A Combined Immunofluorescence and Fluorescent Viability Cocktail Staining Procedure for Rapid Microscopic Detection and Enumeration of Live Legionella pneumophila

Calcium is supposed to play an important role in the control of protoplasmic streaming in slime mold plasmodia. The motive force for protoplasmic streaming is generated by the interaction of actin and myosin. This contraction is supposed to be controlled by intracellular Ca-fluxes similar to the triggering system in skeleton muscle. The histochemical localisation of calcium however is problematic because of the possible diffusion artifacts especially in aquous media.To evaluate this problem calcium localisation was studied in small pieces of shock frozen (liquid propane at -189°C) plasmodial strands of Physarum polycephalum, which were further processed with 3 different methods: 1) freeze substitution in ethanol at -75°C, staining in 100% ethanol with 1% uranyl acetate, and embedding in styrene-methacrylate. For comparison the staining procedure was omitted in some preparations. 2)Freeze drying at about -95°C, followed by immersion with 100% ethanol containing 1% uranyl acetate, and embedding. 3) Freeze fracture, carbon coating and SEM investigation at temperatures below -100° C.

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Electron Microscopic Detection and Characterization of Nonhuman Primate Enteric Viruses

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100054145 ◽

1982 ◽

Vol 40 ◽

pp. 306-307

Author(s):

G. C. Smith ◽

R. L. Heberling ◽

S. S. Kalter

Keyword(s):

Electron Microscopy ◽

Animal Model ◽

Nonhuman Primate ◽

Clinical Condition ◽

Intestinal Tract ◽

Enteric Viruses ◽

Electron Microscopic ◽

Microscopic Detection

A number of viral agents are recognized as and suspected of causing the clinical condition “gastroenteritis.” In our attempts to establish an animal model for studies of this entity, we have been examining the nonhuman primate to ascertain what viruses may be found in the intestinal tract of “normal” animals as well as animals with diarrhea. Several virus types including coronavirus, adenovirus, herpesvirus, and picornavirus (Table I) were detected in our colony; however, rotavirus, astrovirus, and calicivirus have not yet been observed. Fecal specimens were prepared for electron microscopy by procedures reported previously.

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A pre-embedding double staining procedure used to observe the effect of fixation on the activity of intercellular adhesion molecule on T and B cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100159436 ◽

1990 ◽

Vol 48 (3) ◽

pp. 378-379

Author(s):

Jane E. Ramberg ◽

Shigeto Tohma ◽

Peter E. Lipsky

Keyword(s):

B Cells ◽

Adhesion Molecule ◽

Colloidal Gold ◽

Intercellular Adhesion ◽

Intercellular Adhesion Molecule ◽

Staining Procedure ◽

Double Staining ◽

T And B Cells ◽

Microtiter Plates ◽

Streptavidin Peroxidase

Intercellular adhesion molecule (ICAM-1) appears to be a ligand for LFA-1 dependent adhesion in T cell mediated cytotoxcity. It is found on cells of both hematopoietic and non-hematopoietic origin. While observing the activity of ICAM-1 on the surfaces of interacting T and B cells, we found that we could successfully carry out a pre-embedding double staining procedure utilizing both colloidal gold and peroxidase conjugated reagents.On 24-well microtiter plates, mitomycin-treated T4 cells were stimulated with 64.1 (anti-CD3) for one hour before the addition, in some instances, of B cells. Following a 12-48 hour incubation at 38°C, the cells were washed and then immunostained with a colloidal gold conjugated RFB-4 (anti-CD22); biotinylated R6.5 (anti-ICAM-1); followed by streptavidin/peroxidase. This method allowed us to observe two different antigens without concern about possible cross-reaction of reagents. Because we suspected ICAM-1 and R6.5 were sensitive to fixation, we tried varying concentrations of fresh paraformaldehyde before R6.5, after R6.5 and after streptavidin/peroxidase. All immunostaining and washing was done on ice with ice cold reagents.

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Solvent-assisted osmium staining of butyl acrylate and ethylene-propylene-diene in a styrene acrylonitrile matrix

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100150332 ◽

1993 ◽

Vol 51 ◽

pp. 900-901 ◽

Cited By ~ 1

Author(s):

Gudrun A. Hutchins

Keyword(s):

Butyl Acrylate ◽

Room Temperature ◽

Osmium Tetroxide ◽

Staining Procedure ◽

Reactive Site ◽

Ethylene Propylene ◽

Minimal Distortion ◽

Styrene Acrylonitrile ◽

Engineering Polymers ◽

Effective Reaction

In order to optimize the toughening effect of elastomers in engineering polymers, it is necessary to characterize the size, morphology and dispersion of the specific elastomer within the polymer matrix. For unsaturated elastomers such as butadiene or isoprene, staining with osmium tetroxide is a well established procedure. The residual carbon-carbon double bond in these materials is the reactive site and forms a 1,2-dilato complex with the OsO4. Incorporation of osmium tetroxide into the elastomer not only produces sufficient contrast for TEM, but also crosslinks the elastomer sufficiently so that ultramicrotomy can be accomplished at room temperature with minimal distortion.Blends containing saturated elastomers such as butyl acrylate (BA) and ethylene propylene diene monomer (EPDM) cannot be stained directly with OsO4 because effective reaction sites such as C=C or -NH2 are not available in sufficient number. If additional reaction sites can be introduced selectively into the elastomer by a chemical reaction or the absorption of a solvent, a modified, two-step osmium staining procedure is possible.

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