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A comparison of phase contrast microscopy and an immunofluorescence test for the detection of Giardia spp. in faecal specimens from cattle and wild rodents.

As part of a study on the presence of Giardia cysts in water, a commercial direct immunofluorescence (DIF) test for the cysts was evaluated and compared to phase contrast microscopy. Forty faecal samples were collected from cattle; 31 contained Giardia. All 31 samples were positive by DIF whereas only 17 were detected by microscopy. The detected Giardia cysts were identified as belonging to the G. duodenalis group. 216 faecal samples were collected from wild rodents (Apodemus flavicollis, A. sylvaticus and Clethrionomys glareolus); 103 contained Giardia. Cysts were detected in 97 samples by DIF and in 57 by microscopy; 51 samples were positive by both methods. Nineteen rodents harboured cysts which morphologically resembled G. duodenalis. Specific identification was possible only by phase contrast microscopy, but the results suggested that DIF was superior for the detection of Giardia cysts in faeces of the animals tested.

Animals↗
PubMed1991

Light and electron microscopic observations on ciliated vacuoles and cysts in the oviductal and endocervical epithelia of the rabbit.

Ciliated vacuoles and intraepithelial cysts have been observed in oviductal and endocervical epithelia of rabbits. In this study, rabbits under various hormonal conditions were studied by light and transmission electron microscopy and tissue culture in an attempt to determine their distribution and origin. Ciliated vacuoles most frequently lay in the basal cytoplasm, below or beside the nucleus, and very close to the basal lamina. A few were apically located. Their average diameter was 8.8 by 5.1 microns. Cilia and microvilli projected into the vacuolar lumen. These vacuoles were located intracellularly as evidenced first by the degeneration of both their cilia and microvilli and the moderately dense matrix that often filled the vacuolar lumen, as observed by electron microscopy. Secondly, phase microscopy of the living endocervical epithelium allowed us to observe the beating of the cilia within the vacuoles, not on the surface of such cells. Thirdly, ruthenium red stained the surface glycocalyx of ciliated and secretory cells, but not that of the cilia and microvilli within the vacuoles. The intraepithelial cysts were not observed in all tissue blocks. The largest numbers were found in ovariectomized animals treated for 3 and 5 days with estradiol. More were seen in the isthmus and cervix than in the fimbria and ampulla. The cysts were located most often within the epithelium along the sides of, and at the bases of, the mucosal folds. They were lined by flattened epithelium of various combinations of secretory and ciliated cells. An unusual cell type was associated with some of the cysts and ciliated vacuoles. Its cytoplasm contained aggregates of mitochondria and vesicles whose contents varied in density. Although the genesis of the ciliated vacuoles is not certain, our results indicate that they may arise from aberrant positioning of proliferating procentrioles or from a defect in targeting or transporting the centrioles to the apical plasma membrane to serve as basal bodies. Fusion of adjacent ciliated vacuoles with lumina lined by secretory cells having deep apical invaginations appeared to contribute to the formation of cysts.

Animals↗
PubMed1996

Establishment of human colonic epithelial cells in long-term culture.

Study of normal colonic function is important in understanding the cellular mechanisms of carcinogenesis and other diseases of the colon. However, colonic pathophysiological studies have been limited due to the lack of long-term cultures of normal human colonic epithelial cells. The purpose of the present study was to develop methods of isolating viable human colonic epithelial cells for the establishment of nontransformed colonic epithelial cell lines. Human colonic epithelial cells were isolated from surgically resected normal human colons. We found that the use of a short enzymatic digestion gave a consistently higher number (>90%) of viable human colonic epithelial cells. These isolated colonocytes were grown on plastic, collagen-coated filters, or feeder layers using different media formulations. Those colonocytes from the initial primary cultures that were most "epithelial" in appearance were cloned and passaged to establish long-term cultures of nontransformed human colonic epithelial cells. The epithelial nature and secretory function of these established cell lines were confirmed by morphological criteria (light microscopy,, phase contrast microscopy, and electron microscopy). We found that the long-term cultures remained immunopositive to anti-cytokeratin antibodies and immunonegative to anti-vimentin antibodies. Using a soft agar assay we found that the colonocytes did not form colonies, suggesting that the long-term culturing did not cause these cells to become transformed. Under serum-free conditions, we found that epidermal growth factor and transforming growth factor-alpha were equally potent in their mitogenic effects for these colonocytes. Some of the subcultured cells could be maintained for at least 8 months and still retain their epithelial characteristics. We believe that this methodology will serve as a valuable tool for the isolation and culturing of human colonic epithelial cells for studies of normal and malignant colonic disease processes.

Agar↗