Rapid semiquantitative method for screening large numbers of virus samples by negative staining electron microscopy.
01 Aug 1970-Applied and Environmental Microbiology (American Society for Microbiology)-Vol. 20, Iss: 2, pp 259-262
TL;DR: A figure is presented which represents the limit of detectability for virus by negative staining, and is compared with results obtained by the pseudoreplica method and a measureddrop method.
Abstract: A rapid, semiquantitative method for screening large numbers of virus samples by negative staining electron microscopy is presented. Results obtained by this method are compared with results obtained by the pseudoreplica method and a measureddrop method. A figure is presented which represents the limit of detectability for virus by negative staining.
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TL;DR: A cell system was developed for the reproducible detection of human T-lymphotropic retroviruses (HTLV family) from patients with the acquired immunodeficiency syndrome (AIDS) or with signs or symptoms that frequently precede AIDS (pre-AIDS), and it provides large amounts of virus for detailed molecular and immunological analyses.
Abstract: A cell system was developed for the reproducible detection of human T-lymphotropic retroviruses (HTLV family) from patients with the acquired immunodeficiency syndrome (AIDS) or with signs or symptoms that frequently precede AIDS (pre-AIDS). The cells are specific clones from a permissive human neoplastic T-cell line. Some of the clones permanently grow and continuously produce large amounts of virus after infection with cytopathic (HTLV-III) variants of these viruses. One cytopathic effect of HTLV-III in this system is the arrangement of multiple nuclei in a characteristic ring formation in giant cells of the infected T-cell population. These structures can be used as an indicator to detect HTLV-III in clinical specimens. This system opens the way to the routine detection of HTLV-III and related cytopathic variants of HTLV in patients with AIDS or pre-AIDS and in healthy carriers, and it provides large amounts of virus for detailed molecular and immunological analyses.
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TL;DR: Viruses above about 60 nm in diameter may be rapidly sized to a few nanometers in their natural hydrated state as they pass one by one through a single pore in a newly developed nanometer-particle analyzer based on the resistive-pulse technique of the Coulter Counter and the use of submicron diameter pores made by the Nuclepore process.
Abstract: Viruses above about 60 nm in diameter may be rapidly sized to a few nanometers in their natural hydrated state as they pass one by one through a single pore in a newly developed nanometer-particle analyzer based on the resistive-pulse technique of the Coulter Counter and the use of submicron diameter pores made by the Nuclepore process. Size measurements for several type C oncornaviruses are: Rauscher murine leukemia, 122.3 +/- 2 nm; simian sarcoma, 109.7 +/- 3 nm; Mason-Pfizer monkey, 140.0 +/- 2.5 nm; RD-114, 115 +/- 5 nm; and feline leukemia, 127.4 +/- 2 nm, relative to standard 109-nm latex spheres. The T2 bacteriophage has a volume of (5.10 +/- 0.15) X 10(-16) cm3. Concentrations of viruses near 10(9) to 10(11)/ml that are fairly clear of debris are routinely measurable in a few minutes to an accuracy near 15%. A lower practical count limit is near 5 X 10(7) viruses per ml.
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TL;DR: 6 of 20 cotton-top tamarins inoculated with Epstein- Barr virus (EBV) developed diffuse malignant lymphoma resembling reticulum cell or immunoblastic sarcoma of man, and the marmoset has enhanced susceptibility to oncogenesis by EB virus.
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129 citations
Cites methods from "Rapid semiquantitative method for s..."
...The number of viral particles was estimated by electron microscope counts of negatively stained preparations concentrated 100-fold by ultracentrifugation (11)....
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TL;DR: A simple technique has been developed for the study of the external form and structure of virus particles by mixing virus preparations with 1% phosphotungstic acid adjusted to pH 7.5 and spraying directly onto electron microscope supporting films made from evaporated carbon.
1,389 citations
"Rapid semiquantitative method for s..." refers methods in this paper
...The negative staining technique (2, 4, 5) is extremely useful as a rapid means of visualizing virus particles, and the literature contains several suggestions for its use in virus quantitation (1, 3)....
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613 citations
365 citations
TL;DR: Since the pH conditions for high stain absorption are normally destructive, morphology is satisfactorily preserved only when the phosphotungstic acid is applied in concentrations of 10 per cent or greater or when the use of destructive reagents is preceded by a preliminary fixation under mild conditions.
Abstract: Methods are described for determining the relative mass of particles in electron microscope specimens through the measurement of photographic densities in recorded images. These methods were applied to a quantitative study of the amounts of electron stains that could be associated with the particles of tomato bushy stunt virus (BSV) and tobacco mosaic virus (TMV). In the pH range above 2 where the viruses are stable, the amount of stain absorbed is too small to produce adequate contrast in the electron microscope. Maximum stain absorption was achieved at pH about 1 where with several reagents and combinations of reagents the mass of BSV could be increased to about four times that of the unstained particles. Optimum results were obtained with phosphotungstic acid alone or in combination with Pt, Th, or La ions. Since the pH conditions for high stain absorption are normally destructive, morphology is satisfactorily preserved only when the phosphotungstic acid is applied in concentrations of 10 per cent or greater or when the use of destructive reagents is preceded by a preliminary fixation under mild conditions. Maximum staining of TMV increased the mass of the particles to about two times that of the unstained. Estimates of the mass of heavily stained BSV particles indicate that their density is 3.3 gm./cm.(3) The high internal hydration of BSV probably accounts for the greater stain absorption and penetration compared to those of TMV which has very low or zero internal hydration. Anomalous images resulting from the use of electron stains are shown and discussed.
218 citations
"Rapid semiquantitative method for s..." refers methods in this paper
...The negative staining technique (2, 4, 5) is extremely useful as a rapid means of visualizing virus particles, and the literature contains several suggestions for its use in virus quantitation (1, 3)....
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TL;DR: Particle counts made on herpes virus preparations after negative staining with phosphotungstate are shown to be comparable with counts made by the collodion filtration technique and by a method using dark ground optical microscopy.
133 citations