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Korean J Parasitol > Volume 23(2):1985 > Article

Original Article
Korean J Parasitol. 1985 Dec;23(2):269-284. English.
Published online Mar 20, 1994.  http://dx.doi.org/10.3347/kjp.1985.23.2.269
Copyright © 1985 by The Korean Society for Parasitology
Electron-microscopic studies on fine structure and enzyme activity in the axenic and conventional strains of Entamoeba histolytica
Tai Soon Yong,Pyung Rim Chung and Keun Tae Lee
Department of Parasitology, College of Medicine, Yonsei University, Seoul, Korea.
Abstract

The metabolism of Entamoeba histolytica would be affected by various environmental factors, and alteration of the environment was known to affect the fine structure of E. histolytica.

The present study was designed electronmicroscopically to investigate the ultrastructure and enzyme activities in the axenic and conventional strains of E. histolytica.

The trophozoites of axenically cultivated HK-9 strain and conventional YS-27 and YS-49 strains of E. histolytica were collected and fixed with 4% paraformaldehyde/0.1 M cacodylate buffer (pH 7.4). After washing them by centrifugation, 1% warm agar was added in the sediment. Solidified agar with the trophozoites was cut into 1 mm3 cubes, and incubated in the various substrates to observe enzyme activities. Then, the specimen was post-fixed with 3% glutaraldehyde/0.1 M cacodylate buffer (pH 7.4) and 1% osmium tetroxide/0.1 M cacodylate buffer (pH 7.4), dehydrated in ascending ethanol series and embedded in epoxy resin. These were sectioned on an ultramicrotome and observed with a transmission electron microscope. The procedures for the observation of the fine structure were same as the above, except for the incubation in the substrate. The sections were stained with uranyl scetate and lead citrate.

For the observation of the surface of the amoebae, scanning electron microscopy was carried out. The results obtained in the present study are summarized as follows:

1. The fuzzy coat around double-layered plasma membrane of E. histolytica was more irregularly and densely distributed in the conventional strains (YS-27, YS-49 strains) than in the axenic strain (HK-9 strain).

2. The endosomes, button bodies and chromatin material were surrounded by a double-layered nuclear membrane having scattered nuclear pores. The paranuclear body, mono- or double-layered vacuoles, vacuolar membrane whorls, rosette-like cylindrical bodies, aggregation of cylindrical bodies and helical bodies were found in the cytoplasm of the amoebae. Helical bodies and glycogen granules were generally abundant, while a few smooth endoplasmic reticula were observed in the cytoplasm.

3. Alkaline phosphatase activity was mainly demonstrated in the plasma membrane, limiting membranes of vacuoles and smooth endoplasmic reticula. ATPase activity was observed in the nucleus, limiting membranes of vacuoles and vacuolar membrane whorls.

4. Acid phosphatase activity was commonly demonstrated in the limiting membranes an contents of vacuoles, lysosome-like organelles, plasma membrane and the button bodies in the nucleus. The activity was more weakly demonstrated in the HK-9 strain than in the other conventional strains of E. histolytica. No peroxidase activity was observed in the amoeba strains employed in the present study.

5. With a scanning electron microscope, no distinct structural differences were observed between the amoeba strains. All the trophozoite forms of the amoebae showed crater-like depressions and rugged features on the outer surface.

Figures


Figs. 1~3
Fig. 1. Trophozoite form of conventionally cultivated E. histolytica (YS-27 strain), showing a nucleus(N), rosette-like cylindrical bodies(R), various types of vacuoles (V) in the cytoplasm with scattered helical bodies (↓). (× 4,800)

Fig. 2. A conventional strain of E. histolytica(YS-49) with an anomalous nucleus(N), rosette-like cylindrical bodies (R), and vacuoles (V) in the cytoplasm. Enteric bacteria phagocytosed in a vacuole were shown.(↓) (× 4,800)

Fig. 3. An axenic strain of E. histolytica (HK-9), showing a nucleus with 2 endosomes(End), a concentric button body(Bb) and electron-dense peripheral chromatin material (C). (× 11,300)

*Abbreviations in figures: ACb: aggregation of cylindrical bodies, B: bacteria, Bb: button body, C: chromatin material, Cb: cylindrical body, End: endosome, Gly: glycogen, Hb: helical body, Np: nuclear pore, R: rosette-like cylindrical body, SER: smooth endoplasmic reticulum, V: vacuole, Vmw: vacuolar membrane whorl



Figs. 4~7
Fig. 4. A nucleus of HK-9 strain, containing circular arrangement of button bodies (Bb), an endosome (End) and chromatin material (C). Nuclear membrane (Nm) with regularly arranged nuclear pores(↓) was observed. Paranuclear body(Pb) near the nucleus and SER (smooth endoplasmic reticulum) were also noticed, (× 12,000)

Fig. 5. Trophozoite of conventional YS-49 strain with cytoplasmic projections (pseudopodia; Ps), engulfing bacteria (B). Note the vacuoles(V) containing bacteria and foods, (× 6,800)

Fig. 6. Autolytic vacuoles (AV) were more frequently observed in the cytoplasm of axenic HK-9 strain. Helical bodies(Hb) were scattered commonly in the cytoplasm, (× 10,800)

Fig. 7. Axenic strain of E. histolytica(HK-9 strain) containing relatively clear vacuoles(V) in the cytoplasm (× 6,800)

*Abbreviations in figures: ACb: aggregation of cylindrical bodies, B: bacteria, Bb: button body, C: chromatin material, Cb: cylindrical body, End: endosome, Gly: glycogen, Hb: helical body, Np: nuclear pore, R: rosette-like cylindrical body, SER: smooth endoplasmic reticulum, V: vacuole, Vmw: vacuolar membrane whorl



Figs. 8~13
Fig. 8. Double-layered vacuolar membrane whorl (Vmw) in the cytoplasm of a conventional YS-27 strain. (× 50,000)

Fig. 9. Two aggregation masses of cylindrical bodies (Acb) were seen in the cytoplasm of YS-49 strain (unstained), (× 13,700)

Fig. 10. Irregular and thick fuzzy coat(Fc) around the plasma membrane (Pm) of YS-49 strain. Note the glycogen masses in the cytoplasm (Gly). (× 23,200)

Fig. 11. Relatively regular and thin fuzzy coat(Fc) on the plasma membrane (Pm) of HK-9 strain, (× 23,000)

Fig. 12. A conventional strain of E. histolytica(YS-27) engulfing an enteric bacterium (B). Note invaginated plasma membrane(Pm) and the glycogen masses(Gly) in the cytoplasm, (× 47,200)

Fig. 13. A conventional strain of E. histolytica(YS-27) with a double layered food vacuole(Vm) containing a phagocytosed bacterium (B). (× 41,000)

*Abbreviations in figures: ACb: aggregation of cylindrical bodies, B: bacteria, Bb: button body, C: chromatin material, Cb: cylindrical body, End: endosome, Gly: glycogen, Hb: helical body, Np: nuclear pore, R: rosette-like cylindrical body, SER: smooth endoplasmic reticulum, V: vacuole, Vmw: vacuolar membrane whorl



Figs. 14~19
Fig. 14. Alkaline phosphatase activity was demonstrated around the plasma membrane (Pm) and vacuolar membranes (V) of YS-27 strain, but no activity was revealed in the nucleus, (× 7,200)

Fig. 15. Alkaline phosphatase activity was also demonstrated around the plasma membrane (Pm), vacuolar membranes (V) and smooth endoplasmic reticulum (SER) of YS-27 strain. Note two-layered vacuolar membrane(↓). (× 10,900)

Fig. 16. Alkaline phosphatase activity was confirmed mainly in the vacuolar membranes and their contents (V) in the cytoplasm of YS-49 strain. Poor activity was demonstrated around the plasma membrane (Pm). (× 15,900)

Fig. 17. Alkaline phosphatase activity was shown in the nucleus(N) and the cytoplasmic vacuolar membranes (V) of HK-9 strain. Poor activity was demonstrated around the plasma membrane (Pm). (× 3,700)

Fig. 18. Acid phosphatase activity was remarkably demonstrated in the vacuole (V) and lysosome-like organelle (L) and weakly revealed around the plasma membrane (Pm) and in the nucleus (N) of YS-27 strain, (× 11,200)

Fig. 19. Acid phosphatase activity was demonstrated remarkably at the button bodies (Bb) in the nucleus of YS-27 strain. The vacuoles (V) and plasma membrane (Pm) also revealed the activity, (× 9,800)

*Abbreviations in figures: ACb: aggregation of cylindrical bodies, B: bacteria, Bb: button body, C: chromatin material, Cb: cylindrical body, End: endosome, Gly: glycogen, Hb: helical body, Np: nuclear pore, R: rosette-like cylindrical body, SER: smooth endoplasmic reticulum, V: vacuole, Vmw: vacuolar membrane whorl



Figs. 20~25
Fig. 20. Acid phosphatase activity in the YS-49 strain of E. histolytica: Positive reaction was seen in the vacuolar membranes (V) and their contents, and weakly around the plasma membrane (Pm). (× 7,700)

Fig. 21. Acid phosphatase activity in the HK-9 strain: Positive reaction was confirmed in the nucleus(N), vacuoles (V) and plasma membrane (Pm). But activity was generally poor as compared with other conventional strains. (× 3,500)

Fig. 22. ATPase activity in the YS-27 strain of E. histolytica: Vacuoles(V) and vacuolar membrane whorl (↓) showed positive reactions. No ATPase activity was confirmed around the plasma membrane (Pm). (× 9,800)

Fig. 23. ATPase activity in the YS-49 strain: Activity was observed in the nucleus (N) and some vacuoles (V). (× 6,000)

Fig. 24. ATPase activity in the HK-9 strain: Note positive reactions mainly in the nucleus(N) and a few vacuoles (V). (× 4,500)

Fig. 25. Peroxidase activity in the YS-49 strain of E. histolytica: No demonstrable peroxidase activity was observed in all strains employed in this study. (× 4,500)

*Abbreviations in figures: ACb: aggregation of cylindrical bodies, B: bacteria, Bb: button body, C: chromatin material, Cb: cylindrical body, End: endosome, Gly: glycogen, Hb: helical body, Np: nuclear pore, R: rosette-like cylindrical body, SER: smooth endoplasmic reticulum, V: vacuole, Vmw: vacuolar membrane whorl



Figs. 26~27
Fig. 26. Scanning electron-microscopic feature of the HK-9 strain of E. histolytica: Note crater-like depressions and rugged features (↓). (× 5,300)

Fig. 27. Magnification of Fig. 26, showing the crater-like depressions (↓). (× 14,300)

*Abbreviations in figures: ACb: aggregation of cylindrical bodies, B: bacteria, Bb: button body, C: chromatin material, Cb: cylindrical body, End: endosome, Gly: glycogen, Hb: helical body, Np: nuclear pore, R: rosette-like cylindrical body, SER: smooth endoplasmic reticulum, V: vacuole, Vmw: vacuolar membrane whorl



Fig. 28
Schematic drawing including ultrastructural features of a trophozoite of Entamoeba histolytica.

*Abbreviations in figures: ACb: aggregation of cylindrical bodies, B: bacteria, Bb: button body, C: chromatin material, Cb: cylindrical body, End: endosome, Gly: glycogen, Hb: helical body, Np: nuclear pore, R: rosette-like cylindrical body, SER: smooth endoplasmic reticulum, V: vacuole, Vmw: vacuolar membrane whorl


References
1. Akao S. Toxoplasma gondii: localization of peroxidase activity. Exp Parasitol 1971;29(2):250–254.
  
2. Boeck WC, et al. Am J Hyg 1925;5:371–407.
3. Cho KM, et al. Yonsei Rep Trop Med 1972;3(1):37–59.
4. Cho KM, et al. Yonsei Rep Trop Med 1973;4:5–18.
5. Diamond LS. Techniques of axenic cultivation of Entamoeba histolytica Schaudinn, 1903 and E. histolytica-like amebae. J Parasitol 1968;54(5):1047–1056.
  
6. Diamond LS, Mattern CF, Bartgis IL. Viruses of Entamoeba histolytica. I. Identification of transmissible virus-like agents. J Virol 1972;9(2):326–341.
 
7. Eaton RD, Meerovitch E, Costerton JW. A surface-active lysosome in Entamoeba histolytica. Trans R Soc Trop Med Hyg 1969;63(5):678–680.
  
8. Eaton RD, Meerovitch E, Costerton JW. The functional morphology of pathogenicity in Entamoeba histolytica. Ann Trop Med Parasitol 1970;64(3):299–304.
 
9. el-Hashimi W, Pittman F. Ultrastructure of Entamoeba histolytica trophozoites obtained from the colon and from in vitro cultures. Am J Trop Med Hyg 1970;19(2):215–226.
 
10. Eranko O, et al. Acta Anat Suppl 1952;17:1–8.
11. Levine L, Shifrin N. Distribution of adenosine triphosphatase during anaphase in cricket primary spermatocytes. Nature 1974;249(454):256–258.
  
12. Fastag de Shor A, et al. Arch Invest Med 1974;5(2) suppl:357–364.
13. Feria-Velasco A, TRevino N. The ultrastructure of trophozoites of Entamoeba histolytica with particular reference to spherical arrangements of osmiophilic cylindrical bodies. J Protozool 1972;19(1):200–211.
 
14. Feria-Velasco A, Tapia-Arizmendi G, Gonzalez-Del Pliego M. Study of the trophozoite surface of Entamoeba histolytica with high resolution cytochemical studies and scanning electron microscopy. Arch Invest Med (Mex) 1980;11(4):461–475.
15. FLETCHER KA, MAEGRAITH BG, JARUMILINTA R. Electron microscope studies of trophozoites of Entamoeba histolytica. Ann Trop Med Parasitol 1962;56:496–499.
 
16. Flickinger CJ. J Ultrast Research 1968;23:260–271.
 
17. Gomori G. Arch Path 1941;32:189–199.
18. Graham RC Jr, Karnovsky MJ. The histochemical demonstration of uricase activity. J Histochem Cytochem 1965;13(6):448–453.
  
19. Kobayashi S, Takeuchi T, Asami K, Fujiwara T. Entamoeba histolytica: ultrastructural localization of Ca2+-dependent nucleotidases. Exp Parasitol 1982;54(2):202–212.
  
20. Lowe CY, Maegraith BG. Electron microscopy of Entamoeba histolytica in culture. Ann Trop Med Parasitol 1970;64(3):283–291.
 
21. Lowe CY, Maegraith BG. Electron microscopy of an axenic strain of Entamoeba histolytica. Ann Trop Med Parasitol 1970;64(3):293–298.
 
22. Lowe CY, Maegraith BG. Electron microscopy of Entamoeba histolytica in host tissue. Ann Trop Med Parasitol 1970;64(4):469–473.
 
23. Ludvik J, Shipstone AC. The ultrastructure of Entamoeba histolytica. Bull World Health Organ 1970;43(2):301–308.
 
24. LUFT JH. Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol 1961;9:409–414.
  
25. Lushbaugh WB, Miller JH. Fine structural topochemistry of entamoeba histolytica Schaudinn, 1903. J Parasitol 1974;60(3):421–433.
  
26. Meirelles MN, et al. J Parasit 1984;31(1):135–140.
27. Michel R, Westphal A. [Cytochemical studies on Entamoeba histolytica after cultivation in the presence and absence of bacteria]. Z Tropenmed Parasitol 1970;21(4):372–382.
 
28. Mirelman D, Bracha R, Sargeaunt PG. Entamoeba histolytica: virulence enhancement of isoenzyme-stable parasites. Exp Parasitol 1984;57(2):172–177.
  
29. Proctor EM, Gregory MA. The ultrastructure of axenically cultivated trophozoites of Entamoeba histolytica with particular reference to an observed variation in structural pattern. Ann Trop Med Parasitol 1972;66(3):335–338.
 
30. Proctor EM, Gregory MA. The observation of a surface active lysosome in the trophozoites of Entamoeba histolytica from the human colon. Ann Trop Med Parasitol 1972;66(3):339–342.
 
31. Proctor EM, Gregory MA. Ultrastructure of E. histolytica--strain NIH 200. Int J Parasitol 1973;3(4):457–460.
  
32. REYNOLDS ES. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 1963;17:208–212.
  
33. Rosenbaum RM, Wittner M. Ultrastructure of bacterized and axenic trophozoites of Entamoeba histolytica with particular reference to helical bodies. J Cell Biol 1970;45(2):367–382.
  
34. Serrano R, Deas JE, Warren LG. Entamoeba histolytica: membrane fractions. Exp Parasitol 1977;41(2):370–384.
  
35. Siddiqui WA, Rudzinska MA. The fine structure of axenically-grown trophozoites of Entamoeba invadens with special reference to the nucleus and helical ribonucleoprotein bodies. J Protozool 1965;12(3):448–459.
 
36. Silard R, Krohmalnik G, Stoicescu V, Steriu D. Comparative electron microscopic study of an imported pathogenic strain and an autochthonous nonpathogenic strain of E. histolytica with particular reference of nuclear inclusions. Arch Roum Pathol Exp Microbiol 1981;40(4):341–351.
 
37. Sobel HJ, Schwarz R, Marquet E. Nonviral nuclear inclusions. I. Cytoplasmic invaginations. Arch Pathol 1969;87(2):179–192.
 
38. Sykes DE, et al. J Cell Biol 1977;75:86a.
39. Takeuchi T, Kobayashi S, Masuda M, Tanabe M, Miura S, Fujiwara T. Entamoeba histolytica: localization and characterization of ca2+-dependent nucleotidases. Int J Parasitol 1981;11(3):209–215.
  
40. Weinbach EC, Diamond LS. Entamoeba histolytica. I. Aerobic metabolism. Exp Parasitol 1974;35(2):232–243.
  
41. Wittner M, et al. Bull N.Y. Acad Med 1968;44:1152–1163.
42. Wittner M, Rosenbaum RM. Role of bacteria in modifying virulence of Entamoeba histolytica. Studies of amebae from axenic cultures. Am J Trop Med Hyg 1970;19(5):755–761.
 
43. Zaman V. The intranuclear bodies of Entamoeba. Int J Parasitol 1973;3(2):251–253.
  
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