The in vitro-invasion of mouse erythrocytes by Toxoplasma gondii could be detected and analysed by electron microscopy. The sequence of events observed during erythrocyte invasion led to the assumption of an actively penetrating parasite into the non-phagocytic host cell.
The effects of dimethylsulfoxide (DMSO, final concentration 5%) and the deep-freezing process on the infectivity (ID50), motility, and ultrastructure of nontreated and DMSO-treated Trypanosoma cruzi suspensions (PSG-3 buffer with 10% horse serum) were investigated prior to and after cryopreservation in liquid nitrogen. DMSO equilibration caused distinct suppression of motility and characteristic, fine structural alterations in numerous organelles, such as myelin-like structures in the cytoplasm and/or inside the mitochondrial apparatus, enlargement of the perinuclear space, endoplasmic reticulum, and mitochondrial cristae, as well as condensation of the kinetoplast with loss of its lamellar structure. There was no evidence of loss of infectivity in DMSO-treated parasites. DMSO-treated and deep-frozen organisms showed, however, very similar fine structural alterations, although damage occurring during freezing and thawing was more pronounced. Apart from the frequently enlarged kinetoplast and the loosening of its mitochondrial matrix, numerous trypanosomes revealed total disintegration of the kinetoplast-mitochondrion complex with loss of its whole matrix. Deep-frozen trypanosomes were significantly less infective to mice than nontreated organisms, and their motility was strongly suppressed. These results suggest that cryopreservation and thawing of T. cruzi may lead to severe damage of the mitochondrial apparatus and thus to heavy disorders of metabolic function, exhaustion of the metabolic pool, and finally, to death of such damaged trypanosomes, despite the use of DMSO as a cryoprotective agent.
The changes observed in trophozoites of Toxoplasma gondii after deep-freeze preservation were examined by electron microscopy. Toxoplasmas (strain BK) from peritoneal exudate of infected NMRI mice were supended in Ringer's solution, deep-frozen in liquid nitrogen with 5% dimethylsulphoxide (DMSO), and compared after thawing with control samples with and without the addition of DMSO. Slight structural changes such as widening of endoplasmic reticulum, formation of fissures in the cytoplasm, and loosening of chromatin were only observed in some of the free toxoplasmas of the DMSO control. Among the deep-frozen parasites, about 1/5 of the free stages showed no or only slight morphological changes. In contrast to this, almost all intracellular forms found in macrophages showed lesions. The most remarkable change was a partial destruction of the inner cell membrane complex. The outflow of ribosome-containing protoplasm with ballon-like swelling of the outer elementary membrane was observed as a consequence of this frequent lesion. The outflow of protoplasm induced a drastic decrease in the electronic density of the whole cytoplasm. Other characteristic degenerative signs were vacuolation of cytoplasm up to formation of great optically empty spaces, widening of the perinuclear space, swelling of mitochondria, disintegration of rhoptria, micronemata, and Golgi zone, coarse-plaque loosening, and displacement of electron-dense areas of the nucleus up to disintegration with maintenance of the karyoplasm. In some almost completely disintegrated trophozoites, enlarged mitochondria with remarkable electronic density were observed. Apart from the cell membrane, the conoid was the longest-persisting organelle. The alterations observed after deep-freezing permit the conclusion that the free cells, which were only slightly impaired or not at all, remained infective.
The DNA content of culture forms and tissue stages of pathogenic E. histolytica strain SFL 3 were measured photometrically after the nuclei had been stained with the fluorochrome BAO. As a control, the DNA guartity of E. histolytica strain HK 9 and E. invadens were determined by the same method and compared with reference values. Tissue stages were obtained from hamsters experimentally infected by intrahepatic injection of SFL 3 amoebae. Further studies concerning possible changes in the DNA content of tissue stages involved the following methods: (a) isolation of tissue stages from the liver, followed by distinct suspension periods. (b) Infected liver pieces were directly transferred into culture medium; amoebae emigrating therefrom were cultivated. The study demonstrated that tissue stages contained up to 4 times more DNA than did culture forms. After 3 h cultivation, the DNA content of tissue stages decreased to the level of culture forms. Possible reasons for this change are discussed.
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