Wojciech Sawicki

Vital Staining with Neutral Red and Trypan Blue of 3H-Thymidine-Labeled Cells Prior to Autoradiography

Mouse cells cultivated in vitro were continuously labeled with tritiated thymidine for 48 hr. In representative groups of labeled cells, the percentages of dead cells were obtained by vital staining with either neutral red or trypan blue. After fixation and auto-radiography, the fraction of nonlabeled cells was determined in the same group of cells which had been used for interpretation of viability. Neither neutral red nor trypan blue used prior to autoradiography caused spurious grain formation in the emulsion.

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.

Non-thermal effect of microwave radiation in vitro on peritoneal mast cells of the rat.

Microwaves are known to affect biological systems in two ways: either by in creasing the temperature by absorption of radiation energy (thermal effect), or by alteration of molecular and ionic structure of tissues, or both. Paramagnetic resonance (12) is one cause of alteration in the structure of tissues. However, most of the effects of microwave radiation are attributed to the rise in temperature (9). The animal body may be considered as a system consisting of a number of layers possessing various dielectric constants. Consequently, the temperature of animals irradiated in vivo does not rise uniformly in various organs or even within the same organ. Hence, the exact control of temperature in such a system is extremely difficult, if a t all possible, and the uncontrolled rise of temperature may mask a true non-thermal effect of microwave radiation. Peritoneal mast cells of the rat produce and accumulate a t least three important substances: heparin, histamine and 5-hydroxytryptamine (2). These substances play a key role in the physiology and pathology of connective tissue, and may be liberated from the mast cells under the influence of numerous physical and chemical stimuli, which usually cause the disruption of these cells (16). So far, only colchicine and microwaves acting on the whole body alter the mast cell morphology in a way other than disruption. Thus, colchicine changes the shape of mast cells (ll), while microwaves applied in vivo stimulate giant mast cell formation (19). The present investigation is concerned with an autoradiographic study of uptake of labeled sulfate by peritoneal mast cells irradiated in vitro with microwaves. The dry mass of such cells as well as the intensity of their metachromasia were also determined. The results indicate that mast cells irradiated with microwaves remained alive, but that the intensity of metachromatic reaction, the dry mass and the rate of labeled sulfate uptake were markedly decreased.

Hoechst 33342 Staining Coupled with Conventional Histological Technique

Hoechst 33342 was injected either intravenously or intraperitoneally into mice which were killed 1 or 24 hr or 7, 14 or 28 days later. Various organs were fixed and paraffin embedded. Visual inspection showed that independently of the route of dye administration or survival time, distinct fluorescence of nuclei was observed in organs other than cerebral cortex. Even formic acid decalcification of bone failed to abolish the fluorescence of osteocytes. In vivo staining with Hoechst 33342 is proposed as an alternative for staining after sectioning. Cells from spleens of Hoechst 33342-injected mice or stained in vitro were injected intramuscularly into mice. Hoechst 33342-stained splenocytes could be found in deparaffinized sections at the site of injection 24 hr later.

Albumen Embedding and Individual Mounting of One or Many Mammalian Ova on Slides for Fluid Processing

For studies of ova with the light or electron microscope, as well as for autoradiographic and histochemical studies, these cells need to be sectioned. The handling of individual, often hard-to-obtain, cells through fluid processing by micropipettes is time-consuming and can easily cause damage or loss of valuable specimens. A number of interesting methods have been described for handling ova or free-floating cells. In these methods cells are commonly handled in containers with fine-mesh, wire cloth bottoms, when a number of cells are involved. Unfortunately they all require special equipment not readily or easily available (Buchanan 1965; Rinaldi et al. 1966; Izquierdo 1967; Shands 1968). In our method, egg white provides a supporting matrix for mouse ova and allows one or several specimens to be mounted on a slide.

High-speed autoradiography of 3H-thymidine-labelled nuclei

SummaryHigh-speed autoradiography with stripping film of 3H-thymidine-labelled cells was tested. The tests involved: (a) various times of immersion of emulsion-covered cell preparations in the mixture of dioxane-PPO-POPOP, at 20°C, (b) exposure of cell preparations and blanks for various times at either −70°C or +20°C, with different humidity levels. Autoradiographs of good quality could be produced by 2-min immersion in the scintillator, exposure time ≥1 h at either temperature and relative humidity 20–30%. A linear relationship between autoradiographic efficiency and exposure time of 1–7 h was found at either temperature, although the efficiency of autoradiographs exposed at −70°C was by approximately 30% higher than that of autoradiographs exposed at +20°C. Background values of autoradiographs dried with a fan and exposed for 1/4–7h at either −70°C or +20°C were 0.6–0.8 grain/100 μm2. Theoretical calculations and experimental data showed that high-speed autoradiographs are 30–50 times more efficient as compared with conventional stripping film autoradiographs, thus allowing a shortening of the respective exposure time. Theoretical aspects of efficiency and resolution of high-speed autoradiography are considered.

Lysolecithin-induced Fusion of Rabbit Spermatozoa with Hamster Somatic Cells

Summary Rabbit spermatozoa have been successfully fused with hamster somatic cells by the use of lysolecithin. The primary effect of lysolecithin on spermatozoa seems to be related to an alteration of cell membranes. Many of the spermatozoal nuclei were modified in appearance, and observations of the ultrastructure show that a small percentage proceed to a state of partial disaggregation. Incorporation of tritiated thymidine administered in vitro indicated the initiation of DNA synthesis by disaggregated spermatozoal chromatin in the cytoplasm of cultured heterokaryocytes.

Effect of Exposure Temperature on Over-All Efficiency of Autoradiographs and on Fogging of Emulsion

Systematic investigations of the influence of various temperatures of exposure on the autoradiographic efficiency and on the rate of fogging of Kodak AR 10 stripping film were carried out. The oxygen content and humidity of the exposed autoradiographs as well as the time of exposure were constant. Exposure of autoradiographs or storage of film at room temperature caused an increase of overall autoradiographic efficiency by approximately 40%, but although the rate of film fogging was also increased, it did not exceed the safe values of background. Both autoradiographic efficiency and rate of fogging were not altered significantly when a temperature of 4 C or -18 C for exposure was used. Therefore, room temperature of exposure is recommended for ordinary autoradiography. For a special autoradiographic technic when low temperature of exposure has to be used, the temperature -18 C may be safely applied without significant impairment of autoradiographic efficiency.