thirty toads of 40-50 g and twenty frogs of 60-75 g were used. The animals were usually caught at night or very early in the morning on the University Campus and used immediately. Under ether anaesthesia the heart was exposed, and blood taken from it with a clean syringe without anticoagulants. Smears were made from the blood before it clotted. Freshly prepared, air-dried smears were fixed in absolute methanol for 10 minutes, stained in Giemsa solution (diluted 1:1 in distilled water) for 10 minutes, washed in tap water for 5-10 minutes, allowed to dry in air and mounted in a neutral medium. The following histochemical investigations were undertaken: (i) For lipid, Sudan black B staining (Sheehan & Storey, 1947). (ii) For glycogen, periodic acid Schiff (PAS) staining (McManus, 1946; Hayhoe, Quaglino & Flemans, 1960). (iii) For peroxidase, the method of Washburn (1928) and Jacobs (1958). (iv) For acid phosphatase, the method of Kaplow & Burstone (1964). (v) For alkaline phosphatase, the method of Ackerman (1962). RESULTS
Giemsa-stained blood smears from both amphibians revealed a complex population of nucleated erythrocytes, polymorphic granulocytes, monocytes and lymphocytes. Erythrocytes In the frog these cells are ellipsoidal. The round or slightly ovoid centrally placed nucleus is surrounded by agranular, slightly eosinophilic cytoplasm. In the toad the erythrocytes are elliptical, but less elongated than in the frog:
232
A. E. CAXTON-MARTINS
Fig. 1. Frog blood smear after Giemsa staining showing the more elongated, elliptical nucleated erythrocytes. x 1000.
|; .. ...........w
Fig. 2. Blood smear showing the less elongated, elliptical nucleated erythrocytes of the toad after Giemsa staining. x 1000.
otherwise the cells are similar. A few erythrocytes were seen to contain a parasitic body within their cytoplasm which displaced the nucleus to an eccentric position; such erythrocytes were more elongated than usual. Thrombocytes In both species these cells are ellipsoidal with a round, centrally placed nucleus. The cytoplasm is agranular and slightly eosinophilic.
Cytochemistry of amphibian blood cells
233
3 Fig. 3. Intense peroxidase activity in erythrocytes of toad blood smear indicated by a generalized increase in density of the cytoplasm. One erythrocyte shows the presence of a parasitic body in its cytoplasm with the nucleus displaced from the usual central position. x 1000.
Leucocytes In both species granulocytes (neutrophils, eosinophils and basophils) and nongranular leucocytes (monocytes and lymphocytes) closely resemble their mammalian counterparts.
Cytochemistry Lipid This is visualized as a blackish precipitate in the granulocytes and monocytes of both species. Erythrocytes, thrombocytes and lymphocytes were not reactive.
Glycogen Bright red particles indicate the presence of this metabolite. Granulocytes and monocytes were usually strongly reactive, while some lymphocytes showed a few positive granules within their cytoplasm. Neither erythrocytes nor lymphocytes were reactive.
Peroxidase Very intense peroxidase activity, indicated by a yellow-brown precipitate, was present in the cytoplasm of erythrocytes, thrombocytes, granulocytes and monocytes. Lymphocytes, however, gave no reaction in either species.
Acid phosphatase Bluish granules were seen in the cytoplasm of granulocytes and monocytes in both species. Erythrocytes, thrombocytes and lymphocytes showed no activity.
234
A. E. CAXTON-MARTINS
Alkaline phosphatase A faint bluish precipitate was seen in the cytoplasm of granulocytes and monocytes in both species. Erythrocytes, thrombocytes and lymphocytes were not reactive. DISCUSSION
The elliptical shape of amphibian erythrocytes has been known for several years (see Jordan, 1919), but it does not appear to have been reported before that the erythrocytes of the frog are more elongated than those of the toad. The distribution of glycogen and lipids agrees with earlier findings in man (Hayhoe, 1953; Hayhoe, Quaglino & Flemans, 1960) and in the crocodile and wall gheko (Caxton-Martins, 1977). Peroxidase activity was intense in all blood cells except lymphocytes in both the amphibians studied here, suggesting that erythrocytes, granulocytes and monocytes are derived from a common progenitor cell in these animals. The distribution of acid and alkaline phosphatases in granulocytes and monocytes is similar to that in other vertebrate classes. SUMMARY
Lipid, glycogen, peroxidase, acid and alkaline phosphatases were studied in the cells of blood smears in the toad and frog. While lipid and glycogen were largely confined to granulocytes and monocytes, peroxidase activity was intense in all cell types except lymphocytes in both species. The author wishes to express his profound gratitude to Professor T. Adesanya Ige Grillo for financial assistance as well as for providing excellent laboratory facilities. He is also grateful to Miss M. S. Akinosho and Mr F. N. Mekoma for secretarial and technical assistance. REFERENCES ACKERMAN, G. A. (1962). Substituted naphthol AS phosphate derivatives for the localization of leucocyte alkaline phosphatase activity. Laboratory Investigation 11, 563. BAINTON, D. F. & FARQuHAR, M. G. (1968). Differences in enzyme content of azurophil and specific granules of polymorphonuclear leukocytes. I. Histochemical staining of bone marrow smears. Journal of Cell Biology 39, 286-298. CAXTON-MARTINS, A. E. (1977). Cytochemistry of blood cells in peripheral smears of some West African reptiles. Journal of Anatomy. 124, 393-400. EFRATI, P., NIR, E. & YAARI, A. (1970). Morphological and cytochemical observations on cells of the haemopoietic system of Agama stellio (Linnaeus). Israel Journal of Medical Sciences 6, 23-31. HAYHOE, F. G. J. (1953). The cytochemical demonstration of lipids in blood and bone-marrow cells. Journal of Pathology and Bacteriology 65, 413-421. HAYHOE, F. G. J., QUAGLINO, D. & FLEMANS, R. J. (1960). Consecutive use of Romanowsky and periodic-acid-Schiff techniques in the study of blood and bone marrow cells. British Journal of Haematology 6, 23-25. JACOBS, A. (1958). Staining for leucocyte peroxidase. Lancet 1, 697. JORDAN, H. E. (1919). The histology of the blood and the red bone marrow of the leopard frog, Rana pipiens. American Journal of Anatomy 25, 437. KAPLOW, L. S. & BURSTONE, M. S. (1964). Cytochemical demonstration of acid phosphatase in haematopoietic cells in health and in various haematological disorders using azo-dye techniques. Journal of Histochemistry and Cytochemistry 12, 805. MCMANUS, J. F. A. (1946). Oxidation of mucin with periodic-acid producing the capacity of stain with Schiff's reagent. Nature 158, 202.
Cytochemistry of amphibian blood cells
235
MERKAL, R. S. & MORA, E. C. (1962). Cytochemistry of erythrocytes and leukocytes of the white Leghorn chicken. Experimental and Molecular Pathology 1, 497-508. SHEEHAN, H. L. & STOREY, G. W. (1947). An improved method of staining leucocyte granules with Sudan Black B. Journal ofPathology and Bacteriology 59, 336-337. WASHBURN, A. H. (1928). A combined peroxidase and Wright's stain for routine blood smears. Journal of Laboratory and Clinical Medicine 14, 246-250.
J. Anat. (1978), 125, 2, pp. 231-235 With 3 figures Printed in Great Britain
Cytochemistry of blood cells in two West African amphibians A. E. CAXTON-MARTINS
Division of Human Biology and Behaviour, Faculty of Health Sciences, University of Ife, Ile-Ife, Nigeria
(Accepted 28 December 1976) INTRODUCTION
A great deal of information is available concerning the cytochemistry of blood cells in mammals (see Bainton & Farquhar, 1968), birds (Merkal & Mora, 1962) and reptiles (Efrati, Nir & Yaari, 1970). To the best knowledge of the author, however, there is no such information available for amphibia and fish, and so a study was undertaken of the cell populations in blood smears of two West African amphibians - the toad (Bufo regularis) and the frog (Rana temporaris) - with particular reference to lipid, glycogen and peroxidase, acid phosphatase and alkaline phosphatase activities. MATERIALS AND METHODS
Thirty toads of 40-50 g and twenty frogs of 60-75 g were used. The animals were usually caught at night or very early in the morning on the University Campus and used immediately. Under ether anaesthesia the heart was exposed, and blood taken from it with a clean syringe without anticoagulants. Smears were made from the blood before it clotted. Freshly prepared, air-dried smears were fixed in absolute methanol for 10 minutes, stained in Giemsa solution (diluted 1:1 in distilled water) for 10 minutes, washed in tap water for 5-10 minutes, allowed to dry in air and mounted in a neutral medium. The following histochemical investigations were undertaken: (i) For lipid, Sudan black B staining (Sheehan & Storey, 1947). (ii) For glycogen, periodic acid Schiff (PAS) staining (McManus, 1946; Hayhoe, Quaglino & Flemans, 1960). (iii) For peroxidase, the method of Washburn (1928) and Jacobs (1958). (iv) For acid phosphatase, the method of Kaplow & Burstone (1964). (v) For alkaline phosphatase, the method of Ackerman (1962). RESULTS
Giemsa-stained blood smears from both amphibians revealed a complex population of nucleated erythrocytes, polymorphic granulocytes, monocytes and lymphocytes. Erythrocytes In the frog these cells are ellipsoidal. The round or slightly ovoid centrally placed nucleus is surrounded by agranular, slightly eosinophilic cytoplasm. In the toad the erythrocytes are elliptical, but less elongated than in the frog:
232
A. E. CAXTON-MARTINS
Fig. 1. Frog blood smear after Giemsa staining showing the more elongated, elliptical nucleated erythrocytes. x 1000.
|; .. ...........w
Fig. 2. Blood smear showing the less elongated, elliptical nucleated erythrocytes of the toad after Giemsa staining. x 1000.
otherwise the cells are similar. A few erythrocytes were seen to contain a parasitic body within their cytoplasm which displaced the nucleus to an eccentric position; such erythrocytes were more elongated than usual. Thrombocytes In both species these cells are ellipsoidal with a round, centrally placed nucleus. The cytoplasm is agranular and slightly eosinophilic.
Cytochemistry of amphibian blood cells
233
3 Fig. 3. Intense peroxidase activity in erythrocytes of toad blood smear indicated by a generalized increase in density of the cytoplasm. One erythrocyte shows the presence of a parasitic body in its cytoplasm with the nucleus displaced from the usual central position. x 1000.
Leucocytes In both species granulocytes (neutrophils, eosinophils and basophils) and nongranular leucocytes (monocytes and lymphocytes) closely resemble their mammalian counterparts.
Cytochemistry Lipid This is visualized as a blackish precipitate in the granulocytes and monocytes of both species. Erythrocytes, thrombocytes and lymphocytes were not reactive.
Glycogen Bright red particles indicate the presence of this metabolite. Granulocytes and monocytes were usually strongly reactive, while some lymphocytes showed a few positive granules within their cytoplasm. Neither erythrocytes nor lymphocytes were reactive.
Peroxidase Very intense peroxidase activity, indicated by a yellow-brown precipitate, was present in the cytoplasm of erythrocytes, thrombocytes, granulocytes and monocytes. Lymphocytes, however, gave no reaction in either species.
Acid phosphatase Bluish granules were seen in the cytoplasm of granulocytes and monocytes in both species. Erythrocytes, thrombocytes and lymphocytes showed no activity.
234
A. E. CAXTON-MARTINS
Alkaline phosphatase A faint bluish precipitate was seen in the cytoplasm of granulocytes and monocytes in both species. Erythrocytes, thrombocytes and lymphocytes were not reactive. DISCUSSION
The elliptical shape of amphibian erythrocytes has been known for several years (see Jordan, 1919), but it does not appear to have been reported before that the erythrocytes of the frog are more elongated than those of the toad. The distribution of glycogen and lipids agrees with earlier findings in man (Hayhoe, 1953; Hayhoe, Quaglino & Flemans, 1960) and in the crocodile and wall gheko (Caxton-Martins, 1977). Peroxidase activity was intense in all blood cells except lymphocytes in both the amphibians studied here, suggesting that erythrocytes, granulocytes and monocytes are derived from a common progenitor cell in these animals. The distribution of acid and alkaline phosphatases in granulocytes and monocytes is similar to that in other vertebrate classes. SUMMARY
Lipid, glycogen, peroxidase, acid and alkaline phosphatases were studied in the cells of blood smears in the toad and frog. While lipid and glycogen were largely confined to granulocytes and monocytes, peroxidase activity was intense in all cell types except lymphocytes in both species. The author wishes to express his profound gratitude to Professor T. Adesanya Ige Grillo for financial assistance as well as for providing excellent laboratory facilities. He is also grateful to Miss M. S. Akinosho and Mr F. N. Mekoma for secretarial and technical assistance. REFERENCES ACKERMAN, G. A. (1962). Substituted naphthol AS phosphate derivatives for the localization of leucocyte alkaline phosphatase activity. Laboratory Investigation 11, 563. BAINTON, D. F. & FARQuHAR, M. G. (1968). Differences in enzyme content of azurophil and specific granules of polymorphonuclear leukocytes. I. Histochemical staining of bone marrow smears. Journal of Cell Biology 39, 286-298. CAXTON-MARTINS, A. E. (1977). Cytochemistry of blood cells in peripheral smears of some West African reptiles. Journal of Anatomy. 124, 393-400. EFRATI, P., NIR, E. & YAARI, A. (1970). Morphological and cytochemical observations on cells of the haemopoietic system of Agama stellio (Linnaeus). Israel Journal of Medical Sciences 6, 23-31. HAYHOE, F. G. J. (1953). The cytochemical demonstration of lipids in blood and bone-marrow cells. Journal of Pathology and Bacteriology 65, 413-421. HAYHOE, F. G. J., QUAGLINO, D. & FLEMANS, R. J. (1960). Consecutive use of Romanowsky and periodic-acid-Schiff techniques in the study of blood and bone marrow cells. British Journal of Haematology 6, 23-25. JACOBS, A. (1958). Staining for leucocyte peroxidase. Lancet 1, 697. JORDAN, H. E. (1919). The histology of the blood and the red bone marrow of the leopard frog, Rana pipiens. American Journal of Anatomy 25, 437. KAPLOW, L. S. & BURSTONE, M. S. (1964). Cytochemical demonstration of acid phosphatase in haematopoietic cells in health and in various haematological disorders using azo-dye techniques. Journal of Histochemistry and Cytochemistry 12, 805. MCMANUS, J. F. A. (1946). Oxidation of mucin with periodic-acid producing the capacity of stain with Schiff's reagent. Nature 158, 202.
Cytochemistry of amphibian blood cells
235
MERKAL, R. S. & MORA, E. C. (1962). Cytochemistry of erythrocytes and leukocytes of the white Leghorn chicken. Experimental and Molecular Pathology 1, 497-508. SHEEHAN, H. L. & STOREY, G. W. (1947). An improved method of staining leucocyte granules with Sudan Black B. Journal ofPathology and Bacteriology 59, 336-337. WASHBURN, A. H. (1928). A combined peroxidase and Wright's stain for routine blood smears. Journal of Laboratory and Clinical Medicine 14, 246-250.
