Negative Contrast Preparations Research Articles

Evidence of poxvirus in dusky dolphin (Lagenorhynchus obscurus) and Burmeister's porpoise (Phocoena spinipinnis) from coastal Peru.

In 1990, 74 dusky dolphins (Lagenorhynchus obscurus) and 10 Burmeister's porpoises (Phocoena spinipinnis) were examined for the presence of hyperpigmented marks and pinhole lesions on the skin (tattoo lesions) at the fishing terminal of Pucusana, central Peru. Prevalences of tattoo lesions were 8.1% and 30% in the dolphins and porpoises, respectively. Intracytoplasmic poxviruses were demonstrated by transmission electron microscopy in ultrathin sections of three of eight samples of infected epidermis from both species. The reason for the negative results in others is unclear but may be related to stages of infection with low virus density or even incorrect classification of some lesions as genuine viral tattoos. An irregular arrangement of tubules on the outer viral membrane, similar to those in orthopoxviruses, was visible in negative contrast preparations for P. spinipinnis. This is the first record of poxvirus in porpoises (Phocoenidae) and also the first report for dusky dolphins, and generally for cetaceans of the southern hemisphere.

Effect of preparatory techniques on the gross morphology of Mycoplasma hominis.

A method is presented whereby cells of Mycoplasma hominis can be prepared with minimal distortion for electron microscopy. After the addition of glutaraldehyde to broth cultures, incubation is continued for 1 h. The cells are then collected by centrifugation, washed in distilled water, and used for negative-contrast preparations.

Electron microscopy of Ibaraki virus.

Ibaraki virus propagated in BK and BHK-21 cell culture, was investigated by means of negative contrast and ultrathin section electron microscopy. In negative contrast preparations, the virus was shown to possess an icosahedral configuration. The virion measured about 55 mμ in diameter and had no envelope. The capsid of the virus probably consists of a single layer of 32 capsomeres, arranged in 5 ∶ 3 ∶ 2 symmetry. One or more virions enclosed in a pseudo-envelope were observed occasionally. In sections of infected culture cells, virus particles with electron dense cores were associated with intracytoplasmic viral matrices. Unenveloped core particles, as well as more complex membrane-bound forms were occasionally observed. Acquisition of a limiting unit membrane envelope appears to occur during release by budding from cell membranes. Obviously the virus does not multiply in the nucleus.

Arenoviruses in Vero cells: ultrastructural studies.

Thin-section electron microscopy was carried out on Vero green monkey kidney cell cultures infected with some viruses of the newly constituted arenovirus group. Junin, Machupo, Amapari, Pichinde, Parana, Tamiami, and Latino viruses were morphologically identical and indistinguishable from lymphocytic choriomeningitis virus, the prototype virus of the group. Virus particles were round, oval, or pleomorphic, 60 to 280 nm in diameter, and matured via budding from plasma membranes. Most characteristically, particles contained various amounts of homogeneous, 20- to 25-nm, dense granules; these granules in large masses also formed distinctive intracytoplasmic inclusions. In negative-contrast preparations from infected Vero cell culture supernatant fluids, several of the viruses appeared as pleomorphic membrane-bound forms with rather pronounced surface projections. Most particles were between 90 and 220 nm in diameter, although some reached 350 nm in their longest dimension. Internal structure was not resolved by negative-contrast electron microscopy. All observations supported the current delineation of a distinct arenovirus group.

Epizootic hemorrhagic disease virus of deer: an electron microscopic study

A mouse brain adapted strain of epizootic hemorrhagic disease (EHD) virus of deer was studied by ultrathin section and negative contrast electron microscopy. In sections of infected BHK-21 cells and neurons of newborn mouse brains, aggregates of virus particles, some appearing to be lacking their outer coats, were observed within membrane-bound vesicles. The development of virus particles was associated with intracytoplasmic viral matrices, consisting of moderately electron-dense granules. The viral particles measured about 58.8 nm in diameter in ultrathin sections and 62.3 nm in negative contrast preparations. Tubular structures, closely associated with viral matrices, were frequently found in infected cells. In one deer intracerebrally inoculated with virulent EHD virus, viral matrices, virus particles, and tubular structures, identical with those in infected BHK-21 cells and mouse neurons, were found in the cytoplasm of endothelial cells of blood vessels. The morphological details of EHD virus are compared with those of other viruses, and the differences in structure are discussed.

Electron Microscopic Study of Erythrocyte Receptors with Labeled Antisera to Membrane Components

Abstract Hemoglobin-free membranes and erythrocytes of group O, after reaction with ferritin-labeled rabbit antibody to purified virus receptor substance, were examined by electron microscopy. Antibody was found to bind specifically to discrete sites, in thin sections of cells and ghosts, as well as in negative contrast preparations of washed membranes. The distribution of reactive sites was similar to that described by others for ferritin-labeled isoantibody. The same antibody labeled with fluorescein reacted with washed membranes to give a granular type of fluorescence. The relationship of the virus receptor substance-reactive sites to those containing blood group antigens is discussed.

Characterization of Nodamura virus, an arthropod transmissible picornavirus

Nodamura virus, an arthropod-transmissible virus which is resistant to ether and chloroform, was characterized by electron microscopic, physical, and additional immunological studies. Examination of thin sections of limb muscles of infected infant mice revealed extremely large numbers of virus particles (28 nm in diameter) dispersed and in crystalline array within sarcoplasm. Two kinds of viroplasmic inclusions and severe destruction of skeletal muscle cell architecture were associated with the presence of virus. Kupffer cells in the liver of these animals also contained large aggregates of virus particles. Negative contrast preparations contained virus particles 29 nm in diameter with cubic symmetry. The virus was morphologically indistinguishable from picornaviruses. The density of infectious virus was 1.34 g/ml; it was insensitive to pH 3.7, and its sensitivity to heat (50 °) was not stabilized by molar MgCl 2. Since serum neutralization tests against 10 swine enteroviruses were negative, as were previously reported attempts at serological identification, Nodamura virus remains a picornavirus unrelated to any known virus, but with a demonstrated capacity for transmission by and multiplication in arthropods.

Aspects Of The Eb Virus

Publisher Summary This chapter focuses on the structure and maturation of Epstein–Barr (EB) virus. The herpes-type virus particles found in all strains of cultured hemopoietic cells examined so far are structurally indistinguishable with a morphology characteristic, both in thin sections and in negative contrast preparations, for the members of the herpes family. Immature particles are found in both the nucleus and cytoplasm of the cells and showed a hexagonal profile when appropriately orientated to the plane of section. The chapter discusses the effects of EB virus on cells. EB virus has so far only been found in established long-term cultures of human blastoid cells from various sources. Biological activity has only been demonstrated by certain unusual manipulations. Thus, the inoculation of virus preparations together with dimethylsulfoxide (DMSO) into thymectomized newborn hamsters produced transmissible encephalitis, and after serial intracranial passage in these animals the virus is subsequently found to cause fatal encephalitis on inoculation into monkeys, mice, guinea pigs, and rabbits. Neuropathogenicity has been confirmed for the virus without the use of DMSO or thymectomy by intracranial inoculation in newborn kittens. Striking evidence has been obtained that EB virus is the etiological agent of human infectious mononucleosis.

Electron microscope study of the disruption of red-cell membranes

This paper reports the results of an electron microscope study of the disruption of red-cell ghosts in distilled water and in strong salt solutions as observed in negative contrast preparations. Proteins released from the membrane by these treatments include a fibrous component, which appears to play some part in maintaining the structural integrity of the intact ghost. Preliminary estimates indicate that 5–10% of nonhaemoglobin protein is released from the membrane by washing twice in distilled water, and a further 10–20% is released if the pellet of distilled-water-washed vesicles is resuspended in strong NaBr solution.

Morphological observations on a virus associated with sheep pulmonary adenomatosis (jaagsiekte)

The presence of a virus morphologically resembling herpes virus is described in negative contrast preparations and in ultrathin sections from tissue cultures of ovine alveolar macrophages originating from cases of sheep pulmonary adenomatosis (jaagsiekte).

Colorado tick fever virus: an electron microscopic study.

Colorado tick fever (CTF) virus was observed by ultrathin section and negative contrast electron microscopy. In sections of infected cultured cells and of mouse brain, virus particles (75 mμ in diameter) with electron dense cores were associated with intracytoplasmic granular matrices, arrays of intracytoplasmic filaments, and fine kinky threads. Occasionally, virus particles were enveloped by membranes of cytoplasmic organelles. Intranuclear filaments in dense arrays were frequently found in infected cells. In negative contrast preparations the virus was round, 80 mμ in diameter, with regularly spaced surface projections suggestive of cubic symmetry. Partial removal of surface components permitted the observation of an inner capsid 50 mμ in diameter. CTF virus was directly compared with reoviruses, which in several ultrastructural characteristics it resembles.

Ultrastructure of lymphocystis virus.

Lymphocystis virus obtained from bluegills (Lepomis macrochirus) was cultured in the permanent bluegill cell line BF-2 and examined by electron microscopy in ultrathin sections of cell cultures and in negative-contrast preparations from cells and from centrifuged culture medium. According to negative-contrast preparations, the icosahedral virions have an overall diameter close to but not exceeding 300 mmu. Delicate filaments seem to issue from the vertices. In collapsed virions, an ordered array of morphological units was seen. Positively contrasted virions in ultrathin sections show a shell with three dark (heavy metal-stained) layers alternating with and separated by two clear layers. The acquisition of an additional outer membrane during release from the cell, as found in African swine fever virus, was never seen. Morphologically, lymphocystis virus is considered to be closely related to Tipula iridescent virus.

Anatomical features of Vibrio fetus: Electron microscopic survey.

SUMMARY: Vibrio fetus was examined in shadow-cast, negative-contrast and thin-sectioned preparations; Four morphological types were noted in shadowed specimens: comma, coccoid, S-shaped and filamentous. In negative-contrast preparations organisms were differentiated into two types on the appearance of their cytoplasmic mass. A large spherical cytoplasmic inclusion was frequently evident at the flagellated pole of freshly harvested organisms. Various morphological variants, ‘sports’, e.g. multiflagellate, were easily detected. A non-motile isolate was either devoid of flagella or exhibited only a short stub at the pole. In thin-section preparations anatomical features noted were: a rugose loosely fitting membranous integument; a complex cytoplasmic membrane of honeycomb-like structure; a fibrillar nucleoplasm and fibrillar cytoplasmic ground substance with associated ribosomes; lamellated or cytoplasmic granules bounded by a smooth membrane. At the flagellated pole the cytoplasmic ground substance was generally rarefied and a cone-shaped flagellar basal granule was present. The flagella were enlarged at their proximal terminus. Mesosome-like elaborations of the cytoplasmic membrane were not observed. It was concluded that Vibrio fetus closely resembles members of the genus Spirillum.

ULTRASTRUCTURE OF A MYCOPLASMA (NEGRONI) ISOLATED FROM HUMAN LEUKEMIA.

Hummeler, Klaus (The Children's Hospital of Philadelphia, Philadelphia, Pa.), Natale Tomassini, and Leonard Hayflick. Ultrastructure of a mycoplasma (Negroni) isolated from human leukemia. J. Bacteriol. 90:517-523. 1965.-A strain of mycoplasma isolated in tissue cultures from human leukemic bone marrow was cultivated in cell-free media for 23 passages. Concentrates of broth cultures were investigated under the electron microscope in thin sections and negative contrast. The ultrastructure of the mature mycoplasma cells and their elementary bodies is described. It was found that the elementary bodies, particularly in the negative contrast preparations, bear a close resemblance to viruslike particles seen in tissues and plasmas of leukemic patients.

Fractionation of Shope papilloma virus in cesium chloride density gradients

Virus particles prepared from extracts of glycerinated Shope papilloma tissue of wild cottontail rabbits by means of three alternate low and high speed sedimentations were not uniform in the electron microscope. Variations in size and electron density were seen in negative-contrast preparations. When such particles were centrifuged to equilibrium in a cesium chloride density gradient they separated into four layers corresponding to densities of about 1.29, 1.32, 1.33, and 1.34. Ultraviolet absorption studies, infectivity tests, and the homogeneity of the particles were consistent with the thesis that the most dense particles contained the highest proportion of deoxyribonucleic acid (DNA) and were probably the complete virus. The intermediate layers appeared to have less DNA, and the top layer little or none. In electron micrographs the most dense particles were uniform. Their surfaces were composed of an estimated 60 to 70 capsomeres. The particles in the intermediate layers showed various degrees of emptiness at their centers. The lightest particles appeared to be loose aggregates of capsomeres. When the particles were exposed to fresh cesium chloride, they appeared to become more fragile, many of them disintegrating into free capsomeres on the electron microscope grid. These subunits of the protein coat had a structure suggestive of hollow cylinders or cups. Subunits of the viral core could not be definitely characterized in density gradients or visualized by means of the electron microscope.