Isolation and characterization of viruses from fetal calf serum

Summary Ten lots of specially procured fetal calf serum collected under sterile conditions and not filtered and 16 lots of commercial fetal calf serum were tested for both human and bovine viral contamination. The presence of viruses was evaluated by observing for cytopathogenic effect (CPE), hemadsorption with guinea pig erythrocytes, and interference with cytopathogenic challenge viruses in both embryonic bovine trachea (EBTr) and human diploid lung (HDL) cells. Isolates were characterized by their cytopathogenicity, morphology, serology, and ability to propagate and produce cPE in a variety of bovine and nonbovine cells. One isolate was unequivocally identified as bovine herpes virus 1, and the other was presumptively identified as a bovine virus-diarrhea virus. This study was supported by The National Institutes of Health under Contract PH 43-66-539.     

Short Fixation-Shock Freezing and Freeze-Drying versus Chemical Fixation and Dehydration: Computer Assisted Image Analysis of Morphological Variables and Immunogold Labeling Density on Pituitary Secretory Granules

We have performed a computer assisted image analysis evaluation of the effects of two preparation protocols on morphological variables and immunolabeling density of secretory granules using rat adenohypophysis as a model. Glutaraldehyde (GA) fixation for 2 hr and chemical dehydration was compared with short GA fixation (15 min) followed by cryofixation and freeze-drying (CF-FD). The 2 hr GA fixed specimens showed spherical nuclei and secretory granules regardless of their size, contrasting with the more irregularly shaped nuclei and secretory granules after short GA-CF-FD. In the latter group of specimens a correlation could be found between smaller nuclear areas and more irregular shapes. The differences in morphological variables between the two preparation protocols might be due to a better protein stabilization and a reduced collapse of macromolecules after the 2 hr GA fixation, strengthened by the chemical dehydration. The specific immunolabeling with antiserum to growth hormone was much greater but more varied after short time GA-CF-FD than after long GA fixation. Epon surface topography over the granule area also differed: smooth after short time GA-CF-FD and furrowed after long GA fixation. Our results, demonstrating important differences in morphological parameters and immunolabeling density between two common preparation protocols, seem critical for more reliable interpretation in quantitative immunoelectron microscopy. We also emphasize the need for computer assisted image analysis and measurements in immunoelectron microscopy to ensure objective evaluations.     

Immunofluorescent Staining of Epon Embedded Kidney Sections Through Simultaneous use of Two Different Fluorochrome-Conjugated Antisera

Kidney biopsies can be examined in Epon sections for comparison of immunofluorescence and histology. This is possible by an incubation method which has now been modified to allow simultaneous localization of two antigens using fluorescein and rhodamine-conjugated antibodies on the same semithin sections of formalin fixed tissue. Consecutive sections from the same blocks can also be cut for electron microscopy. The method is now used in our immunopathological diagnostic procedures for examination of kidney biopsies.     

Seasonal dynamics of small vagile predators on a marine shallow soft bottom

The seasonal on- and offshore migrations of the epibenthic community on a marine shallow soft bottom in Gullmar Fjord (Sweden) was recorded for a period of three years. Quantitative sampling was carried out in shallow water (≤ 1 m depth) using drop-nets. A new sampling device was also introduced which allowed quantitative sampling in deeper water. The brown shrimp Crangon crangon L., juvenile plaice Pleuronectes platessa L. and the gobiid fish species Pomatoschistus minutus (Pallas) and P. microps (Krøyer) constituted the dominant inhabitants of shallow water, contributing >90% of the total number. The annual production was 2.2–2.9 g m⁻² yr⁻¹ dry wt with the brown shrimp as the dominant contributor. The abundance in shallow water fluctuated strongly between the seasons, while a relatively small seasonal variation was recorded in deeper water (2.5–10 m depth). The shallow/deep water abundance ratio for the epibenthic community heavily increased during the summer months, for the brown shrimp the ratio exceeded 200 in July. However, the heavy concentration in shallow water did not correspond to the reduced density of animals in deeper water. The boardering deeper water seemed to serve only as a transition area for animals arriving from or migrating to still deeper areas. Heavy mortality in the shallow nursery due to predation by the small epibenthic carnivores may also explain the low abundance in deeper areas during the autumn migration.     

Interferon production by mammalian cells grown in a serum-free medium.

Interferon, produced by rabbit heart cells grown in a serum-free medium, failed to protect rabbit heart serum-free cells, but protected rabbit heart serum-containing-medium cells against vaccinia and vesicular stomatitis virus. Interferon produced in serum-free cells had a greater species specificity than that produced in serum-containing media. The difference in activity was shown to be due to lack of adsorption by serum-free-medium cells.     

Quantitative Ultrastructural Immunocytochemistry Using a Computerized Image Analysis System

Secretory granules in human pituitary adenoma cells have been examined indirectly for hormone epitopes by immunogold labelling of resin-embedded ultra thin sections. The specific binding of different immunoglobulin-gold complexes to the antigrowth hormone antibodies over the secretory granules was measured using a computerized image analysis system. This facilitated the assessment of the preferential binding to the target granules of gold particles with three different average particle diameters (Au₇, Au₁₁, Au₁₇). The time of pretreatment of sections with H₂0₂ or a buffer was found to influence the staining considerably. The scanning electron microscopic findings of protruded secretory granules with a mountain-like surface might be relevant to the uneven distribution of immunolabels seen over the secretory granules in the adenohypophysis.     

Transportation and Storage of Cryosections in PBS-Dulbecco

Tissue pieces that cannot be fixed and embedded before immuno-staining must be cryosectioned and stained quickly. We describe an easy and reliable technique to keep cryosections with immune complexes and complement stored for several weeks until the final immiinostaining. The same technique was used for long distance transportation of cryosections and it was extensively tested for human kidney biopsies with excellent results.     

A Procedure for Ecological Tiered Assessment of Risks (PETAR)

Several procedures for Ecological Risk Assessment (ERA) have been suggested. The use of these existing procedures often relies on availability of existing data and/or on large resources for acquisition of new ones. This paper presents a three-tiered procedure for retrospective evaluation of risks adapted to limited resources and scarce background information of relevance for risk assessments, such as in developing countries. The tiers require successively more detailed investigations. The approach assures that resources available for site-specific investigations are directed towards well-formulated questions raised during previous stages of the assessment. The first tier, the preliminary assessment, is a qualitative evaluation of existing information on anthropogenic stressors, sources of stressors and expected ecological effects. The second tier is a regional risk assessment; a semi-quantitative evaluation of ecological risks, over large geographical areas, which results in a ranking of sources and stressors having the greatest potential for ecological impact and ranking of subareas inside the study area more likely to be impacted. The final tier is a site-specific and quantitative risk assessment, at a smaller scale and requiring more resources, that incorporates methodologies for establishing causality between exposure to multiple stressors and effects on specific endpoints of ecological and societal relevance.