Gutteridge W E, Cover B, Gaborak M
Parasitology. 1978 Apr;76(2):159-76. doi: 10.1017/s0031182000047740.
Isolation of blood and intracellular forms of Trypanosoma cruzi was made mainly from rats (90-110 g) which had received 580 rad of whole-body gamma-irradiation not more than 24 h before subcutaneous inoculation with 10(7) trypomastigotes of the Sonya strain of T. cruzi. Unirradiated chinchillas (250-350 g) were, however, used for some experiments. Blood forms were isolated using a technique involving differential centrifugation to remove most of the erythrocytes and DEAE-cellulose chromatography to remove the remaining blood cells. Overall recoveries were usually in the range 30-70%. Parasites were mainly (approximately 98%) broad forms and were motile, metabolically active (as judged by respiratory and radio-tracer incorporation studies) and had lost none of their infectivity for mice. Intracellular forms were isolated from hind-limb muscle tissue. This was disrupted in an MSE tissue homogenizer and the homogenate incubated with DNase, collagenase and trypsin. Parasites, contaminated only by a few blood cells, were then obtained by differential centrifugation. For purer preparations, a terminal sucrose gradient step was used. Recoveries ranged between 40 and 70%. About 1-3% of the parasites isolated were epimastigotes and trypomastigotes; the remainder are probably best collectively termed 'amastigotes', though they were pointed and most had a short, free flagellum. They were undamaged as judged by light and electron microscopy and metabolically active as judged by respiratory and radio-tracer incorporation studies. However, the infectivity for mice of both these purified preparations and the initial cell homogenates could be accounted for by the epimastigotes and trypomastigotes present in them. Preliminary biochemical studies with isolated parasites have shown that blood, intracellular and culture forms of T. cruzi have a respiratory system which is in part sensitive to CN- and that all forms synthesize nucleic acids and proteins when incubated in vitro. There appears, however, to be a lack of DNA synthesis in blood stages, and thus it is not surprising that these forms do not divide.
克氏锥虫的血液型和细胞内型主要从大鼠（90 - 110克）中分离获得，这些大鼠在皮下接种10⁷个克氏锥虫索尼娅株的锥鞭毛体前不超过24小时接受了580拉德的全身γ射线照射。然而，一些实验使用了未照射的栗鼠（250 - 350克）。血液型通过一种技术分离，该技术包括差速离心以去除大部分红细胞，以及DEAE - 纤维素色谱法以去除剩余的血细胞。总体回收率通常在30% - 70%范围内。寄生虫主要（约98%）为宽型，具有运动能力，代谢活跃（通过呼吸和放射性示踪剂掺入研究判断），并且对小鼠仍具有全部感染力。细胞内型从后肢肌肉组织中分离。将该组织在MSE组织匀浆器中破碎，匀浆液与DNA酶、胶原酶和胰蛋白酶一起孵育。然后通过差速离心获得仅被少量血细胞污染的寄生虫。为了获得更纯的制剂，使用了终末蔗糖梯度步骤。回收率在40%至70%之间。分离出的寄生虫中约1% - 3%为前鞭毛体和锥鞭毛体；其余的可能最好统称为“无鞭毛体”，尽管它们呈尖形且大多数有短的游离鞭毛。通过光学显微镜和电子显微镜判断，它们未受损，通过呼吸和放射性示踪剂掺入研究判断，它们代谢活跃。然而，这些纯化制剂和初始细胞匀浆对小鼠的感染力可由其中存在的前鞭毛体和锥鞭毛体来解释。对分离出的寄生虫进行的初步生化研究表明，克氏锥虫的血液型、细胞内型和培养型具有部分对CN⁻敏感的呼吸系统，并且所有类型在体外孵育时都能合成核酸和蛋白质。然而，血液阶段似乎缺乏DNA合成，因此这些类型不分裂也就不足为奇了。
