Electron Microscopy of Dna Molecules "Stained" With Heavy Metal Salts

Abstract

Single DNA molecules can be rendered visible in the electron microscope by "staining" with water-soluble salts of heavy metals. The best results were obtained with lanthanum nitrate, uranyl acetate, and lead perchlorate. The molecules appear as filaments approximately 20 A wide. Their length was not determined, but it could be shown that it varied with the molecular weight of the DNA used. The same heavy metal salts will preferentially "stain" the nucleic acid in a protein-DNA complex. Evidence is provided for the possibility of a partial separation of a double-stranded molecule into single strands on adsorption to the supporting film.Mounting interest in the role of nucleic acids in biology and increasing knowledge of their molecular structure have prompted numerous attempts to supplement the information gained through indirect methods by direct observation of single molecules in the electron microscope. Several techniques have been used to obtain a suitable distribution of single molecules and to increase contrast in the preparation. After drying from a drop of dilute solution on a supporting film of collodion, Formvar, or carbon, DNA is usually found in large aggregates, in which little detail is visible. Spraying the solution from an atomizer onto a hydrophilic surface will result in a more homogeneous distribution of the DNA over a larger area and will allow the visualization of single molecules. Hall (10) and Hall and Litt (11) used the surface of freshly cleaved mica, which has the additional advantage of providing a very smooth background for subsequent shadowing, but transfer of the preparation to a grid presents some difficulties. Birbeck and Stacey (6) obtained satisfactory results by using a polymer with positively charged groups as the supporting film, so that DNA could be sprayed directly on the grid. Neither method, however, seems to be well suited for work with material of the very high molecular weight found in native DNA from many sources.A new technique recently published by Beer (4, 5) offers definite advantages in this respect. By streaking a film-covered grid over the surface of a DNA solution and by carefully draining off, in the same direction, the surplus adhering to the grid, he succeeded in attaching to the film single molecules in the form of nearly straight and parallel strands.Still another interesting approach is that of Kleinschmidt and Zahn (16,17), who spread DNA in a monomolecular film of protein on a water surface. The protein is necessary since DNA alone will not form a film, and the protein, furthermore, keeps the molecules of DNA separated so that they can be individually resolved.In all these investigations shadowing with heavy metals has been used to render the nucleic acid visible in the microscope. This procedure, however, limits the resolution to such a degree that it is difficult if not impossible to distinguish clearly a single molecule from two molecules in close contact. In addition, molecules are not visible when their long dimension is parallel to the

Ultrastructural and electron energy‐loss spectroscopic analysis of an extracellular filamentous matrix of an environmental bacterial isolate

“…Therefore, as a non-interfering alternative lead acetate has been used as staining agent. Lead acetate as well as lead perchlorate were successfully applied to stain pure DNA, giving similar results in contrast as those of conventional uranyl acetate (Stoeckenius, 1961). Thus, background intensities, which were corrected according to the 'potence' underground function, were mainly based on high mass-thickness of the underlying carbon-Butvar™ layer of approximately 75 nm A.…”

The Cyclic Helix and Cyclic Coil Forms of Polyoma Viral Dna

“…These lengths correspond to a molecular weight of 3.0 X 106, if we assume that the mass per unit length of DNA molecules in the protein film is the same as in the B-form in fibers of NaDNA. [19][20][21] Electron microscopy thus confirms that polyoma DNA molecules exist in linear and cyclic forms of equal molecular weight. The proportion of I and II by electron microscopy corresponds to that found by sedimentation analysis, as described below.…”

“…20 Calvin, M., Science, 135, 879 (1962). 21 Ishida, M. R., and R. Sager, in preparation. 22 Sager, R., unpublished.…”

Avantages et inconvénients de l'acétate d'uranyle en virologie comparée: étude de quatre bactériophages caudés

“…I1 est sent gukre utilids. La coloration positive par emP'oY' en PH acid' et forme des crista'x partir de pH 5.6 (34). Son emploi tend a se 'Requ le 29 aoct, 1973. g~n~r a~i s e r pour p~isieurs raisois: ce colorant ZPresente a la 23e Reunion annuelle de la SociCte permet de prendre des microp~otograp~ies trks canadienne des Microbiologistes, Edmonton, Alta., juin 1973.…”