Russell L. Steere

Transmission-electron microscopic observations of freeze-etched polyacrylamide gels

Abstract Structures of polyacrylamide gels were examined by transmission-electron microscopy of freeze-etched specimens. This experimental approach allows higher resolution than that achieved in previous studies and reveals structural details that correlate with empirical concerning the molecular sieving effect of polyacrylamide gels. The influnces of the total acrylamide concentration, the cross-linking ratio, the different cross-linkers and the catalyst concentration were investigated. A comparison of polyacrylamide gel with agar gel revealed a structural difference in the two materials.

Freeze-etching simplified

Summary A new freeze-etching module, which permits easy preparation of 4 simultaneous replicas and more than 40 replicas per day, has been developed. In standard use, rough fracturing in a vacuum with the tip of a pointed knife replaces the use of a razor blade. A sharp-pointed carbon rod permits routine satisfactory evaporation of platinum-carbon shadow films. In order to improve shadow quality and prevent excessive contamination of the vacuum system, shielded shadowing guns that are easily removed, dismantled, cleaned, and reloaded have been devised. Satisfactory specimens of yeast cells, barteria, algae, nematodes, mouse brain, parasitized red blood cells, and virus-infected tobacco cells have been prepared in the new module.

Tobacco Mosaic Virus: Purifying and Sorting Associated Particles According to Length

End-to-end aggregation of tobacco mosaic virus and associated particles during extraction and purification may be prevented by transfer of the virus to 0.001M buffer solution of ethylenediaminetetraacetic acid, pH 7.5, as rapidly as practicable. Colored components were removed with charcoal and diatomaceous earth, and salts by rapid passage through a column of granulated 8 percent agar gel. The virus particles were sorted according to length by passage through a 1 percent granulated agar-gel column to isolate the 300- and 200-m� fractions and through a 5 percent agar gel to isolate the shorter particles.

Freeze-etching and direct observation of freezing damage

Summary Freeze-etching, as a method of preparing specimens for electron microscopy, is perhaps the best method for visualizing the fine structure of freezing damage to cells. New equipment permits the preparation of multiple specimens and double replicas, and rapid temperature changes. These make possible direct comparisons of different suspending media, different freezing rates, and apposed fracture surfaces. Also, recrystallization and condensation studies can be made. Freeze-etching is particularly useful for studies of membranes because the fracture plane often follows membrane surfaces and, even when large ice crystals destroy much of the detail within a cell, many membranes remain intact.

Complementary freeze-fracture, freeze-etch specimens

A procedure is described for the preparation and comparison of complementary freeze‐fracture‐freeze‐etch specimens. These complementary replicas reveal the value of etching some specimens even when the cryoprotectant concentration amounts to as much as 25% glycerol and 25% sucrose. As expected, the peplomers on the outer surface of the surrounding membrane of potato yellow dwarf virus are clearly revealed on the etched specimens but nearly invisible in the complementary freeze‐fracture specimen. Unexpected differences in the internal structure of vesicles within a freeze‐etched chloroplast have been revealed. The cross‐fractured surfaces of some other vesicles have a rough structure, whereas the complementary freeze‐etch surfaces, also shadowed at 123 K, are extremely smooth, suggesting that the contents must have been liquid at 175 K. Large ice crystals form within tobacco mosaic virus (TMV) crystals in infected cells frozen rapidly without cryoprotectant. Cryoprotectant consisting of 25% glycerol and 25% sucrose prevents formation of ice crystals when these specimens are frozen under the same conditions. When cryoprotectant concentrations are decreased from the above level, ice crystals within the TMV crystals are often clearly demonstrated in the freeze‐etch specimen but not in the complementary freeze‐fracture specimen. These results suggest that the complementary freeze‐fracture‐freeze‐etch procedure and the extremely fragile TMV crystalline inclusions are ideal for examining the value of various cryoprotectants and the effect of freezing rates, etching temperatures, and etching times on the final image.

Plasmodium falciparum: Freeze-fracture of the gametocyte pellicular complex

Freeze-fracturing has been used to study the architecture of the pellicular complex of the gametocytes of Plasmodium falciparum. The gametocyte is surrounded by three membranes and a layer of subpellicular microtubules. During freeze-fracturing, each of the three membranes is split along its hydrophobic interior to yield a total of six fracture faces. The most obvious feature of each fracture face is the presence of globular intramembranous particles on their surfaces. The six fracture faces differ from one another in arrangement, size, and density of these intramembranous particles. In gametocytes, unlike in sporozoites, the intramembranous particles are always distributed randomly and lack any definite pattern or orientations. A unique feature of gametocytes revealed by the freeze-fracturing technique is the presence of several transverse sutures on the middle membrane that encircle the gametocyte and give it a segmented appearance.

Plasmodium berghei: Architectural analysis by freeze-fracturing of the intraoocyst sporozoite's pellicular system

Abstract The technique of freeze-fracturing has been used to study the architecture of the pellicular complex of the intraoocyst sporozoite of Plasmodium berghei . The sporozoite is surrounded by three plasma membranes and a layer of subpellicular microtubules. During freeze-fracturing, each of the three membranes can split along its hydrophobic interior to yield a total of six fracture faces. The most obvious feature of each fracture face is the presence of globular intramembranous particles on the surface. The six fracture faces differ from one another in arrangement, size, and density of these intramembranous particles. Two of the fracture faces exhibit a unique arrangement of particles in well-organized parallel rows along the long axis of the sporozoite. This arrangement has not been reported in either the erythrocytic or the exoerythrocytic forms of Plasmodium spp. Another unique feature in the sporozoite revealed through freeze-fracturing is a single suture line that traverses the long axis of the inner two membranes of the parasite.

A resistance monitor with power cut-off for automatic regulation of shadow and support film thickness in freeze-etching and related techniques

A resistance monitor with sensors and automatic power cut‐off has been developed to control the thickness of Pt‐C shadow and C replica films in freeze‐etching and related techniques. The monitor and sensors, in conjunction with newly modified evaporators, should considerably reduce the amount of C and Pt required and should prove useful in other applications employing vacuum evaporation of thin films of Pt, C, or other conducting materials.

Equilibrium Sedimentation of Uniform Rods of Tobacco Mosaic Virus

Equilibrium was achieved in centrifugal fields with tobacco mosaic virus by the Beams magnetically suspended ultracentrifuge. An attempt was made to account for the earths gravitational component by using tilted cells in the rotor. The determinations of particle weight on highly uniform rods of tobacco mosaic virus in the apparent absence of nonideal behavior gave a value of (41.6 � 0.1) 106.

Liquid gradient zone electrophoresis in removable capsules for accurate sampling

Abstract A unique new apparatus provides a simple method for performing zonal density gradient and pH gradient electrophoresis in a capsule. The capsule (a column with removable top and bottom caps) with preformed gradient is inserted into the U-tube, top and bottom caps are removed, and electrophoresis is performed. For sampling, caps are replaced and the capsule is withdrawn from the U-tube. Gradient is then pumped out through a cone-shaped cap and capillary, through a monitor, and into a fraction collector. The instrument also makes possible simultaneous electrophoresis in several separate columns. Convenient and rapid interchange of capsules permits nearly continuous operation.