Differential staining of nerve cells and fibres for sections of paraffin-embedded material in mammalian central nervous system

A differential staining method is described of myelinated fibres and nerve cell bodies applicable to sections of mammalian, including human, central nervous system specimens embedded in paraffin wax. Experimental and human necropsy material fixed in acetic paraformaldehyde in phosphate buffer was used. Sections of 15–20 m in thickness were obtained, attached to slides, deparaffinized and hydrated. After hydration, sections are oxidized (30 s) in 2% potassium permanganate, bleached (1 min) in 5% oxalic acid and rinsed in distilled water. Staining is for 2–5 h in the following solution: 0.06% thionin, 1% formaldehyde, 10% acetic acid in distilled water. Sections are subsequently washed in distilled water, dehydrated through 96% and absolute ethanol, cleared in eucalyptol and mounted in Eukitt. Using the method described in the present paper, a differential coloration of myelin and neurons is obtained. Myelinated fibres appear red, whereas nerve cell bodies and glial nuclei are stained blue. This procedure provides a high contrast between myelin and cells suitable for observation and photography of sections. Simultaneous and differential coloration of both myelin and cells is easily and directly obtained with constant and homogeneous results.     

How to measure image quality in tissue-based diagnosis (diagnostic surgical pathology)

Background

Automated image analysis, measurements of virtual slides, and open access electronic measurement user systems require standardized image quality assessment in tissue-based diagnosis.

Aims

To describe the theoretical background and the practical experiences in automated image quality estimation of colour images acquired from histological slides.

Theory, material and measurements

Digital images acquired from histological slides should present with textures and objects that permit automated image information analysis. The quality of digitized images can be estimated by spatial independent and local filter operations that investigate in homogenous brightness, low peak to noise ratio (full range of available grey values), maximum gradients, equalized grey value distribution, and existence of grey value thresholds. Transformation of the red-green-blue (RGB) space into the hue-saturation-intensity (HSI) space permits the detection of colour and intensity maxima/minima. The feature distance of the original image to its standardized counterpart is an appropriate measure to quantify the actual image quality. These measures have been applied to a series of H&;E stained, fluorescent (DAPI, Texas Red, FITC), and immunohistochemically stained (PAP, DAB) slides. More than 5,000 slides have been measured and partly analyzed in a time series.

Results

Analysis of H&;E stained slides revealed low shading corrections (10%) and moderate grey value standardization (10 – 20%) in the majority of cases. Immunohistochemically stained slides displayed greater shading and grey value correction. Fluorescent stained slides are often revealed to high brightness. Images requiring only low standardization corrections possess at least 5 different statistically significant thresholds, which are useful for object segmentation. Fluorescent images of good quality only posses one singular intensity maximum in contrast to good images obtained from H&;E stained slides that present with 2 – 3 intensity maxima.

Conclusion

Evaluation of image quality and creation of formally standardized images should be performed prior to automatic analysis of digital images acquired from histological slides. Spatial dependent and local filter operations as well as analysis of the RGB and HSI spaces are appropriate methods to reproduce evaluated formal image quality.

     

Five years of experience teaching pathology to dental students using the WebMicroscope

Background

We describe development and evaluation of the user-friendly web based virtual microscopy - WebMicroscope for teaching and learning dental students basic and oral pathology. Traditional students microscopes were replaced by computer workstations.

Methods

The transition of the basic and oral pathology courses from light to virtual microscopy has been completed gradually over a five-year period. A pilot study was conducted in academic year 2005/2006 to estimate the feasibility of integrating virtual microscopy into a traditional light microscopy-based pathology course. The entire training set of glass slides was subsequently converted to virtual slides and placed on the WebMicroscope server. Giving access to fully digitized slides on the web with a browser and a viewer plug-in, the computer has become a perfect companion of the student.

Results

The study material consists now of over 400 fully digitized slides which covering 15 entities in basic and systemic pathology and 15 entities in oral pathology. Digitized slides are linked with still macro- and microscopic images, organized with clinical information into virtual cases and supplemented with text files, syllabus, PowerPoint presentations and animations on the web, serving additionally as material for individual studies. After their examinations, the students rated the use of the software, quality of the images, the ease of handling the images, and the effective use of virtual slides during the laboratory practicals. Responses were evaluated on a standardized scale. Because of the positive opinions and support from the students, the satisfaction surveys had shown a progressive improvement over the past 5 years. The WebMicroscope as a didactic tool for laboratory practicals was rated over 8 on a 1-10 scale for basic and systemic pathology and 9/10 for oral pathology especially as various students’ suggestions were implemented. Overall, the quality of the images was rated as very good.

Conclusions

An overwhelming majority of our students regarded a possibility of using virtual slides at their convenience as highly desirable. Our students and faculty consider the use of the virtual microscope for the study of basic as well as oral pathology as a significant improvement over the light microscope.

     

Quality evaluation of virtual slides using methods based on comparing common image areas

Background

There are many scanners of glass slides on the market now. Quality of digital images produced by them may be different and pathologists who examine virtual slides on a monitor may subjectively evaluate it. However, objective comparison of quality of digital slides captured by various devices requires assessment algorithms, which will be automatically executed.

Methods

In this work such an algorithm is proposed and implemented. It is dedicated for comparing quality of virtual slides which show the same glass slide captured by two or more scanners. In the first step this method looks for the largest corresponding areas in the slides. This task is realized by defining boundaries of tissues and providing the relative scale factor. Then, a certain number of smaller areas, which show the same fragments of both slides, is selected. The chosen fragments are analyzed using Gray Level Co-occurrence Matrix (GLCM). For GLCM matrices some of the Haralick features are calculated, like contrast or entropy. Basing on results for some sample images, features appropriate for quality assessment are chosen. Aggregation of values from all selected fragments allows to compare the quality of images captured by tested devices.

Results

Described method was tested on two sets of ten virtual slides, acquired by scanning the same set of ten glass slides by two different devices. First set was scanned and digitized using the robotic microscope Axioscope2 (Zeiss) equipped with AxioCam Hrc CCD camera. Second set was scanned by DeskScan (Zeiss) with standard equipment. Before analyzing captured virtual slides, images were stitched and converted using software which utilizes advances in aerial and satellite imaging.The results of the experiment show that calculated quality factors are higher for virtual slides acquired using first mentioned device (Axioscope2 with AxioCam).

Conclusions

Results of the tests are consistent with opinion of the pathologists who assessed quality of virtual slides captured by these devices. This shows that the method has potential in automatic evaluation of virtual slides’ quality.

     

Quantitative aspects of the cytochemical Feulgen-DNA procedure studied on model systems and cell nuclei

Summary Quantitative aspects of DNA losses during fixation and pararosaniline(SO₂)-Feulgen staining of microscopic preparations were studied. The preparation of a new cytochemical model, consisting of DNA-protein layers (with thicknesses between 0.1 and 5.0 m) on microscopic glass slides is described and potentialities and limitations of this model are discussed.Polyacrylamide films into which high molecular weight calf thymus DNA or chicken erythrocyte nuclei had been constrained served as another model. As biological objects chicken erythrocyte nuclei and rat liver nuclei either in suspension or on microscopical glass slides were used.The experimental results indicate a loss of about 5% of the DNA due to the fixation procedure applied. Hydrolysis in 5 N HCl at room temperature, staining with the pararosaniline-Schiff medium and rinsing with sulfurous acid induced losses of DNA too, varying in amount depending on the type of preparation used. About 10% of the original DNA content is lost in total from chicken erythrocyte nuclei and rat liver nuclei dried on microscopical glass slides, from chicken erythrocyte nuclei constrained in polyacrylamide films, and from DNA-protein layers on microscopic glass slides. For nuclei fixed and stained in suspension the total losses amount to about 40%. The differences in losses between various types of preparations are discussed. Biochemically, the content of DNA originally present per chicken eythrocyte nucleus was determined to be 2.52 pg, a value, which is in good accordance with reliable biochemical data published already. It is shown that calibration of cytochemical staining intensities into biochemical units or absolute amounts of material by use of a model system, is only reliable when it is known or to be expected that both the loss of material due to fixation and staining, and the stoichiometric relation between material present and dye molecules is identical. The same holds for the application of internal biological reference systems.Supported by grant no. 28-394 of the Praeventiefonds, The HagueIn receipt of a grant from Het Ministerie van Onderwijs en Wetenschappen, Afdeling Buitenlandse Betrekkingen, The Netherlands     

Spreading and staining of human metaphase chromosomes on aminoalkylsilane-treated glass slides

Summary The properties of aminoalkylsilane-treated glass slides for the preparation of metaphase spreads and their staining quality have been studied and compared with those of slides which had only been cleaned in ethanol/ether. The parameters investigated were: (1) the average area of metaphases from cultures of blood from both healthy donors and haematology patients; (2) the influence of the positively charged coating on the quality of quinacrine- and Giemsa-banding patterns; (3) non-specific background staining for these banding methods; (4) the number of metaphases as compared to the number of interphase cell nuclei per area of preparation; and (5) the Feulgen-staining intensities of chromosomes and chicken erythrocyte nuclei.The quality of metaphase preparations and the differential staining of chromosomes is better on aminoalkysilane-treated glass slides than that of preparations on routinely cleaned normal microscope slides. In the preparations on aminoalkylsilance-treated slides, the distribution of the cells over the glass surface is more homogeneous; and no influence could be detected on the relative frequency of metaphases as compared to the number of non-divided cell nuclei; the average area per metaphase is increased by about 10% and consequently the number of overlapping chromosomes is decreased.Preparations on aminoalkylsilane-treated glass, after Q-, G- and DAPI-banding procedures, always showed less binding of the staining compounds to the glass slide (a cleaner background) than those on routinely cleaned microscope glass slides. The Feulgen-pararosaniline staining intensities of human metaphase chromosomes and chicken erythrocyte nuclei are the same on aminoalkylsilane-treated slides and on routinely cleaned glass slides. Furthermore, the reproducibility and constancy of quinacrine banding was improved by development of an equilibrium staining method which does not require a washing procedure. The medium, containing 0.002% quinacrine, allows optimal staining results to be obtained for microphotography purposes within 30 min of staining (for visual inspection at least 90 min is required) and is used as the embedding medium.In combination with aminoalkylsilane-treated glass slides, this procedure leads to a clean background and reproducible banding patterns of excellent quality, the results being better and more constant than those of methods described before.     

Glucosinolate degradation products,alkanes and fatty acids from plants and cell cultures of Descurainia sophia

Allyl and 3-butenyl isothiocyanate with two nitriles and an epithiobutane derivative were estimated. These glucosinolate degradation products were found in callus, seed, and dried plant but not in suspension cultures. Seventeen alkanes and five fatty acids were also identified and estimated in plant material and cultures. 4-Methylthiobutyl and 2-phenylethyl isothiocyanates were also detected in seeds. Incubation of cultures at 4° increased levels of the fatty acids but not isothiocyanates.Abbreviations GC Gas chromatography - MS Mass Spectroscopy - 2,4-D 2,4-Dichlorophenoxy acetic acid - NAA - Naphthalene acetic acid     

Stoffwechsel von Isoflavonen und Cumöstanen in Zell- und Callussuspensionskulturen von Phaseolus aureus Roxb.

Summary The isoflavone daidzein, the coumestanes coumestrol and soyagol as well as 2,4,4-trihydroxychalcone were isolated from callus and cell suspensions of root tip tissue from Phaseolus aureus Roxb. Upon prolonged culturing callus suspensions gradually became cell suspensions, a process which was accompanied by a decrease in the accumulation of phenolics. Upon transfer of the cells into 3 different media containing -indolyl acetic acid, kinetin or -naphthalene acetic acid, a drastic increase in the amount of coumestrol was measured.The data are discussed in relation to the observed differentiation of the cultures after application of the various hormones. The cultures were shown to metabolize daidzein and other phenylpropanoid compounds. A pronounced binding of daidzein to polymeric, ethanol-insoluble material is discussed in relation to our earlier findings in isoflavone metabolism.     

Expression of Mitochondrial Cytochrome C Oxidase Chaperone Gene (COX20) Improves Tolerance to Weak Acid and Oxidative Stress during Yeast Fermentation

Introduction

Saccharomyces cerevisiae is the micro-organism of choice for the conversion of fermentable sugars released by the pre-treatment of lignocellulosic material into bioethanol. Pre-treatment of lignocellulosic material releases acetic acid and previous work identified a cytochrome oxidase chaperone gene (COX20) which was significantly up-regulated in yeast cells in the presence of acetic acid.

Results

A Δcox20 strain was sensitive to the presence of acetic acid compared with the background strain. Overexpressing COX20 using a tetracycline-regulatable expression vector system in a Δcox20 strain, resulted in tolerance to the presence of acetic acid and tolerance could be ablated with addition of tetracycline. Assays also revealed that overexpression improved tolerance to the presence of hydrogen peroxide-induced oxidative stress.

Conclusion

This is a study which has utilised tetracycline-regulated protein expression in a fermentation system, which was characterised by improved (or enhanced) tolerance to acetic acid and oxidative stress.     

Dynamics and attenuation characteristics of periphyton upon artificial substratum under various light conditions and some additional observations on periphyton upon Potamogeton pectinatus L.

The seasonal variation in periphyton dynamics has been studied upon artificial substratum (microscopic glass slides) under various light conditions during the periods May–October 1986 and May–September 1987, in Lake Veluwe. Some additional observations on the periphyton development upon leaves of Potamogeton pectinatus L. have been made simultaneously. Four different light conditions were created in an experimental setup by manipulating the photon flux density through artificial shading.Periphyton upon artificial substratum exhibited a relatively high abundance with a distinct seasonal pattern. Periphyton accrual rates were highest at the beginning of June and in August and September upon slides which were incubated for two weeks. Periphyton mass increased during May and June, decreased or remained about the same during July and subsequently increased until an upper plateau was reached upon slides which were incubated from the beginning of May onwards.Generally, periphyton mass was lower upon slides than upon P. pectinatus. The seasonal variation in periphyton mass was more pronounced upon P. pectinatus leaves than upon the slides.Attenuation by periphyton upon slides ranged from 5 to 65% after two weeks of incubation. Periphyton upon slides which had been incubated for more than two weeks demonstrated an attenuation of more than 85%.Water quality parameters other than photon flux density were probably more important in determining the periphyton dynamics, since only minor differences were observed in periphyton mass between the various light conditions. Chlorophyll-a content was higher with increased shading on various sampling dates.Periphyton, especially older periphyton consisted largely of settled silt and clay particles and to a lesser extent of detrital matter on both substrata. Living epiphytes were only a relatively small fraction.It is concluded that a reduction of resuspension of sediment particles, giving less suspended matter in the water column, will result in lower periphytic mass. Consequently, the quantity of photosynthetically active radiation reaching the submerged macrophytes is expected to increase considerably.     

A simple method for incubation of tissue sections in immunohistochemistry

Summary The construction of a simple incubatin chamber is presented by which a homogeneous incubation of tissue sections with calculable amounts of immunoreagents is guaranteed. The incubation of large area sections, and parallel incubations of 10 slides are possible. As an example of application a case of alpha-1-antitrypsin deficiency in the liver is demonstrated.     

Effect of cytokinin on morphological changes of suspension cultured cells of the moss, Barbula unguiculata

Suspension cultured cells of the moss, Barbula unguiculata, grow actively in both light and dark culture. Light-grown cells contain chlorophyll and exhibit an undifferentiated callus form. When cells are transferred to a dark condition, they develop into protonemata. Protonemata formation in the dark can be inhibited by the addition of 5 M benzyladenine or 6-furfurylaminopurine but is not affected by the addition of 5 M 2,4-dichlorophenoxyacetic acid or naphthalene acetic acid.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - kinetin 6-furfurylaminopurine - NAA naphthalene acetic acid     

Online teaching of inflammatory skin pathology by a French-speaking International University Network

Introduction

Developments in technology, web-based teaching and whole slide imaging have broadened the teaching horizon in anatomic pathology. Creating online learning material including many types of media such as radiologic images, whole slides, videos, clinical and macroscopic photographs, is now accessible to most universities. Unfortunately, a major limiting factor to maintain and update the learning material is the amount of resources needed. In this perspective, a French-national university network was initiated in 2011 to build joint online teaching modules consisting of clinical cases and tests. The network has since expanded internationally to Québec, Switzerland and Ivory Coast.

Method

One of the first steps of the project was to build a learning module on inflammatory skin pathology for interns and residents in pathology and dermatology. A pathology resident from Québec spent 6 weeks in France and Switzerland to develop the contents and build the module on an e-learning Moodle platform under the supervision of two dermatopathologists. The learning module contains text, interactive clinical cases, tests with feedback, virtual slides, images and clinical photographs. For that module, the virtual slides are decentralized in 2 universities (Bordeaux and Paris 7). Each university is responsible of its own slide scanning, image storage and online display with virtual slide viewers.

Results

The module on inflammatory skin pathology includes more than 50 web pages with French original content, tests and clinical cases, links to over 45 virtual images and more than 50 microscopic and clinical photographs. The whole learning module is being revised by four dermatopathologists and two senior pathologists. It will be accessible to interns and residents in the spring of 2014. The experience and knowledge gained from that work will be transferred to the next international resident whose work will be aimed at creating lung and breast pathology learning modules.

Conclusion

The challenges of sustaining a project of this scope are numerous. The technical aspect of whole-slide imaging and storage needs to be developed by each university or group. The content needs to be regularly updated and its accuracy reviewed by experts in each individual domain. The learning modules also need to be promoted within the academic community to ensure maximal benefit for trainees. A collateral benefit of the project was the establishment of international partnerships between French-speaking universities and pathologists with the common goal of promoting pathology education through the use of multi-media technology including whole slide imaging.

     

Vital DNA staining of agarose-embedded protoplasts and cell suspensions of Nicotiana plumbaginifolia

The DNA of agarose-embedded protoplasts of Nicotiana plumbaginifolia was stained with Hoechst 33342 by immersing microscope slides, coated with immobilized protoplasts, into Erlenmeyer flasks containing consecutively dye solution, pH-correcting washing solutions and culture medium. After staining, protoplasts regenerated cell walls, started to divide and proliferated to calli. The culture system with immobilized protoplasts permits rapid change of culture media and accurate control of experimental conditions. The staining technique offers the opportunity for continuous observation of chromosomal behaviour and cell dynamics in individual plant cells.The same staining procedure was successfully applied to DNA of plant cells in suspension. Flow cytometric analysis revealed a retarding effect of the dye on the cell cycle, but within hours the cells recovered and showed their normal growth characteristics as compared to the controls.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxy acetic acid - DAPI 4'6-diamidino-2-phenylindole - FDA fluorescein diacetate - LMT low melting temperature - MES 2(N-morpholino)ethanesulfonic acid - MS Murashige and Skoog-medium - NAA -naphthaleneacetic acid - PCV packed cell volume - Tris Tris(hydroxymethyl)amino methane     

The periodic acid-schiff reaction in neutrophil leukocytes-influence of fixation and amylase digestion

Summary The periodic acid-Schiff (PAS) reaction in normal human neutrophil leukocytes was studied morphologically and microspectrophotonietrically after different fixation methods and after treatment with -amylase and -amylase.The best fixation methods with respect to the morphological distribution of the stain and to the quantitatively measured amount of PAS positive material preserved in the cells were fixation with absolute methanol, absolute ethanol, acetic alcohol formalin and Rossman's fluid as well as fixation by freeze substitution. The amount of PAS reactive material in the cells after these fixation methods was in the same range as in unfixed cells covered with a semi-permeable formvar membrane. The PAS positive material could be removed by treatment with -amylase after most fixatives, the most important exception being acetic alcohol formalin, after which fixative no reduction was obtained in the amount of PAS positive material. This may be due to the fixative creating an -amylase resistant protein-glycogen binding, -amylase digestion gave only a slight reduction in the amount of PAS reactive material.The results indicate that the main part of the PAS reactive material in normal human neutrophil leukocytes consists of glycogen. It is probably preserved to about 90 per cent or more by the above mentioned fixatives.

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Zusammenfassung Die Perjodsäure-Schiff-(PAS-)Reaktion in normalen menschlichen neutrophilen Leukocyten wurde morphologisch und mikrospektrophotometrisoh nach verschiedenen Fixierungen und nach - und -Amlyase-Behandlung untersucht.Die besten Fixierungsmittel waren — in Hinsicht auf die Verteilung und die quantitativ bestimmbare Menge der erhaltenen PAS-positiven Substanz — absolutes Methanol, absolutes Äthanol, acetic-alcohol-formalin und Rossmans Fixierungsmittel. Fixierung mittels Gefrier-Substitution hat sich ebenfalls bewährt.Die Menge der nachweisbaren PAS-positiven Substanz lag nach diesen Vorbehandlungen in der gleichen Größenordnung, wie bei nicht fixierten, mit einer semipermeablen Formvarmembran überzogenen Zellen.Die PAS-positive Substanz war durch -Amylase-Behandlung nach den meisten Fixierungen entfernbar; die wesentliche Ausnahme bildet acetic-alcohol-formalin, hiernach war keine Entfernung der PAS-positiven Substanz möglich. Die Ursache wird in einer -Amylase-resistenten Protein-Glykogen-Bindung gesehen. -Amylase führte zu einer nur geringfügigen Abnahme PAS-positiven Materials.Die Ergebnisse zeigen, daß der größere Teil des PAS-positiven Materials in normalen menschlichen Leukocyten Glykogen ist. Es wird mindestens zu 90% durch die oben erwähnten Methoden fixiert.

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This work has been supported by grants from the Swedish Medical Research Council and the Wallenberg Foundation.

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The Fibrinogen was kindly supplied by the Kabi AB, Stockholm, Sweden.     

Isolation and classification of acetic acid bacteria from high percentage vinegar fermentations

Summary A method for growing acetic acid bacteria from high percentage submerged vinegar fermentations was established by overflowing soft agar, containing acetic acid, with fermenting reactor fluid. Mixed cultures were found in a submerged process that was working well (1), in a submerged process that had broken down (2), and in a vinegar generator (3). The strains differed in part from each other with respect to tolerance towards acetic acid, ethanol, pH and in other physiological criteria. All strains that were isolated from (1) and some from (3) were specialized for acetate media as they needed acetic acid and low pH values (2.1–3.8) in addition to yeast extract and glucose or ethanol. We suppose that they belonged to the acetic acid-producing strains active in the process. None of the strains derived from (2) was of this acetophilic type. All except one of the stains from (2) belonged to the species Acetobacter hansenii, the other cultures were of A. pasteurianus.     

Phytomonitoring of pulverized fuel ash leachates by the duckweed Lemna minor

The duckweed Lemna minor is one of the smallest vascular plants with a known strong capacity for metal accumulation. L. minor is proposed as a phytomonitor for coal ash drainage systems and for bio-assay studies directed to complexation and speciation. The duration of the experiment can be restricted to fourteen days; it is then possible to determine accurate data of differences in growth of the clone forming plant by using image processing techniques. Leaching of pulverized fuel ash (PFA) with acetic acid according to EPA instruction resulted in effects attributed to the acetic acid itself rather than to the metals in solution. Toxic effects of both leachates, natural and artificial, are discussed. The order of toxicity of metals studied so far in separate metal experiments is Cd > Cu > Zn > As(Arsenite) > Se(Selenite) > Ge > B > Mo.