Processing Stripping Film Autoradiographs of Citrus Buds; Avoidance of Entrapped Air between Tissues and Developed Emulsion

Autoradiography was used on paraffin sections for investigating DNA synthesis in vegetative and flower buds of Citrus sinensis (L.) Osbeck. We preferred stripping film to liquid emulsion to obtain a more uniform thickness of the silver grain layer, as this would not vary more than ±10%. Furthermore, Baserga and Nemeroff (1962) have observed a lower sensitivity of fluid emulsion in comparison to stripping film together with occasionally erratic grain count and a slightly higher incidence of mechanical fogging. However, on using the customary procedure it was found that close contact between the surface of the section and the developed emulsion was not maintained when dehydration and clearing was done in the usual manner. The following modifications were therefore introduced to prevent the formation of air pockets between these two layers.     

Soluble compound electron microscope (EM) autoradiography: a resolution source to test redistribution of soluble tritiated compounds during processing

The development of a resolution source that can be labeled with either a soluble or insoluble tritiated compound, and of a method for applying a dry, uniform monolayer of emulsion is reported. Influences due to redistribution of the soluble isotope during emulsion coating were measured by comparing the grain density distributions around the resolution source for soluble tritiated proline (3H-PRO) with that obtained for cross-linked tritiated bovine serum albumin (3H-BSA). The grain density distributions resulting from a standard method of emulsion application (partly gelled/loop method) are compared to that obtained from a dry stripping film. It was found that only the dry stripping film gave a grain distribution which was statistically not different for the soluble and insoluble specimens.     

Effect of Drying on Unexposed Autoradiographic Emulsion in Relation to Background

Unexposed blanks prepared from Kodak AR 10 stripping film were dried by 3 methods: (1) fast, open drying with a fan for 25 min, (2) slow drying in a desiccator for 6 hr, and (3) very slow drying in a desiccator for 24 hr. The number of background grains depended on the mode of drying. Fast drying (method 1) gave 0.7 grain per 100 μ², slow drying (method 2) gave 0.33 grain; very slow drying (method 3), only 0.17 grain. The increase of background after fast drying is assumed to be caused by the rapid shrinkage of wet emulsion. This causes an increase in the intraemulsion pressure which, in turn, sensitizes the silver bromide crystals to cause an increase in the number of developable grains.     

Autoradiographic Differentiation of Tritium and Another β-Emitter by A Combined Colour-Coupling and Double Stripping-Film Technique

This technique distinguishes cells labelled with ³H, with ₁₄C, or with both isotopes together, in the same histological preparation. The technique depends on the application of two layers of autoradiographic stripping film, separated by a thin layer of celloidin. The first layer (in contact with the tissue) records predominantly the distribution of ³H in the sample, the second exclusively that of ¹⁴C. The silver grains in one layer are coloured by dye-coupling, which enables the grains in the two layers to be differentiated without the need for separate focussing. The merits of stripping film over liquid emulsion are: rigid control of the thickness and uniformity of the film is assured; an inert celloidin layer of 0.1 μ or less can be applied between the two films; and the thickness of each film can be chosen to suit emission characteristics of the radioisotopes.     

Pyronin Y For Staining Stripped Autoradiographs Prepared from Plastic Embedded Sections of Rat Tissues Containing Plutonium

Autoradiography prepared by the stripping film technique from 1 μm sections of semithin plastic embedded liver and bone tissues were poststained for examination with a 1% pyronin Y solution. The use of this dye avoids heating the tissue section and the overlying photographic emulsion. It also allows the staining of the tissue section without excessive uptake of the stain by the gelatin of the stripping film.     

A simple procedure for obtaining autoradiographs of G-banded chromosomes

Chromosomal preparations from cells flash-labeled with ³H-dT were Giemsa-banded following trypsin digestion, allowed to air-dry, and then were coated with a layer of 1% Formvar. The slides were subsequently coated with radiographic emulsion (NTB2) and processed for autoradiography. The resulting chromosomes had distinct G-banding and radiographic labeling patterns. Chemographic grain formation in the emulsion, normally caused by Giemsa stain, was prevented by the film of Formvar, allowing a very low background grain level.     

Factors which Affect Efficiency of Autoradiography with Tritiated Thymidine

The overall efficiency of autoradiography with tritium-labeled thymidine has been found to be influenced by the following conditions: (1) exposure in an atmosphere of CO₂ and the use of the stripping-film technique, both of which increase the autoradiographic efficiency when compared to exposure in air or to dip-coating technique; (2) latent image fading, which increases with increasing exposure. Up to 2 wk of exposure, however, this disadvantage is counterbalanced by the fading of the mechanical background produced during stripping or dip-coating; (3) the thickness of the inert coating interposed between the labeled locus and the sensitized emulsion. A layer of inert coating can be obtained that will arrest all beta particles from tritium, while having no effect on more energetic emitters like C¹⁴; (4) the amount of tritiated thymidine given, with relatively large amounts producing an increase in the mean grain count per labeled cell but not in the percentage of cells identifiable as labeled; and (5) the type of fixative and the staining procedure used. Feulgen stain reduces the mean grain count in cells labeled with radioactive thymidine, while fixation with acetic alcohol (1:3) reduces the grain count in cells labeled with precursors of ribonucleic acid.     

ANALYSIS OF TRITIUM INCORPORATION INTO INDIVIDUAL CELLS BY AUTORADIOGRAPHY OF SQUASH PREPARATIONS

The relation between tritium content of individual cells and grain count obtained in autoradiographs of squashed cells was investigated. The tissues used were root meristems of Tradescantia paludosa and intestinal epithelium of the mouse. The relation between grain count and tritium content is affected by self-absorption which depends on the thickness of the labeled cell. Therefore, squashed preparations were sectioned to determine the uniformity of thickness of nuclei. In a preparation of mouse cells, thicknesses were 1.18 ± 0.35 µ, and in a preparation of Tradescantia cells, 2.97 ± 0.35 µ. The effects of similar and larger variations in thickness upon grain count were studied in material squashed with different pressures; no marked correlation was found. The lack of correlation is explained by the geometric relation between labeled nuclei and the emulsion. By counting grains and directly measuring tritium content in a glass proportional counting tube in the same preparation, the yield of grains per disintegration was measured in Tradescantia cells and found to be 1 grain for 10.9 disintegrations with AR 10 autoradiographic film and 1 grain for 19.3 disintegrations for NTB nuclear track liquid emulsion. Latent image fading may pose a problem with long exposures; the conditions of its occurrence are as yet not well known.     

Biodeterioration of cinematographic cellulose triacetate by Sphingomonas paucimobilis using indirect impedance and chemiluminescence techniques

The bacterium Sphingomonas paucimobilis, isolated from cinematographic films in an earlier project, was able to biodeteriorate the cellulose triacetate material (acetylation degree of 2.7). Film colonization was monitored by the indirect impedance technique and the production of carbon dioxide. The presence of ruggedness and irregularities on the surface of the film, produced when the plasticizer was extracted, accelerated the biodeterioration of the material. In contrast, cinematographic films with their layered structure made of photographic gelatine emulsion were protected and no colonization was observed. The cinematographic film without photographic emulsion reached a 5% level of biodeterioration after six weeks of incubation, confirming the possibility of biodeterioration of archival cinematographic materials if conservation conditions are not adequate. Through viscosity measurements, a decrease in relative viscosity was observed on the biodeteriorated sample, with respect to the original material, confirming a lower molecular weight as a result of enzymatic activity of S. paucimobilis. Also, using chemiluminescence, the film surface oxidation on the biodeteriorated sample was observed. This technique was very sensitive in detecting material oxidation by reactive oxygen species generated by bacteria, and could be useful to study microbiological biodeterioration in polymeric materials.     

Dry, High Resolution Autoradiography

Microtomed sections of freeze-dried, paraffin-embedded tissues are placed on pieces of thin sheet-Teflon backed by a felt pad. The sections are then pressure-mounted on dry photographic emulsion. After suitable exposure, the sections are firmly cemented to the emulsion with 0.45% cellulose acetate in a 10:1 mixture of 2-butanone and acetone. This prevents the specimens from falling off or moving during photographic processing, though the tissue can be stained through the cellulose acetate binder. The method has been tested with tissues containing tritium-labelled DNA, and it produced resolution comparable to that obtained with standard liquid emulsion or stripping film techniques.     

Dry,High Resolution Autoradiography

Microtomed sections of freeze-dried, paraffin-embedded tissues are placed on pieces of thin sheet-Teflon backed by a felt pad. The sections are then pressure-mounted on dry photographic emulsion. After suitable exposure, the sections are firmly cemented to the emulsion with 0.45% cellulose acetate in a 10:1 mixture of 2-butanone and acetone. This prevents the specimens from falling off or moving during photographic processing, though the tissue can be stained through the cellulose acetate binder. The method has been tested with tissues containing tritium-labelled DNA, and it produced resolution comparable to that obtained with standard liquid emulsion or stripping film techniques.     

Über die Beziehungen der Ektodesmen zur Stoffaufnahme durch Blätter

Summary When droplets of radioactive solutions are applied to the cuticle, micro-autoradiograms show that the guard cells and the anticlinal walls of the leaf epidermis ofSpinacia oleracea and ofViola tricolor are the favoured sites of absorption. Scattered black spots in the stripping film could also be observed above the periclinal walls of the epidermis.A comparison of such micro-autoradiograms with the distribution of the ectodesmata in the leaf epidermis of the same two species demonstrates that the areas of favoured foliar absorption correspond to the areas of the densest accumulation of the ectodesmata, while the ectodesmata in the less absorbing periclinal walls are more loosely dispersed. Thus, though ectodesmata cannot be localized exactly by the occurrence of silver grains in the stripping film, identity exists between the areas of foliar absorption and the distribution of ectodesmata in the leaves which proves that ectodesmata are functioning as pathways of transport in foliar absorption.The experiments reported cofirm earlier statements that guard cells participate essentially in foliar absorption. The micro-autoradiograms also clearly show that absorption does not take place through the pores of the guard cells.

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Bark stripping in cork oak (Quercus suber L.): effect of an antitranspirant application on gas exchange and water relations of the stripped surface

Quercus suber L. is the primary source of industrial cork, which can be legally collected every 9 years. The main objective of this work was to test the efficiency of an application of an antitranspirant, at three different concentrations, after the bark stripping. For this purpose, several measurements of the gas exchange, water potential, total chlorophylls and the carotenoids contents were determined in cork oak trees, at two times in a day, morning and afternoon. The antitranspirant film was applied immediately after stripping. Transpiration rate showed a significant increase in the afternoon. The parameters, water potential, photosynthetic rates, stomatal conductance and the intrinsic water use efficiency, showed a significant decrease from morning to afternoon. The difference between pigments concentration was not significant throughout the day. Water potential and transpiration rate were high in the treatments with lower antitranspirant concentration. However, the treatment with a higher paraffin concentration showed larger photosynthesis rate. This result suggests that the loss of water observed for the stripping surface can be minimized by a larger concentration of the antitranspirant.     

Commercial Varieties of Nuclear Fast Red; Their Behaviour in Staining After Autoradiography

Dye marketed as nuclear fast red by various British firms (mixtures of anthraquinones by B. D. H. and G. T. Gurr, a quinone-imine dye by E. Gurr and Kodak) were investigated as nuclear stains particularly in autoradiography where the uptake of haematoxylin hindered visualization of the grains; in this case the uptake of S³⁵ by the epiphyseal plate of the tibiae of mice. A 0.1% solution of the anthraquinone dye CI 60760 (Nuclear fast red; G. T. Gurr) in saturated potassium aluminium sulphate gave a good nuclear stain and was used in the staining sequence: Alcian blue—nuclear fast red—tartrazine. These dyes barely coloured the emulsions used (both stripping film and gel-form emulsion) so that visualization of the silver grains and histological detail were good. The strains were applied after development and other processing of the emulsion was complete.     

Bark stripping of hinoki cypress by sika deer in relation to snow cover and food availability on Mt Takahara,central Japan

Bark stripping of hinoki cypress (Chamaecyparis obtusa (Sieb. et Zucc.) Endl.) by sika deer (Cervus nippon Temminck) was monitored for 4years on approximately 300 trees at three sites on Mt Takahara, central Japan. We investigated the effects of snow cover and food availability on seasonal and yearly changes in bark stripping. Bark stripping occurred during snowy periods (January and February) when less food was available because of the snow cover. The most serious bark stripping was observed in 1996 when the snow was the deepest and food resources were the poorest among the 4years of the study. In contrast, bark stripping did not occur in 1999 when the snow was thin and, consequently, more food was available. These results suggest that snow cover and food avail-ability influence the occurrence and intensity of bark stripping both seasonally and annually.     

Stability Assessment of Injectable Castor Oil-Based Nano-sized Emulsion Containing Cationic Droplets Stabilized by Poloxamer–Chitosan Emulsifier Films

The objectives of the present work were to prepare castor oil-based nano-sized emulsion containing cationic droplets stabilized by poloxamer–chitosan emulgator film and to assess the kinetic stability of the prepared cationic emulsion after subjecting it to thermal processing and freeze–thaw cycling. Presence of cryoprotectants (5%, w/w, sucrose +5%, w/w, sorbitol) improved the stability of emulsions to droplet aggregation during freeze–thaw cycling. After storing the emulsion at 4°C, 25°C, and 37°C over a period of up to 6 months, no significant change was noted in mean diameter of the dispersed oil droplets. However, the emulsion stored at the highest temperature did show a progressive decrease in the pH and zeta potential values, whereas the emulsion kept at the lowest temperatures did not. This indicates that at 37°C, free fatty acids were formed from the castor oil, and consequently, the liberated free fatty acids were responsible for the reduction in the emulsion pH and zeta potential values. Thus, the injectable castor oil-based nano-sized emulsion could be useful for incorporating various active pharmaceutical ingredients that are in size from small molecular drugs to large macromolecules such as oligonucleotides.     

Effects of Lecithin Addition in Oil or Water Phase on the Stability of Emulsions Made with Whey Proteins

The effects of lecithin addition in oil or water phase on the stability of oil-in-water emulsions made with 0.1 wt% whey protein and 10 wt% n-tetradecane at neutral and acidic pH were studied by monitoring the gravitational creaming and phase separation. The effects of lecithin addition on the interfacial behavior of β-lactoglobulin were also studied to compare with the results of emulsion stability. At neutral pH, crude phosphatidylcholine (PC) from egg yolk or soybean increased the stability of the emulsion made with protein and lowered the interfacial tension of protein films more effectively than pure egg PC. A more remarkable effect on both the emulsion stability and the interfacial tension was found when crude PC was added in the oil phase rather than in the water phase. The purity of lecithins and the way to add them are suggested to be very important to make a stable emulsion with protein. On acidic pH (4.5 or 3.0), the increased creaming or phase separation in a whey protein-stabilized emulsion, but the lowered interfacial tension of β-lactoglobulin films, were found upon the addition of pure or crude PC in oil or water phase. These results suggest that in acidic pH, densely packed films may be formed on a planar oil–water interface, but not on adsorbed layers around oil droplets in an emulsion.     

Phenidone-ascorbic acid development in electronmicroscopic autoradiography

Summary Phenidone-ascorbic acid development was calibrated for electronmicroscopic autoradiography, using Ilford L4 as photographic emulsion and microdol-x as reference developer. Grain yield and efficiency were studied on pale gold sections of uniformly labeled tritium methacrylate. For determination of the resolution, a radioactive line source was prepared by crosssectioning of an epon-embedded film of tritium labeled albumin. The spatial relationship between silver grains and silver bromide crystals was investigated by shadowing the emulsion with platinumcarbon before development. In shadowed autoradiographs both, silver grains and silver bromide crystal were visible.Phenidone was about twice as sensitive as microdol-x and had a half distance value (Salpeter et al., 1969) of 175 mm. Most of the silver grains of both developers were located within the perimeters of their parent silver bromide crystals. In the case of phenidone more than 80% of the excited crystals gave rise to just one silver deposit. These parameters, together with grain size and shape, and counting feasibility make phenidone a useful developer for quantitative EM-autoradiography.     

Is there "Metabolic" DNA in the Mouse Seminal Vesicle?

This study was designed to answer the question: Is H³-thymidine uptake by nuclei of the mouse seminal vesicle evidence for DNA synthesis and mitosis, or does it signify some "metabolic" function of DNA unrelated to chromosome duplication? Mice were given an intraperitoneal injection of H³-thymidine. Six hours later Feulgen squashes of the seminal vesicle epithelium were made and covered with autoradiographic stripping film. The silver grains above labeled nuclei were counted, and the Feulgen dye contents of these same nuclei were determined photometrically after removal of the grains from the emulsion. Unlabeled nuclei were also measured. The dye contents of non-radioactive nuclei form a unimodal distribution, indicating that polyploidy is absent from this tissue. The radioactive nuclei fall into two groups. In the first, the average dye content is the same as that of the cold nuclei (2C). In the second, the values range from 2C to 4C. In the 2C to 4C group the grain count is proportional to the dye content, showing that incorporation is correlated with synthesis. The radioactive 2C nuclei arose by mitosis during the course of the experiment. This is shown by the following facts: (1) They frequently occur in pairs. (2) They average smaller than unlabeled 2C nuclei. (3) Their average grain count is approximately half that of the 4C nuclei. (4) Labeled division figures are found. (5) A mitotic rate estimated from the number of labeled 2C nuclei accords reasonably well with one based on the number of observed mitoses. Since the incorporation of thymidine accompanies DNA synthesis and precedes mitosis, there is no reason to postulate a special "metabolic" DNA in this tissue.