Nucleoside Incorporation into Strain L Cells: Inhibition by Pleuropneumonia-Like Organisms

Nardone, Roland M.; Todd, Jean Elizabeth; González, Paula Mariela; Gaffney, Edwin V.

doi:10.1126/science.149.3688.1100

Cited by 98 publications

(16 citation statements)

References 9 publications

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“…The structures that we have seen adherent to the surface of culture cells and which we believe to represent mycoplasmas have all of the expected morphological characteristics (13,18,24). The application of radioautographic techniques, widely used at the light microscope level for mycoplasma detection (7,19), supports our morphological identification. Electron microscope autoradiography of both sections and replicas of cell cultures indicates that the suspicious bodies seen on cell surfaces do incorporate ['H]thymidine and are therefore likely to represent mycoplasmas.…”

Section: Discussionsupporting

confidence: 68%

“…Because whole cells are used in replicas, the geometry of the sample is not favorable for the highest resolution of which autoradiography is capable, and the "background" was somewhat higher than is usually considered acceptable for thin-section autoradiography. Even though the autoradiographic resolution is therefore no better than what can be obtained in the usual light microscope preparations (7,19) (13,18,27). Note that in some cases mycoplasmas appear to nestle in a depression on the surface of the culture cell.…”

Section: Resultsmentioning

confidence: 95%

See 1 more Smart Citation

Interaction of Mycoplasmas with Cell Cultures, as Visualized by Electron Microscopy

Brown

Teplitz

Revel

1974

Proc. Natl. Acad. Sci. U.S.A.

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Mycoplasmas were examined on the surfaces of tissue culture cells prepared for transmission and scanning electron microscopy. The pleomorphic bodies seen were proved to be mycoplasmas by the use of thin sections, passage of the infection from one cell line to another, and by autoradiography with [3H]thymidine both on the sections and the replicas. The mycoplasmas were not always evenly distributed over the cell's surface; their arrangement seemed to correlate with the activity or morphology of the cell. The use of replicas and scanning electron microscopy in routine examination of cultures for mycoplasma contamination is discussed.A number of surveys (9,11,15,27) indicate that many cell cultures, particularly when grown in the presence of antibiotics (10,15), are contaminated with mycoplasmas, and undetected infections have been an important source of artifact in many experiments (27).There are established techniques for the detection and identification of mycoplasmas (3,9,14,15,27,29). Many, however, are time consuming and complex. We have now found that very simple electron microscopic techniques such as cell-surface replicas (8,21,22) can provide a rapid and sensitive assay for the presence of mycoplasmas in tissue cultures. If available, scanning electron microscopy (5) can also be used to good advantage. This paper describes the morphology of mycoplasmas as observed in replicas and in preparations for the scanning electron microscope (SEM), presents an electron microscope autoradiographic technique for identification of these organisms in replicas, and discusses preliminary data on the interactions of culture cells with mycoplasmas. MATERIALS AND METHODSWe have examined cells from a number of different types (BHK, 3T3, Cl-1-D, LA9, L929, LD, HeLa, ME180, Chimp, CV-1, and AGMK), often obtaining the same cell line from a number of different sources. These cells were cultured on glass coverslips in the absence of antibiotics. * They were fixed before reaching confluency in 2.5% glutaraldehyde-0.5% osmium tetroxide in 0.1 M cacodylate buffer pH 7.4 at 0C (12). After dehydration in a graded series of ethanols, they were dried from Freon in a critical-point bomb (6). If lower-quality replicas can be tolerated (see Discussion) one can also dry the Abbreviation: SEM, scanning electron microscope. * Mycoplasma contamination can be detected in the presence of antibiotics. However, we have found that cells may need to be grown without antibiotics for several weeks before the mycoplasmas are present in more than marginally detectable numbers.samples from amyl acetate (21). For examination in the scanning electron microscope (ETEC; ETEC Corp., Hayward, Ca.), coverslips were rotary coated with gold. For transmission electron microscopy, samples were shadowed at a 450 angle with platinum-palladium (80:20) and at a 900 angle with carbon; the replicas were floated off the glass with hydrofluoric acid; and the cells were digested away with Clorox (21). The replicas were then rinsed in water, picked up on grids, and e...

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Section: Discussionsupporting

confidence: 68%

Section: Resultsmentioning

confidence: 95%

Interaction of Mycoplasmas with Cell Cultures, as Visualized by Electron Microscopy

Brown

Teplitz

Revel

1974

Proc. Natl. Acad. Sci. U.S.A.

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“…Mouse L929 cells were grown in Eagle's minimum essential medium with 10% fetal calf serum (Kansas City Biological) and were judged to be mycoplasma-free by the following tests: cytoplasmic thymidine incorporation (19), surface appearance in the scanning electron microscope (performed by J. Meek and M. Clark), and conversion of uridine to uracil by cell extracts (20). Enucleation was performed by centrifugation at 370 in medium containing 10 .g/ml of cytochalasin B (Aldrich Chemical), after a pre-spin to remove weakly attached cells, as described elsewhere (21).…”

Section: Methodsmentioning

confidence: 99%

Intracellular localization of mouse DNA polymerase-alpha.

Herrick¹,

Spear²,

Veomett³

1976

Proc. Natl. Acad. Sci. U.S.A.

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Although DNA polymerase-a (DNA nucleotidyltransferase; deoxynucleoside triphosphate:DNA deoxynucleotidyltransferase; EC 2.7.7.7) probably functions in the nucleus, it is usually found predominantly in the nonnuclear fraction of disrupted cells. We have reexamined the intracellular location of this enzyme using cytochalasin-B-induced enucleation, a technique which avoids exposure of nuclei to extra-cellular conditions during cell fractionation. In conditions where viability of separated cell parts is high and recovery is quantitative, we find greater than 85% of total DNA polymerase-a (and DNA polymerase-P) activity in the nucleated cell fragments (karyoplasts), from which we conclude that the location in vivo of DNA polymerase-a is either nuclear or perinuclear. On the other hand, thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7. MATERIALS AND METHODS Mouse L929 cells were grown in Eagle's minimum essential medium with 10% fetal calf serum (Kansas City Biological) and were judged to be mycoplasma-free by the following tests: cytoplasmic thymidine incorporation (19), surface appearance in the scanning electron microscope (performed by J. Meek and M. Clark), and conversion of uridine to uracil by cell extracts (20). Enucleation was performed by centrifugation at 370 in medium containing 10 .g/ml of cytochalasin B (Aldrich Chemical), after a pre-spin to remove weakly attached cells, as described elsewhere (21). Cytoplasts or untreated whole cells were removed by trypsin treatment, collected by a brief centrifugation 5.min at 300 X g), and gently resuspended in phosphate-b ffered saline. The karyoplasts were gently resuspended in the enucleation flasks in phosphate-buffered saline. Concentrations of whole cells, karyoplasts, and cytoplasts in the suspensions were determined by drying drops of known weight on microscope slides; these preparations were subsequently fixed, Feulgen stained, methyl green counter-stained, and all particles from each drop were scored as whole cells, karyoplasts, or cytoplasts. After samples were removed from the suspensions for counting and determination of intactness (trypan blue exclusion), the whole cells and cell parts were collected by a final centrifugation. Examination of supernatants indicated complete recovery (>99%) in the three pellets.For

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“…J.). It was possible to check cells for evidence of mycoplasmal contamination in most experiments by monitoring for cytoplasmic incorporation of tritiated thymidine ([3HJTdR) during autoradiography (20). Less frequently, mycoplasmal contamination was excluded by (a) monitoring microscopically for evidence of cytoplasmic or extracellular fluorescence in cell preparations treated with the fluorescent DNA-binding benzimidole derivative, Hoechst 33258 (26), and (b) standard aerobic and anaerobic culture procedures (1).…”

Section: Cellsmentioning

confidence: 99%