Influence of mounting media on the fading of basic aniline dyes in epoxy embedded tissues.

A simple cytophotometric technique is used to quantitate stain fading of basic aniline dye-stained epoxy-embedded tissues mounted in six different commonly used mountants. Significant fading was detected with all six mountants, although rates varied. The lowest rate of fading was observed with immersion oil and the highest rate of fading with Canada balsam. No significant differences in fading rates of four synthetic mounting preparations were observed.     

Influence of light and mounting medium on the fading of Feulgen stain.

Serial microspectrophotometric estimations of the absorbance of Feulgen-stained interphase nuclei of human fibroblasts were made on slides mounted in a variety of mounting media and stored in light or in darkness. All preparations showed some fading, at rates which varied with different mountants, were greater in the light than in the dark, and were greater in the monochromatic illumination of the microspectrophotometer under working conditions than in normal laboratory light conditions. Fading was minimised by using XAM improved white neutral mounting medium (G.T. Gurr), storing slides in darkenss, and by reading samples as quickly as possible.     

The effect of selected mountants and stains on the measurement of fungal spores

Spores from five genera of filamentous fungi were measured by light microscopy using thirteen different mounting and staining techniques. The measurements obtained for the different methods for each organism were compared. Measurements taken in mountants and stains such as 40% KOH, Lugol's iodine, and cold lactophenol cotton blue showed significant differences with four of the five isolates. Comparisons between living and herbarium specimens suggested that for other mountants this would not give rise to significant differences. Possible explanations for these results are reviewed, their significance for critical taxonomic studies considered, and recommendations to mycologists are made.     

Structural basis for the anticoagulant activity of heparin. 2. Relationship of anticoagulant activity to the thermodynamics and fluorescence fading kinetics of acridine orange-heparin complexes.

Complexing heparin or dermatan sulfate with the fluorescent probe acridine orange provides a means of studying electrostatic as well as static and dynamic conformational aspects of these glycosaminoglycans via the thermodynamic and photochemical (fluorescence fading) properties of these complexes. The cooperative binding constants (Kq), fluorescence fading rate parameters (r'), and anticoagulant activities of heparins fractionated according to anionic density all showed qualitatively the same dependence upon anionic density. When Kq and r' were plotted against anticoagulant activity, empirical relationships were observed. Interestingly, the corresponding values for unfractionated dermatan sulfate fell on the lines defined by the heparin fractions. Temperature-dependence, studies demonstrated that differences in fading rate observed for heparins of different anionic densities are entropic in origin and reflect differences in the ability to assume a special configuration. Differences in activation entropy for fluorescence fading can be empirically correlated with anticoagulant activity. The latter correlation suggests a physical similarity in the roles played by anionic density in both fluorescence fading and anticoagulant activity.     

Thermoluminescence properties of annealed natural quartz after beta irradiation

Here we investigated the effects of annealing, heating rate and fading (after annealing at 800 °C) on the thermoluminescence (TL) glow curves of natural quartz (NQ). All of the samples were annealed at different temperatures between 100 °C and 800 °C and then irradiated with a beta dose of about 34 Gray (Gy), in order to determine the effects of annealing treatments on TL peaks of natural quartz. TL glow curves of the samples were recorded. It was observed that the intensities of TL peaks were strongly sensitive to annealing temperatures at 800 °C. The heating rate and fading effect of TL peaks of natural quartz were examined for the annealed samples at 800 °C for 30 min. It was observed that the intensities of the TL peaks were differently affected from heating rate and fading. Additionally, TL kinetic parameters (activation energy, frequency factor and order of kinetics) of all peaks were determined for annealed samples using a computerized glow curve deconvolution (CGCD) method and Mathematica software. Copyright © 2016 John Wiley & Sons, Ltd.     

Cytofluorometric demonstration of a catecholamine in rat mast cells after the administration of DOPA

Synopsis A catecholamine, probably dopamine, was identified in rat peritoneal mast cells after subcutaneous injection of DOPA. Its identity was established by cytofluorometry of cells treated with hot formaldehyde vapour according to the Falck-Hillarp technique. Injections of 50–200 mg/kgl-DOPA were followed by a dose-dependent increase in fluorescence intensity, measured at the emission maximum for catecholamines. The increase in fluorescence intensity was accompanied by a change in the emission spectrum with displacement of the fluorescence maximum towards a shorter wavelength characteristic for a catecholamine. Recordings of rates of photodecomposition showed a rapid exponential fading of the fluorescence in mast cells of control rats comparable to that of 5-hydroxytryptamine-containing protein droplets, whereas the mast cells of DOPA-treated rats showed a slower fading rate, intermediate between that of dopamine- and 5-hydroxytryptamine-containing models.     

Fluorescence fading and stabilization in cytofluorometry

The factors affecting fluorescence fading in cytofluorometry were investigated using different kinds of nuclear staining, mounting media, and procedure of specimen preparation. Acceleration of fluorescence fading was observed in smear specimens treated with RNase, trypsin, or hypotonic solution before pararosaniline Feulgen nuclear staining. Similar effect was found for other DNA-stainings such as "33258 Hoechst" and Feulgen reactions with different Schiff-type dyes, such as acriflavine-SO2 and cresyl-violet-SO2, when chemically pure DNA was used. Fluorescence decay was rapid for all fluorochromes examined, when glycerin or buffer solution was used as mounting medium. Marked stabilization of fluorescence emission was induced in specimen mounted in non-fluorescent resin, Entellan (Merck), after post-staining fixation with absolute methanol for all tested fluorochromes. The same treatment induced almost complete fluorescence stabilization of fluorescein isothiocyanate (FITC); no detectable fluorescence fading was observed in a specimen stained with indirect immunofluorescence reaction using anti-UV-DNA antibody, during storage for 2 years at room temperature without special protection against light. These observations suggest that factors which bring about conformational stability of macromolecule-dye complexes generally induce fluorescence stabilization.     

Staining reaction for beta-galactosidase in immunocytochemistry and in situ hybridization.

beta-galactosidase, revealed by an indigo blue reaction product, represents a valid tracer in immunohistochemistry. Observations on the instability of the indigo precipitate led us to investigate this phenomenon. We conclude that avoiding of xylene, and mounting of the preparates in Histoclear (a xylene substitute) and Canada balsam (instead of synthetic resin mountants) yields a sharp and stable indigo precipitate. In addition, we propose a sensitive alternative staining procedure for beta-galactosidase, based on TNBT reduction and precipitation. These reactions can be used both in immunohistochemistry and in in situ hybridization.     

Thermoluminescence glow curve deconvolution and kinetic parameter determination of samarium-doped lithium borosilicate glass

Thermoluminescence glow curves of gamma-irradiated samarium-doped lithium borosilicate glass were investigated. The number of overlapping peaks was determined using the repeated initial rise method. The glow curves were deconvoluted into four overlapping peaks. The trapping parameters such as activation energy E, frequency factor (s), and kinetic order (b) for each peak were determined. The obtained results indicated that the lithium borosilicate glass doped with samarium had four electron trap levels with the average activation energies of 0.82, 1.01, 1.21, and 1.31 eV. Thermal fading analysis of the individual peaks based on the deconvolution data was performed. The obtained results showed high thermal fading of the first peak, but high thermal stability of the second and third peaks compared with the other peaks. These results could be used to explain some observed properties such as high thermal fading and light sensitivity for this thermoluminescent material. Moreover, the obtained results may be helpful in minimizing fading corrections in dosimetric applications.     

Synthesis and characterization of high-sensitivity Dy,Eu co-doped CaSO4 thermoluminescent phosphor using coprecipitation technique

In this work, (99 − x)CaSO₄-Dy₂O₃–xEu₂O₃, (where x = 0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5) thermoluminescence phosphors were prepared using a coprecipitation method. The thermoluminescence (TL) dosimetry (TLD) characteristics such as TL sensitivity, dose–response, minimum detectable dose, thermal fading, and the effect of sunlight on the prepared phosphors were investigated. The obtained results indicated that the most sensitive phosphor was obtained at x = 0.05. Large thermal fading of 6% after 1 h and 26% after 24 h from irradiation followed by 71% after 1 month with no additional fading was observed within a time frame exceeding 2 months throughout the remaining duration of the investigation, which also spanned over 2 months. Despite the phosphor's high fading rate, the relative sensitivity of the prepared samples was ~90% compared with TLD-100. The marked effect of day sunlight was also determined. High dose–response within the low-dose range from 0.01 to 5 Gy was observed. The obtained results suggested that the synthesized phosphor is well suited for applications involving radiation biology and radiotherapy dosimetry.     

Comparison of the rates of aneuploidy occurrence in quiescent and dividing cells of humans exposed to adverse environmental factors

Fluorescence in situ hybridization (FISH) was used to compare aneuploidy rates in four autosomes and two sex chromosomes in interphase nuclei of noncultivated (quiescent) and cultivated (induced to divide with phytohemagglutinin (PHA)) leukocytes in people engaged in nuclear-chemical industry and in a control group of people not exposed to mutagenic factors occupationally or at home. The overall rates of numerical chromosome aberrations for all of the six chromosomes studied showed little difference, although a higher rate of loss of the X- and Y-chromosomes was observed in the exposed group. In individuals exposed to several adverse environmental factors, the overall rate of numerical chromosome aberrations in cultivated cells after at least one DNA replication cycle exceeded that in noncultivated cells by 52% (P = 0.01), whereas only a trend for its increase was observed in the control group (23%, P = 0.25). Thus, the effect of adverse environmental factors in humans caused more than a twofold increase in the difference between the rates of aneuploid cells in cultivated and non-cultivated leukocytes in the exposed group as compared to control. It is conjectured that cell division is accompanied by the expression of potential damage of mitotic chromatid segregation apparatus accumulated in vivo. These defects, realized during cell division, bring about numerical chromosome aberrations.     

A cytofluorometric study of the myenteric plexus in the guinea pig

Summary The myenteric plexus of the guinea pig ileum was studied in stretch preparations of the longitudinal muscle layer with adherent plexuses, and in freeze-dried transverse sections from the small intestinal wall. Catecholamines and serotonin (5-HT) were visualized according to the Falck-Hillarp technique. Emission spectra from the resulting fluorophores and recordings of their rates of photodecomposition were analysed. Adrenergic nerve terminals showed a slow fluorescence fading rate and a fluorescence spectrum compatible with their known contents of noradrenaline (NA), while the enterochromaffin cells showed a rapid exponential fading and a fluorescence spectrum compatible with their known contents of 5-HT. In order to unmask any low amounts of 5-HT in the neurons of the plexus, analysis of fluorescence parameters at various time intervals after pretreatment with reserpine followed by MAO-inhibition was performed. With the methods used no evidence of the presence of 5-HT in the myenteric plexus of the guinea pig could be found.We thank Iréne Svensson and Uno Johansson for skilful technical assistance. We are also indebted to Ciba, Pfizer and Draco for generous supplies of Reserpine, Nialamide and Pheniprazine. —This work was supported by grants from the Swedish Medical Research Council (Project 14 X-2235) and Göteborgs Läkaresällskap.     

Multiple-Locus Heterozygosity and the Physiological Energetics of Growth in the Coot Clam, MULINIA LATERALIS, from a Natural Population

The relationship between individual energy budgets and multiple-locus heterozygosity at six polymorphic enzyme loci was examined in Mulinia lateralis. Energy budgets were determined by measuring growth rates, rates of oxygen consumption, ammonia excretion and clearance rates. Enzyme genotypes were determined using starch gel electrophoresis. Growth rate and net growth efficiency (the ratio of energy available for growth to total energy absorbed) increased with individual heterozygosity. The positive relationship between observed growth and multiple-locus heterozygosity was associated with a negative relationship between routine metabolic costs and increasing heterozygosity. Reduction in routine metabolic costs explained 60% of the observed increased growth of more heterozygous individuals. When routine metabolic costs were standardized for differences in feeding rates, these standard metabolic costs explained 97% of the differences in growth rate. Lower standard metabolic costs, associated with increasing heterozygosity, have been proposed as a physiological mechanism for the relationship between multiple-locus heterozygosity and growth rate that has been reported for a variety of organisms, ranging in diversity from aspens to humans. This study demonstrates that reduction of standard metabolic costs, at least in clams, accounts for virtually all of the differences in growth rate among individuals of differing heterozygosity.     

A quantitative study of growth variability of tumour cell clones in vitro

Abstract.   Objectives : In this study, we quantify growth variability of tumour cell clones from a human leukaemia cell line. Materials and methods : We have used microplate spectrophotometry to measure growth kinetics of hundreds of individual cell clones from the Molt3 cell line. Growth rate of each clonal population has been estimated by fitting experimental data with the logistic equation. Results : Growth rates were observed to vary between different clones. Up to six clones with growth rates above or below mean growth rate of the parent population were further cloned and growth rates of their offspring were measured. Distribution of growth rates of the subclones did not significantly differ from that of the parent population, thus suggesting that growth variability has an epigenetic origin. To explain observed distributions of clonal growth rates, we have developed a probabilistic model, assuming that fluctuation in the number of mitochondria through successive cell cycles is the leading cause of growth variability. For fitting purposes, we have estimated experimentally by flow cytometry the average maximum number of mitochondria in Molt3 cells. The model fits nicely observed distributions in growth rates; however, cells in which mitochondria were rendered non-functional (ρ⁰ cells) showed only 30% reduction in clonal growth variability with respect to normal cells. Conclusions : A tumour cell population is a dynamic ensemble of clones with highly variable growth rates. At least part of this variability is due to fluctuations in the initial number of mitochondria in daughter cells.     

Mutation rate in human microsatellites: influence of the structure and length of the tandem repeat.

In 10,844 parent/child allelic transfers at nine short-tandem-repeat (STR) loci, 23 isolated STR mismatches were observed. The parenthood in each of these cases was highly validated (probability >99.97%). The event was always repeat related, owing to either a single-step mutation (n=22) or a double-step mutation (n=1). The mutation rate was between 0 and 7 x 10(-3) per locus per gamete per generation. No mutations were observed in three of the nine loci. Mutation events in the male germ line were five to six times more frequent than in the female germ line. A positive exponential correlation between the geometric mean of the number of uninterrupted repeats and the mutation rate was observed. Our data demonstrate that mutation rates of different loci can differ by several orders of magnitude and that different alleles at one locus exhibit different mutation rates.     

Effect of incubation and preservation on resting egg hatching and mixis in the derived clones of the rotifer Brachionus plicatilis

The marine rotifer Brachionus plicatilis typicus (Clone 8105A, Univ. of Tokyo) was cultured in 500 ml beakers to form resting eggs. Tetraselmis tetrathele was used as a culture food. Just after formation, resting eggs were exposed to various temperature (5–25 °C) and light regimes (24L: OD and OL : 24D). When eggs were exposed to light just after formation, the eggs hatched sporadically over a month. No hatching was observed for six months when eggs were preserved under dark conditions regardless of the temperature. These eggs hatched simultaneously after being exposed to light and eggs preserved at 5 °C showed twice as high hatching rate (40%) as that of eggs preserved at 15–25 °C (24%). Clones from resting eggs that were kept under different temperature and light regimes were reared individually to the third generation. Incubation at 25 °C with lighting produced the highest (5.4% and 5.2 %) rate of mictic females during their 2nd and 3rd generations, respectively. The lowest rates (0 and 1.5%) were found when the eggs were kept at 5 °C in total darkness for six months. A lower rate of amictic female production was found in clones with higher rates of mixis.     

Bromobimanes — fluorescent labeling agents for histochemical detection of sulfur containing neuropeptides in semithin sections

Summary Sulfur containing neuropeptides could be demonstrated in semithin sections of invertebrate nervous tissue, especially of gastropods, by using the bromobimanes as fluorescent labeling agents for thiol groups. Semithin sections showed a brilliant fluorescence of labeled peptides and should be used if an excellent resolution is important. The three bromobimanes (MB: monobromobimane, DB: dibromobimane, MQ: monobromotrimethylammoniobimane) gave positive results under our experimental conditions. Dibromobimane (DB) was selected because the application is more convenient.In gastropods, the bromobimane technique seems to be the most specific and sensitive one compared to the classical neurosecretory staining methods. Neuropeptides with sulfur containing amino acids could be demonstrated in perikarya, axons, and axon swellings easily. We suppose that there are neurons—not stainable by the classical methods—which can be identified as peptidergic ones by the bromobimane technique.A slight reduction of fluorescence intensity (fading) was observed. So, the fading rate was determined for dibromobimane reaction products; a decrease in the fluorescence intensity of 50% was only reached after 1 h using a Neofluar objective 10/0.30. Nevertheless, we suppose that a comparative quantification of the labeled neuropeptides should be possible if special parameters are considered.     

Bromobimanes--fluorescent labeling agents for histochemical detection of sulfur containing neuropeptides in semithin sections

Sulfur containing neuropeptides could be demonstrated in semithin sections of invertebrate nervous tissue, especially of gastropods, by using the bromobimanes as fluorescent labeling agents for thiol groups. Semithin sections showed a brilliant fluorescence of labeled peptides and should be used if an excellent resolution is important. The three bromobimanes (MB: monobromobimane, DB: dibromobimane, MQ: monobromotrimethylammoniobimane) gave positive results under our experimental conditions. Dibromobimane (DB) was selected because the application is more convenient. In gastropods, the bromobimane technique seems to be the most specific and sensitive one compared to the classical neurosecretory staining methods. Neuropeptides with sulfur containing amino acids could be demonstrated in perikarya, axons, and axon swellings easily. We suppose that there are neurons--not stainable by the classical methods--which can be identified as peptidergic ones by the bromobimane technique. A slight reduction of fluorescence intensity (fading) was observed. So, the fading rate was determined for dibromobimane reaction products; a decrease in the fluorescence intensity of 50% was only reached after 1 h using a Neofluar objective 10/0.30. Nevertheless, we suppose that a comparative quantification of the labeled neuropeptides should be possible if special parameters are considered.     

Simulation of the population dynamics and social structure of the Virunga mountain gorillas

An agent-based model was developed to simulate the growth rate, age structure, and social system of the endangered mountain gorillas (Gorilla beringei beringei) in the Virunga Volcanoes region. The model was used to compare two types of data: 1) estimates of the overall population size, age structure, and social structure, as measured by six censuses of the entire region that were conducted in 1971-2000; and 2) information about birth rates, mortality rates, dispersal patterns, and other life history events, as measured from three to five habituated research groups since 1967. On the basis of the research-group data, the "base simulation" predicted a higher growth rate than that observed from the census data (3% vs. 1%). This was as expected, because the research groups have indeed grown faster than the overall population. Additional simulations suggested that the research groups primarily have a lower mortality rate, rather than higher birth rates, compared to the overall population. Predictions from the base simulation generally fell within the range of census values for the average group size, the percentage of multimale groups, and the distribution of females among groups. However, other discrepancies predicted from the research-group data were a higher percentage of adult males than observed, an overestimation of the number of multimale groups with more than two silverbacks, and an overestimated number of groups with only two or three members. Possible causes for such discrepancies include inaccuracies in the census techniques used, and/or limitations with the long-term demographic data set obtained from only a few research groups of a long-lived species. In particular, estimates of mortality and male dispersal obtained from the research groups may not be representative of the entire population. Our final simulation addressed these discrepancies, and provided a better basis for further studies on the complex relationships among individual life history events, group composition, population age structure, and growth rate patterns.