Cytochemistry of the pellicle of bloodstream forms of Trypanosoma (Trypanozoon) brucei.

The cell coat of Trypanosoma (Trypanozoon) brucei has been found to vary in thickness and in differentiation into layers according to preparative techniques for electron microscopy. Measurements of coat and cell membrane are given. Cytochemical staining techniques indicate the presence of a prominent inner, and less prominent outer, layer of carbohydrates within the cell coat. Localization of the carbohydrates in the coat material is shown by trypsin digestion of living trypanosomes. Carbohydrates and protein may be the sole components of the cell coat. Negatively charged groups could not be demonstrated in the cell coat or membrane. An extracellular layer has recently been demonstrated as the outermost component of the pellicle of bloodstream forms of several species of trypanosomes (Fuge, 1968; Vickerman, 1969a, b; Godfrey and Taylor, 1969; Wright et al., 1970). Such an external cell coat may be suspected of playing an important role in the antigen-antibody interactions with the host, in providing structural support to the cell surface, and in having a modifying effect on the passage of metabolites from the environment to the cell membrane. The chemistry of the cell coat is therefore of some interest. This report extends the examination of the trypanosome cell coat to the cytochemical level by application of techniques to localize carbohydrates and by trypsin digestion of the coat. MATERIALS AND METHODS Bloodstream forms of Trypanosoma brucei were obtained from 3 to 5-day-old infections in white mice produced from the same stabilates as those used in an earlier study of "filopodium" formation by these animals (Wright et al., 1970). Trypanosomes were fixed both directly after hematocrit centrifugation and after resuspension in Hanks' salt solution. The basic morphology of the pellicle was determined from trypanosomes fixed by 2 procedures: (1) 20 to 30-min fixation at room temperature in 2.5% glutaraldehyde in Sorensen's 0.1 M phosphate buffer pH 7.4, followed by washing in phosphate-buffered 4% sucrose, and 1 hr postfixation in 1% osmium tetroxide in veronal acetate (0.05 M barbital) buffer; (2) 20 to 30min fixation at room temperature in 5% glutaraldehyde in 0.066 M cacodylate pH 7.4, followed by washing in cacodylate-buffered 5% sucrose, and 1 hr postfixation in 1% osmium in 0.066 M cacodylate. Some pellets were treated with 0.5% Received for publication 26 August 1969. * Assistance of National Research Council of Canada grant A3757 is gratefully acknowledged. uranyl acetate prior to dehydration (Terzakis, 1968). All were embedded in an Epon-Araldite mixture. Sections were stained either with saturated methanolic uranyl acetate followed by lead citrate, or with 2% potassium permangenate followed by lead citrate. For cytochemical examination of the cell coat, only the 5% glutaraldehyde-cacodylate fixation procedure was used. Cytochemical techniques for carbohydrates Sections to be stained by the periodic acid-thiosemicarbazide-silver proteinate technique and the periodic acid-silver methenamine technique were collected on gold grids and stained as recommended by Thiery (1967). Control sections were treated similar to these experimental sections except for the omission of periodic acid oxidation in some, and blockading of aldehyde groups with dimedone in others. Blocking of aldehydes with dimedone was done before, in in place of, or after periodic acid oxidation. The sections were floated on a 2% dimedone solution in 1% acetic acid for 1 hr at 60 C (Swift and Saxton, 1967) and were subsequently well rinsed before completing the staining technique. Pellets of trypanosomes fixed in glutaraldehyde were stained 1.5 hr with positively charged colloidal iron at pH 1.8 prepared according to Gasic et al. (1968). All blocks were postfixed in osmium. Ruthenium red staining was carried out as recommended by Luft (1964); some pellets were fixed in glutaraldehyde-ruthenium for 30 min and postfixed in osmium-ruthenium for hr, while some were fixed in glutaraldehyde-ruthenium for 24 hr and postfixed in osmium-ruthenium for 3 hr.