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Cytochemical detection of a polysaccharide surface coat in trypanosomatids
361 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE. Vol. 69. No. 3. 1975.
BRIEF COMMUNICATION CYTOCHEMICAL
DETECTION
OF A POLYSACCHARIDE TRYPANOSOMATIDS
WANDERLEY Institute
SURFACE
COAT
IN
DE SOUZA
de Biofzsica, Universidade Federal do Rio de Janeiro, Cidade Universitdria,
Rio de Janeiro, Brazil.
Much evidence has been presented for the existence of carbohydrates in the cell membrane and for the part that these play in the formation ofthe cell coat (MARTINEZ-PALOMO, 1970). Recently a polysaccharide surface coat has been described in blood trypomastigotes of Trypanosoma brucei (WRIGHT and HALES, 1970) and T. duttoni (DWYER and D’ALESANDRO, 1974), promastigotes of Leishmania donovani (DWYER, 1974; DWYER et al., 1974) and epimastigotes of T. cruzi from an acellular culture (ALVES and C~LLI, 1974) and in all the forms of T. crzui observed in tissue culture (DE SOUZAand MEYER, 1975). In order to detect the cell coat two methods have been used: a) the agglutination of the parasites when incubated with plant lectins which are specific for certain terminal polysaccharide and glycoprotein residues (DWYER, 1974; ALVES and COLLI, 1974) b) cytochemical techniques adapted for electron microscopy (WRIGHT and HALES, 1970; DWYER et al., 1974; DE SOUZA and MEYER, 1975). Among the cytochemical techniques, the most used have been cationic stains such as ruthenium red (LUFT, 1971) and alcian blue associated with lanthanum nitrate (SHEA, 1971). However these substances do not penetrate into the cell and cannot therefore be applied for the study of intracellular parasites. A quite distinct technique, developed by SELIGMANet al. (1965) and modified by THIERY (1967), consists of an adaptation of the classical PAS method for electron microscopy. It can be applied to ultra thin sections obtained in the normal way by double fixation with glutaraldehyde-OsO, and embedding in epon. This technique has now been adapted to the study of some trypanosomatids in the following way: 1) ultra thin sections were collected on gold grids and oxidized for 30 minutes with 1% (w/v) periodic acid at 37°C.; 2) the sections were rinsed in distilled water (5 mins.) and incubated in 1% (w/v) thiosemicarbazide in 10% (v/v) acetic acid for 2 to 24 hours at 37°C.; 3) after incubation the sections were rinsed in 10 and 5% acetic acid (2 mins. each), distilled water (5 mins.), and then incubated in 1% (w/v) silver proteinate for 40 minutes at 37°C. in a darkroom. After incubation they were rinsed 3 times (5 mins. each) in distilled water and then observed with the electron microscope. This technique should be useful in the study of the cell coat of other protozoa especially those with an intracellular stage in the life cycle. REFERENCES ALVES, M. J. M. & COLLI, W. (1974). J. Protozool., 21, 575. DE SOUZA, W. & MEYER, H. (1975). Z. ParasitKde., 46, 179. DEFER, D. M. (1974). Science, 184, 471. & D’ALESANDRO, P. A. (1974). J. ProtozooE., 21, 430. -, LANGRFITH, S. G. 81 DWYER, N. K. (1974). Z. ParasitKde., 43, 227. LUFT, J. H. (1971). Anat. Rec., 171, 347. MARTINEZ-PALOMO, A. (1970). Int. Rev. Cytol., 29, 29. SELIGMAN, A. M., HANKER, J. S., WASSERKRUG,H., DMOCHOWSKI, H. & KATZOFF, L. (1965). J. Histochem. Cytochem., 13, 629. SHEA, S. M. (1971). J. Cell. Biol., 51, 611. THIERY, J. P. (1967). J. Microsc., 6, 987. WRIGHT, K. A. & HALES, H. (1970). J. Parasit., 56, 671.
BRIEF COMMUNICATION CYTOCHEMICAL
DETECTION
OF A POLYSACCHARIDE TRYPANOSOMATIDS
WANDERLEY Institute
SURFACE
COAT
IN
DE SOUZA
de Biofzsica, Universidade Federal do Rio de Janeiro, Cidade Universitdria,
Rio de Janeiro, Brazil.
Much evidence has been presented for the existence of carbohydrates in the cell membrane and for the part that these play in the formation ofthe cell coat (MARTINEZ-PALOMO, 1970). Recently a polysaccharide surface coat has been described in blood trypomastigotes of Trypanosoma brucei (WRIGHT and HALES, 1970) and T. duttoni (DWYER and D’ALESANDRO, 1974), promastigotes of Leishmania donovani (DWYER, 1974; DWYER et al., 1974) and epimastigotes of T. cruzi from an acellular culture (ALVES and C~LLI, 1974) and in all the forms of T. crzui observed in tissue culture (DE SOUZAand MEYER, 1975). In order to detect the cell coat two methods have been used: a) the agglutination of the parasites when incubated with plant lectins which are specific for certain terminal polysaccharide and glycoprotein residues (DWYER, 1974; ALVES and COLLI, 1974) b) cytochemical techniques adapted for electron microscopy (WRIGHT and HALES, 1970; DWYER et al., 1974; DE SOUZA and MEYER, 1975). Among the cytochemical techniques, the most used have been cationic stains such as ruthenium red (LUFT, 1971) and alcian blue associated with lanthanum nitrate (SHEA, 1971). However these substances do not penetrate into the cell and cannot therefore be applied for the study of intracellular parasites. A quite distinct technique, developed by SELIGMANet al. (1965) and modified by THIERY (1967), consists of an adaptation of the classical PAS method for electron microscopy. It can be applied to ultra thin sections obtained in the normal way by double fixation with glutaraldehyde-OsO, and embedding in epon. This technique has now been adapted to the study of some trypanosomatids in the following way: 1) ultra thin sections were collected on gold grids and oxidized for 30 minutes with 1% (w/v) periodic acid at 37°C.; 2) the sections were rinsed in distilled water (5 mins.) and incubated in 1% (w/v) thiosemicarbazide in 10% (v/v) acetic acid for 2 to 24 hours at 37°C.; 3) after incubation the sections were rinsed in 10 and 5% acetic acid (2 mins. each), distilled water (5 mins.), and then incubated in 1% (w/v) silver proteinate for 40 minutes at 37°C. in a darkroom. After incubation they were rinsed 3 times (5 mins. each) in distilled water and then observed with the electron microscope. This technique should be useful in the study of the cell coat of other protozoa especially those with an intracellular stage in the life cycle. REFERENCES ALVES, M. J. M. & COLLI, W. (1974). J. Protozool., 21, 575. DE SOUZA, W. & MEYER, H. (1975). Z. ParasitKde., 46, 179. DEFER, D. M. (1974). Science, 184, 471. & D’ALESANDRO, P. A. (1974). J. ProtozooE., 21, 430. -, LANGRFITH, S. G. 81 DWYER, N. K. (1974). Z. ParasitKde., 43, 227. LUFT, J. H. (1971). Anat. Rec., 171, 347. MARTINEZ-PALOMO, A. (1970). Int. Rev. Cytol., 29, 29. SELIGMAN, A. M., HANKER, J. S., WASSERKRUG,H., DMOCHOWSKI, H. & KATZOFF, L. (1965). J. Histochem. Cytochem., 13, 629. SHEA, S. M. (1971). J. Cell. Biol., 51, 611. THIERY, J. P. (1967). J. Microsc., 6, 987. WRIGHT, K. A. & HALES, H. (1970). J. Parasit., 56, 671.