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

Abstract

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...

Characterisation of an Endogenous Protein Kinase Activity in Ribonucleoprotein Structures Containing Heterogenous Nuclear RNA in HeLa Cell Nuclei

“…Washed cell pellets were always used immediately. Cultures were monthly checked for the absence of mycoplasma contaminations by scanning electron microscopy [26].…”

A quantitative description of the extension and retraction of surface protrusions in spreading 3T3 mouse fibroblasts.

“…The cells were subcultured every 3 days after suspension in a 0.5-mM ethylenediaminetetraacetic acid (EDTA) solution in phosphate-buffered saline. Tests for mycoplasm infection were carded out by the scanning electron microscope method (9). Throughout the reported experiments, we used the same batch of cells.…”

Arrest and metastasis of blood-borne tumor cells are modified by fusion of plasma membrane vesicles from highly metastatic cells.

“…(FlLr, F1OLr) selected from these two sublines have been described in detail (13,18). All cells were grown in Eagle's minimal essential medium plus 10% fetal calf serum as described and were found to be uncontaminated by mycoplasma species by several assays (19)(20)(21)(22). Plasma membrane vesicles were harvested from the culture medium of actively growing roller bottle cultures of the various B16 sublines and purified as described (17).…”