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Cytochemical reactions of normal and starving Amoeba proteus
206 BRIEF
CYTOCHEMICAL
REACTIONS
NOTES
OF NORMAL
AND
STARVING
AMOEBA PROTEUS
Department
II. LOCALIZATION
OF MIXERALS
IDA M. HELLER’j*
and M. J. KOPAC
ofBiology,
of
Graduate School Arts and Science, New York University, New York, N.Y., U.S.A. Received May 2, 1956
SEVERAL minerals have been studied in normal and starved amoebas by cytochemical means. The results from this work confirm and extend the results obtained in previous work in which the microincinerative method was used for mineral localization [6]. Materials and Methods.--The manner of handling, e.g., culturing, embedding of the normal and starved amoebas, has been described [5]. The fixative used in the following cytochemical procedures, unless otherwise indicated, consisted of 1 part neutral formalin and 9 parts absolute ethanol. Paraffin sections were cut at 4 p. Magnesium.-Sections and whole mounts of normal amoebas as well as whole mounts of amoebas starved for 1, 9, and 19 days were stained according to the Broda procedure [ 11. Calcium.-The Rabl test [la] for soluble calcium was applied to living unstarved amoebas as well as to amoebas starved for 1, 7, 20, and 22 days. The amoebas were fixed in a mixture of equal parts of 4 per cent ammonium oxalate and 25 per cent neutral formalin. A positive test is indicated by the appearance of calcium oxalate crystals in the specimen. The Manigault modification [9] of the CrCtin test [3] for the localization of calcium salts was applied to whole mounts of normal amoebas. Iron.-Sectioned and whole mount amoebas were tested for inorganic iron by the Turnbull blue method [8], as well as by the Prussian blue method [2]. For these tests, some amoebas were fixed in 10 per cent aqueous formalin, buffered at,pH 7, and the others with the alcoholic fixative described above. For the demonstration of organic iron, sections and whole mounts were first hydrolyzed in 4 per cent sulfuric acid in 95 per cent ethanol for 24 to 36 hours at 1 Work carried on as a Predoctoral Fellow stitutes of Health, Federal Security Agency. 2 Present address, Department of Surgery, School, 550 First Ave., New York 16, N.Y. Experimental
Cell Research 11
of the National New
York
Cancer
University
Institute,
National
Post-Graduate
In-
Medical
Cytochemical reactions of Amoeba proteus
207
Fig. 1. Portion of the cytoplasm of a whole mount amoeba which had been fixed in Rabl’s solution. Note the large calcium oxalate crystals. Approx. x 600. Fig. 2. Pqrtion of the cytoplasm of a whole mount amoeba which had been centrifuged prior to fixation in 25 per cent neutral formalin. Note the appearance of the many round refractive bodies. Apprax. x 600. Fig. 3. Centrifugal end of a whole mount amoeba which was fixed for 24 hours in Rabl’s solution. Note the numerous small crystals of calcium oxalate. Approx. x 600. Fig. 4. Centrifugal end of the same amoeba as in Fig. 3, a short time after sulfuric acid was applied. Fig. 5. Whole mount amoeba stratified prior to staining with Quinalizarin, showing a concentration of deeplv staining refractive bodies in the centrifugal end. Note particularly the darkly staining core and paler annulus seen in many of the refractive bodies. -Wrattan filter no. 58. Approx. x 800. Fig. 6. Whole mount amoebas treated with Titan yellow. Note the similarity between the appearance of these refractive bodies to those seen in the above figure. Wrattan filter no. 58. Approx. x 800. Experimenlal Cell Research 11
Ida M. Helfer and M. J. Kopac 35°C and then treated with either the Prussian blue reagent for 5 minutes 181, or with a freshly prepared solution of equal volumes of 1.5 per cent potassium ferrocyanide and 0.5 per cent hydrochloric acid [4]. As a check on the reactivity of the reagents, sections of rat liver and spleen were included in each series. Zinc.-The Mendel and Bradley method [ll] was applied to sections and whole mounts of normal amoebas as well as to whole mounts of amoebas starved for 1, 9, 17, and 19 days. A purple color indicates zinc and possibly copper. Copper.-The Waterhouse method [13] for the detection of copper was tested on both living and whole mount normal amoebas, as well as on whole mounts of amoebas which had been starved for 1, 9, and 20 days. Results.-Spot tests were carried out to determine whether the three dyes of the Broda test, Quinalizarin, Azo blue, and Titan yellow, specifically demonstrate magnesium. It was observed that magnesium chloride formed solid colored precipitates with all three of the alkalinized dyes, while calcium chloride formed flocculent precipitates. The color reactions of calcium and magnesium to Azo blue and Quinalizarin were practically indistinguishable. With alkalinized Titan yellow, the magnesium precipitate was bright red, and that of calcium pink and yellow. When these dyes were tested on filter paper previously impregnated with calcium distinguished or magnesium chlorides, again only the Titan yellow adequately between the two. The Titan yellow applied to sections and whole mounts produced an orange color in the cytoplasm and a deeper orange color in the nucleus and food vacuoles. The refractive bodies were seen to have a bright red core surrounded by a paler annulus (Fig. 6). Amoebas stained with Quinalizarin or Azo blue display the same pattern of color distribution in the refractive bodies (Fig. 5). Amoebas which had been starved for varying lengths of time prior to testing with the Broda stains showed no appreciable difference relative to each other or to the normal controls. Precipitated calcium oxalate was observed in amoebas treated with Rabl’s mixture (Fig. 1). The major portion of the calcium oxalate appears to be due to the calcium derived from the refractive bodies. On application of the reagent most of the refractive bodies disappear and calcium oxalate crystals appear. In amoebas stratified prior to fixation in Rabl’s mixture, the calcium oxalate crystals can be seen in the centrifugal end where the refractive bodies are normally found (cf. Figs. 2 and 3). The crystals were identified as calcium oxalate by their crystalline form and by their solubility. They are soluble in 2 per cent sulfuric acid (Fig. 4) and insoluble in 20 per cent acetic acid. Mast and Doyle [lo] reported the refractive bodies to be soluble in 20 per cent acetic acid. No significant differences in the occurrence or number of calcium oxalate crystals were noted between amoebas starved for 1, 7, 20, and 22 days, nor between them and the unstarved amoebas. Application of the modified C&in test to the centrifuged normal whole mounts [5] indicated the centrifugal end to be pinkish-brown in some or bluish or greenishbrown in others. The alkalinity of the reagent caused disruption and possibly a partial dissolution of the refractive bodies. A few intact refractive bodies were seen; of these, several contained a darkly colored pinkish or bluish interior with a paler annulus. With this reagent, a positive reaction for calcium is indicated by a blue Experimental
Cell Research 11
Cytochemical reactions of Amoeba proteus
209
color; and a positive reaction for magnesium by a yellow-rose color. Several other minerals as well, react giving individual color reactions. The observed coloration is not pure and therefore indicates a mixture of several reactive minerals in the fluid interior of the refractive body. No iron, either free or organically bound was demonstrated in normal amoebas by the procedures applied. Negative results were obtained as well for normal and starved amoebas tested for copper and zinc. Discussion.-Previous work involving microincineration [6] indicated a high concentration of minerals, possibly calcium and magnesium to be associated with the refractive bodies of Amoeba proteus. The cytochemical studies reported here confirm and further define this association. It was demonstrated that the calcium and magnesium are localized primarily in the innermost portion or core of the refractive body. Other minerals in lower concentrations may be present as well. The minerals are in a highly reactive form. The failure to demonstrate zinc and copper in the amoebas is not a surprising result. The total amount of each in the amoeba is probably low. The tests used are not highly sensitive. These procedures were used in spite of this consideration because of the anticipation that these minerals might be concentrated in or on some inclusion. As indicated, this is not so. The absence of cytochemically detectible iron is in agreement with the negative results for iron in microincinerative studies [6]. Summury.-Cytochemical methods of mineral localization were applied to normal and starved Amoeba profeus. Neither iron, zinc, nor copper could be demonstrated by the procedures used. The concentration of these minerals is apparently below the sensitivity limits of the applied tests. Magnesium and calcium, presumably in their ionized form, were found to be concentrated in the fluid core of the refractive bodies. REFERENCES 1. BRODA,B., Mikrokosmos 32, 184 (1939). 2. BUNTING, H., Stain Technol. 24, 109 (1949).
A., Bull. histol. appl. physiol. et pathol. et techn. microscop. 1, 125 (1924). D., Techniques of Histo- and Cytochemistry. Interscience Publishers, Inc., New York, 1949. 5. HELLER, I. M. and KOPAC, M. J., Ezptl. Cell Research 8, 62 (1955). 6. -Esptt. Cell Research 8, 563 (1955). 7. LANGERON, M., Precis de Microscopic. Massonet Cie,Editeurs, Paris, 1949. 8. LISON, L., Histochimie Animale. Gauthier-Villars, Editeur, Paris, 1936. 9. MANIGAULT, P., Ann. inst. oceanog. 18, 385 (1939). (Vide, LANGERON, 1949.) 10. MAST, S. 0. and DOYLE, W. L., Arch. Protistenk. 86, 155 (1935). 11. MENDEL, L. B. and BRADLEY, H. C., Am. J. Physiot. 14, 313 (1905). 12. RABL, C. H. R., Klin. Wochschr. 5, 365 (1926). (Vide LEON, 1936.) 13. WATERHOUSE, D. F., Council for Scientific and Industrial Research, Commonwealth o/ Australia, Melbourne Bull. no. 191, 7 (1945).
3. CRETIN,
4.
GLICK,
Experimental
Cell Research 1 t
CYTOCHEMICAL
REACTIONS
NOTES
OF NORMAL
AND
STARVING
AMOEBA PROTEUS
Department
II. LOCALIZATION
OF MIXERALS
IDA M. HELLER’j*
and M. J. KOPAC
ofBiology,
of
Graduate School Arts and Science, New York University, New York, N.Y., U.S.A. Received May 2, 1956
SEVERAL minerals have been studied in normal and starved amoebas by cytochemical means. The results from this work confirm and extend the results obtained in previous work in which the microincinerative method was used for mineral localization [6]. Materials and Methods.--The manner of handling, e.g., culturing, embedding of the normal and starved amoebas, has been described [5]. The fixative used in the following cytochemical procedures, unless otherwise indicated, consisted of 1 part neutral formalin and 9 parts absolute ethanol. Paraffin sections were cut at 4 p. Magnesium.-Sections and whole mounts of normal amoebas as well as whole mounts of amoebas starved for 1, 9, and 19 days were stained according to the Broda procedure [ 11. Calcium.-The Rabl test [la] for soluble calcium was applied to living unstarved amoebas as well as to amoebas starved for 1, 7, 20, and 22 days. The amoebas were fixed in a mixture of equal parts of 4 per cent ammonium oxalate and 25 per cent neutral formalin. A positive test is indicated by the appearance of calcium oxalate crystals in the specimen. The Manigault modification [9] of the CrCtin test [3] for the localization of calcium salts was applied to whole mounts of normal amoebas. Iron.-Sectioned and whole mount amoebas were tested for inorganic iron by the Turnbull blue method [8], as well as by the Prussian blue method [2]. For these tests, some amoebas were fixed in 10 per cent aqueous formalin, buffered at,pH 7, and the others with the alcoholic fixative described above. For the demonstration of organic iron, sections and whole mounts were first hydrolyzed in 4 per cent sulfuric acid in 95 per cent ethanol for 24 to 36 hours at 1 Work carried on as a Predoctoral Fellow stitutes of Health, Federal Security Agency. 2 Present address, Department of Surgery, School, 550 First Ave., New York 16, N.Y. Experimental
Cell Research 11
of the National New
York
Cancer
University
Institute,
National
Post-Graduate
In-
Medical
Cytochemical reactions of Amoeba proteus
207
Fig. 1. Portion of the cytoplasm of a whole mount amoeba which had been fixed in Rabl’s solution. Note the large calcium oxalate crystals. Approx. x 600. Fig. 2. Pqrtion of the cytoplasm of a whole mount amoeba which had been centrifuged prior to fixation in 25 per cent neutral formalin. Note the appearance of the many round refractive bodies. Apprax. x 600. Fig. 3. Centrifugal end of a whole mount amoeba which was fixed for 24 hours in Rabl’s solution. Note the numerous small crystals of calcium oxalate. Approx. x 600. Fig. 4. Centrifugal end of the same amoeba as in Fig. 3, a short time after sulfuric acid was applied. Fig. 5. Whole mount amoeba stratified prior to staining with Quinalizarin, showing a concentration of deeplv staining refractive bodies in the centrifugal end. Note particularly the darkly staining core and paler annulus seen in many of the refractive bodies. -Wrattan filter no. 58. Approx. x 800. Fig. 6. Whole mount amoebas treated with Titan yellow. Note the similarity between the appearance of these refractive bodies to those seen in the above figure. Wrattan filter no. 58. Approx. x 800. Experimenlal Cell Research 11
Ida M. Helfer and M. J. Kopac 35°C and then treated with either the Prussian blue reagent for 5 minutes 181, or with a freshly prepared solution of equal volumes of 1.5 per cent potassium ferrocyanide and 0.5 per cent hydrochloric acid [4]. As a check on the reactivity of the reagents, sections of rat liver and spleen were included in each series. Zinc.-The Mendel and Bradley method [ll] was applied to sections and whole mounts of normal amoebas as well as to whole mounts of amoebas starved for 1, 9, 17, and 19 days. A purple color indicates zinc and possibly copper. Copper.-The Waterhouse method [13] for the detection of copper was tested on both living and whole mount normal amoebas, as well as on whole mounts of amoebas which had been starved for 1, 9, and 20 days. Results.-Spot tests were carried out to determine whether the three dyes of the Broda test, Quinalizarin, Azo blue, and Titan yellow, specifically demonstrate magnesium. It was observed that magnesium chloride formed solid colored precipitates with all three of the alkalinized dyes, while calcium chloride formed flocculent precipitates. The color reactions of calcium and magnesium to Azo blue and Quinalizarin were practically indistinguishable. With alkalinized Titan yellow, the magnesium precipitate was bright red, and that of calcium pink and yellow. When these dyes were tested on filter paper previously impregnated with calcium distinguished or magnesium chlorides, again only the Titan yellow adequately between the two. The Titan yellow applied to sections and whole mounts produced an orange color in the cytoplasm and a deeper orange color in the nucleus and food vacuoles. The refractive bodies were seen to have a bright red core surrounded by a paler annulus (Fig. 6). Amoebas stained with Quinalizarin or Azo blue display the same pattern of color distribution in the refractive bodies (Fig. 5). Amoebas which had been starved for varying lengths of time prior to testing with the Broda stains showed no appreciable difference relative to each other or to the normal controls. Precipitated calcium oxalate was observed in amoebas treated with Rabl’s mixture (Fig. 1). The major portion of the calcium oxalate appears to be due to the calcium derived from the refractive bodies. On application of the reagent most of the refractive bodies disappear and calcium oxalate crystals appear. In amoebas stratified prior to fixation in Rabl’s mixture, the calcium oxalate crystals can be seen in the centrifugal end where the refractive bodies are normally found (cf. Figs. 2 and 3). The crystals were identified as calcium oxalate by their crystalline form and by their solubility. They are soluble in 2 per cent sulfuric acid (Fig. 4) and insoluble in 20 per cent acetic acid. Mast and Doyle [lo] reported the refractive bodies to be soluble in 20 per cent acetic acid. No significant differences in the occurrence or number of calcium oxalate crystals were noted between amoebas starved for 1, 7, 20, and 22 days, nor between them and the unstarved amoebas. Application of the modified C&in test to the centrifuged normal whole mounts [5] indicated the centrifugal end to be pinkish-brown in some or bluish or greenishbrown in others. The alkalinity of the reagent caused disruption and possibly a partial dissolution of the refractive bodies. A few intact refractive bodies were seen; of these, several contained a darkly colored pinkish or bluish interior with a paler annulus. With this reagent, a positive reaction for calcium is indicated by a blue Experimental
Cell Research 11
Cytochemical reactions of Amoeba proteus
209
color; and a positive reaction for magnesium by a yellow-rose color. Several other minerals as well, react giving individual color reactions. The observed coloration is not pure and therefore indicates a mixture of several reactive minerals in the fluid interior of the refractive body. No iron, either free or organically bound was demonstrated in normal amoebas by the procedures applied. Negative results were obtained as well for normal and starved amoebas tested for copper and zinc. Discussion.-Previous work involving microincineration [6] indicated a high concentration of minerals, possibly calcium and magnesium to be associated with the refractive bodies of Amoeba proteus. The cytochemical studies reported here confirm and further define this association. It was demonstrated that the calcium and magnesium are localized primarily in the innermost portion or core of the refractive body. Other minerals in lower concentrations may be present as well. The minerals are in a highly reactive form. The failure to demonstrate zinc and copper in the amoebas is not a surprising result. The total amount of each in the amoeba is probably low. The tests used are not highly sensitive. These procedures were used in spite of this consideration because of the anticipation that these minerals might be concentrated in or on some inclusion. As indicated, this is not so. The absence of cytochemically detectible iron is in agreement with the negative results for iron in microincinerative studies [6]. Summury.-Cytochemical methods of mineral localization were applied to normal and starved Amoeba profeus. Neither iron, zinc, nor copper could be demonstrated by the procedures used. The concentration of these minerals is apparently below the sensitivity limits of the applied tests. Magnesium and calcium, presumably in their ionized form, were found to be concentrated in the fluid core of the refractive bodies. REFERENCES 1. BRODA,B., Mikrokosmos 32, 184 (1939). 2. BUNTING, H., Stain Technol. 24, 109 (1949).
A., Bull. histol. appl. physiol. et pathol. et techn. microscop. 1, 125 (1924). D., Techniques of Histo- and Cytochemistry. Interscience Publishers, Inc., New York, 1949. 5. HELLER, I. M. and KOPAC, M. J., Ezptl. Cell Research 8, 62 (1955). 6. -Esptt. Cell Research 8, 563 (1955). 7. LANGERON, M., Precis de Microscopic. Massonet Cie,Editeurs, Paris, 1949. 8. LISON, L., Histochimie Animale. Gauthier-Villars, Editeur, Paris, 1936. 9. MANIGAULT, P., Ann. inst. oceanog. 18, 385 (1939). (Vide, LANGERON, 1949.) 10. MAST, S. 0. and DOYLE, W. L., Arch. Protistenk. 86, 155 (1935). 11. MENDEL, L. B. and BRADLEY, H. C., Am. J. Physiot. 14, 313 (1905). 12. RABL, C. H. R., Klin. Wochschr. 5, 365 (1926). (Vide LEON, 1936.) 13. WATERHOUSE, D. F., Council for Scientific and Industrial Research, Commonwealth o/ Australia, Melbourne Bull. no. 191, 7 (1945).
3. CRETIN,
4.
GLICK,
Experimental
Cell Research 1 t