The effects on biological materials of freezing and drying by vacuum sublimation. II. Effect on influenza virus.
(17/105)
The infectivity titre of influenza virus-infected allantoic fluid was determined after a variety of procedures involving cyclic slow freezing and thawing, freezing at various rates with subsequent storage at different temperatures freezing at various rates with subsequent dehydration at various temperatures, and different degrees of dehydration. All these factors were found to influence the survival rate of the virus particles. Five freeze-thaw cycles resulted in a fall in titre from 10(-8.6) to 10(-0.8) cycles 2, 3, and 4 causing much greater losses than cycles 1 and 5. Rapid cooling to -40 degrees C. or slow cooling to -80 or 190 degrees C. did not cause significant titre loss, but rapid cooling to temperatures above -40 degrees or slow cooling to temperatures above -80 degrees C. caused definite titre loss. Loss of titre on storage occurred only at temperatures above -40deg;C. The effect of lyophilization depends both on the preliminary treatment and on the dehydration temperature. Better conservation of titre was obtained after preliminary cooling to -190 or -80 degrees C. than after preliminary cooling to higher temperatures. The most effective sublimation temperatures were 0 and -80 degrees .; the least effective was +20 degrees C. Titre losses in suspensions sublimated at -10, -30, and -60 degrees C. were in general intermediate. No loss in titre occurred after preliminary cooling to -80 or -190 degrees C. and subsequent dehydration at -80 or 0 degrees C. The degree of dehydration definitely affects the survival of virus on storage at 0 degrees C., but sublimation for 4 hours at 0 degrees C. gave complete protection against titre loss on storage at this temperature. Possible explanations of the observations made are suggested, based on known physiochemical phenomena such as supercooling, vitrification, variations in size and shape of ice crystals with different freezing speeds, differential enzyme inactivation, changes in salt concentration, and changes in energy levels. (+info)
On artifacts appearing in the histochemical fixation of glycogen.
(18/105)
1. Fixation artifacts associated with glycogen translocation are prevalent in tissues of parenchymatous type and scarce or non-existent in tissues of loose type. 2. Liver tissue treated with M/3 NaOH solution before fixation did not show an uneven distribution of glycogen. This was interpreted as indicating that the liver, a tissue of parenchymatous type, was changed, so to speak, into a loose type of tissue by alkali treatment. 3. The so called Alkohol-flucht of glycogen was produced in Yoshida's ascites tumor cells by a procedure which changed a loose type of tissue into a parenchymatous one, that is, by packing the tumor cells tightly. 4. The translocation of glycogen in cells appeared to occur when the fixatives penetrated the cells rapidly from a single direction, but failed to occur when the cells were attacked by the fixative from all directions. 5. In dried smears of Yoshida's ascites tumor cells and bone marrow cells, the glycogen particles are translocated to the peripheral regions of the cells, and coalesce there. The production of these artifacts is related in some way to the physicochemical properties of the protoplasm and plasma membrane of the cells. (+info)
The use of OsO4 as fixative for Feulgen-stained preparations.
(19/105)
OsO(4) has many advantages over Carnoy's fixative mixture for the Feulgen nuclear staining in the protozoan Tokophrya infusionum. While Carnoy's fluid used prior to the Feulgen reaction produces shrinkage of the macronucleus and coarse clumping of its chromatin bodies, OsO(4) preserves faithfully the size and shape of the macronucleus and its chromatin material. This finding seems to be of special importance in view of the fact that electron microscopy relies on OsO(4) fixation. The satisfactory preservation of structured detail in Feulgen-stained preparations is of importance for the correlation of histochemical and morphological information. (+info)
Some effects of the microtome knife and electron beam on methacrylate-embedded thin sections.
(20/105)
A technique for the examination of specimens at low electron beam intensity has been presented. Sections micrographed with this technique showed numerous knife scratches and frequently contained bands running parallel to the knife edge. Banding with an average spacing of 0.2 micro appeared to result from periodic distortion produced by impact of the knife. At the beam intensities customarily employed, differential sublimation and probably flow of the methacrylate resulted in obliteration of the bands and all but the deepest knife scratches. In addition, changes in the size, shape, and orientation of certain structures were noted. Artifacts resulting from incineration or sublimation of tissue components fixed in formalin were illustrated, and the suggestion was made that such instability to the electron beam accounted in part for the differences observed in osmium- and formalin-fixed tissues. The deformation revealed in serial sections was discussed, and it was pointed out that shortening in the axis perpendicular to the knife edge was associated with elongation in the axis parallel to the cutting edge, the elongation usually occurring locally without change in the width of the section. It was noted that the material causing contamination of the surface of sections during examination exhibited no structure but caused progressive loss of contrast. (+info)
The preparation of frozen-dried tissue for electron microscopy.
(21/105)
A method is described for the preparation of liver by freezing and drying which avoids the formation of ice crystals visible with the electron microscope. Such dried preparations after postfixation may be stained with numerous reagents. They may also be stained for cytochemical study of ribonucleic and desoxyribonucleic acids, and for glycogen. Details of the methods are given, together with a brief justification of them through a summary of the controls employed. Further details, as well as a description of the submicroscopic morphology following the use of the methods, will be described elsewhere. (+info)
Thin sections. I. A study of section thickness and physical distortion produced during microtomy.
(22/105)
Knowledge of the thickness of sections is important for proper interpretation of electron micrographs. Therefore, the thicknesses of sections of n-butyl methacrylate polymer were determined by ellipsometry, and correlated with the color shown in reflected light. The results are: gray, thinner than 60 mmicro; silver, 60 to 90 mmicro; gold, 90 to 150 mmicro; purple, 150 to 190 mmicro; blue, 190 to 240 mmicro; green, 240 to 280 mmicro; and yellow, 280 to 320 mmicro. These results agree well with optical theory and with previous published data for thin films. Sections, after cutting, are 30 to 40 per cent shorter than the face of the block from which they were cut. Only a small improvement results from allowing the sections to remain in the collecting trough at room temperature. Heating above room temperature, however, reduces this shortening, with a corresponding improvement in dimensions and spatial relationships in the sections. When the thickness of the section is considered in interpreting electron micrographs instead of considering the section to be two-dimensional, a more accurate interpretation is possible. The consideration of electron micrographs as arising from projections of many profiles from throughout the whole thickness of the section explains the apparent lack of continuity often observed in serial sections. It is believed that serial sections are actually continuous, but that the change in size of structure through the thickness of one section and the consideration of only the largest profile shown in the micrograph can account for the lack of continuity previously observed. (+info)
Methods and principles of fixation by freeze-substitution.
(23/105)
Freeze-substitution is based on rapid freezing of tissues followed by solution ("substitution") of ice at temperatures well below O degrees C. A 1 to 3 mm. specimen was thrown into 3:1 propane-isopentane cooled by liquid nitrogen to -175 degrees C. (with precautions). The frozen tissue was placed in substituting fluid at -70 degrees C. for 1 week to dissolve ice slowly without distorting tissue structure. Excess substituting agent was washed out, and the specimen was embedded, sectioned, and stained conventionally. For best morphological and histochemical preservation, substituting fluids should in general contain both chemical fixing agent and solvent for ice, e.g., 1 per cent solutions of osmium tetroxide in acetone, mercuric chloride in ethanol, and picric acid in ethanol. Preservation of structure was poorer after substitution in solvent alone. Evidence was obtained that the chemical agent fixes tissue at low temperatures. The chemical mechanisms of fixation are probably similar to those operating at room temperature: new chemical cross-linkages, which contain the fixing agent, join tissue constituents together. This process is distinguished from denaturation by pure solvents. Freeze-substitution has many advantages, particularly the preservation of structure to the limit of resolution with the light microscope, and the accurate localization of many soluble and labile substances. (+info)
A CYTOLOGICAL AND HISTOLOGICAL STUDY OF ACUTE PREMYELOCYTIC LEUKAEMIA.
(24/105)
Among the acute leukaemias of the granulocytic group, acute premyelocytic leukaemia is distinguished by the severity of its haemorrhages, the frequency of hypofibrinaemia, a rapidly fatal course, and an unusual cellular hyperplasia. Myelograms show an increased proportion (average 80%) of characteristic cells of large diameter, with numerous azurophil granules. The infiltration of other organs is inconstant. (+info)