Development of a sensitive protein A-gold immunoelectron microscopy method for detecting viral antigens in fluid specimens.

Protein A-colloidal gold immunoelectron microscopy (PAG IEM) has been employed to specifically detect rotavirus and enterovirus antigen in negatively stained fluid specimens. Unlike other IEM methods, PAG IEM can detect not only viral antigen associated with morphologically recognizable particles but also viral antigens of unrecognizable ultrastructure. This rapid and sensitive immunoassay was found to be applicable to virus-infected stool specimens as well as partially purified virus preparations. The sensitivity of viral antigen detection by PAG IEM was 2- to 40-fold greater than direct IEM and 200- to 1,000-fold greater than direct electron microscopy. In addition, PAG IEM appears to offer a more reliable and sensitive alternative to standard IEM for detection and quantitation of viral antibody.

Effect of specimen support film in solid phase immunoelectron microscopy.

The type of grid support film used in solid phase immunoelectron microscopy (SPIEM) for poliovirus affected the efficiency of virus trapping. Parlodion/carbon films coated with homologous antiserum were found to be 9 times more efficient in trapping virus than similar grids prepared with Formvar/carbon, and 20 times better than SPIEM grids prepared with pure carbon films. Trapping efficiency could be further improved if support films were pretreated prior to exposure to viral antiserum. Maximum efficiency was obtained with Parlodion/carbon films pretreated with UV light. Almost as efficient were Parlodion/carbon and Formvar/carbon films pretreated with glow discharge ionization.

Ultrastructural evidence for the cellular uptake of rotavirus by endocytosis.

The ultrastructural aspects of rotavirus SA11 infection were studied in MA104 cells, with particular attention directed to the early stages of virus adsorption and penetration. Within the first few minutes postadsorption, rotavirus was seen at the plasma membrane, associated with coated pits and coated vesicles, indicating that SA11 virus is taken into the cell by adsorptive or receptor-mediated endocytosis. Multiple virus particles were seen, beginning at 1 h and continuing throughout infection, in lysosome-like structures. Ultrastructural evidence was obtained to support the hypothesis that rotavirus progeny acquire a temporary envelope by budding into cisternae of the rough endoplasmic reticulum.

Virus morphology as an aid for rapid diagnosis.

Standard methods of virus diagnosis may take many days to complete. As antiviral drugs are being used with more effectiveness, it becomes more important to develop rapid diagnostic methods. It takes only a few minutes to prepare and examine a specimen for electron microscopy (EM), using the negative staining technique. Viruses in the specimen can readily be identified by their morphology. In order to be detected by EM there must be at least 10(7) virus particles per milliliter of sample. This concentration is frequently found in certain types of specimens. The sensitivity of EM is increased 100-fold if homologous antibody is used to aggregate the virus. Visualization of virus-antibody aggregates forms the basis for serotyping by immunoelectron microscopy (IEM).

Can steroid reduce endothelial damage in stored corneas? Effect on cell viability and ultrastructure.

We studied the effect of steroid on the viability and integrity of the endothelium of variously stored corneas of different species of animals and man. Paired corneas of rodents and humans were used,--one cornea (experimental) being treated with steroid and the other (control) not. Rat and guinea pig corneas were kept at 37 degrees C for one hour in phosphate buffered saline with or without hydrocortisone 21-sodium succinate (10-6M). Human corneas were kept at 4 degrees C for 4 days in M-K medium with or without hydrocortisone 21-sodium succinate (10-6M). The viability of the endothelia (rat and guinea pig) was tested with para Nitro Blue Tetrazolium and trypan blue dye exclusion tests. The ultrastructural changes in the control and experimental endothelial (guinea pig and human) were examined by scanning electron microscopy. Viable cells P less than 0.001) were more numerous and ultrastructural alteration less in the endothelia of the steroid treated corneas compared to the controls.

Two-hour embedding procedure for intracellular detection of viruses by electron microscopy.

An embedding method requiring only 2 h to complete and giving excellent ultrastructural preservation has been used for the rapid detection of viruses in tissue cultures. The method has also been applied successfully to mammalian tissue. It provides a rapid technique for identifying viruses isolated in tissue cultures, for screening cultures for adventitious agents, and for examining tissue biopsies for viruses.

Agar diffusion method for negative staining of microbial suspensions in salt solutions.

Microbial suspensions in a saline medium can be prepared for negative staining by a simple agar diffusion method that achieves a high degree of particle concentration.