bluuebunny/crossref_metadata_2025 huggingface dataset:
Formation of the chorion (zona pellucida) in the teleost, Oryzias latipes. II. Polysaccharide cytochemistry of early oogenesis.
Authors: Tesoriero J V
Published: April 2011

It has been proposed that the mechanism of formation of the chorion of Oryzias latipes involves the transfer of precursor material from the Golgi bodies of the oocyte to the chorion by means of a population of dense cored vesicles. Thin sectioned ovarian material was investigated by means of several techniques available for the resolution of periodate reactive material at the ultrastructural level. The techniques employed were modifications of the thiocarbohydrazide, thiosemicarbazide, alkaline bismuth subnitrate and silver methenamine reactions. These all demonstrated a positive reaction in the material of the chorion and that of the dense cored vesicles. This finding is in accord with the hypothesis that the dense cored vesicles are transporting material from the Golgi to the growing chorion.
Response of the lysosomal system of the corneal epithelium to tyrosine-induced cell injury.
Authors: Gipson I K , Anderson R A
Published: April 2011

Rats fed excess tyrosine develop corneal epithelial disease which parallels that found in humans with tyrosine aminotransferase deficiency (tyrosinosis). In the rat, focal lesions develop within the central epithelium and contain crystals (presumably tyrosine) that disrupt cells. We have studied these lesions and localized acid phosphatase and aryl sulfatase at the electron microscope level. Within 60 hr after initiation of diet, cells within the lesions showed an increase in lysosomal enzyme activity. This activity was localized inside some of the crystal ghosts and in numerous lysosomes, including autophagic vacuoles and multivesicular bodies. After 84 hr on diet the entire central corneal epithelium was disrupted and polymorphonuclear leukocytes had infiltrated the area. Crystals were phagocytosed by or developed within polymorphonuclear leukocytes. We hypothesize that crystals form within epithelial cells, disrupt first their lysosomes, and then cells, leading to externalization of lysosomal enzymes. This extracellular lysosomal enzyme release may be responsible for the acute inflammatory response that ensues.
Temperature and acid concentration in the search for optimum Feulgen hydrolysis conditions.
Authors: Kjellstrand P T
Published: April 2011

Exposure and removal of aldehyde groups during Feulgen acid hydrolysis were studied at a wide range of temperature and acid concentrations. Temperatures between 9 and 75degreesC were found to influence only the rate of the hydrolysis reaction over the entire range from high (6 M) to low (0.05 M) HCl concentrations. The temperature dependence was high, and around +5degreesC was sufficient to double the reaction rate. The influence of acid concentrations between 0.02 and 6 M was studied, and the extraction rates that determine the peak values of the Feulgen hydrolysis curve were found to depend in the same way on the (H+) concentration. A diagram is given that makes it possible to determine the time to reach the point during hydrolysis where the maximum amount of aldehyde groups are developed for a wide range of temperatures and acid concentrations. Temperatures slightly above room temperature in combination with high acid concentration is recommended for Feulgen hydrolysis.
Oxidation of tissue polysaccharides by periodic acid in dimethyl sulfoxide and its anhydrous and aqueous mixtures.
Authors: Malinin G I
Published: April 2011

Periodic acid (1% w/v) solvated by anhydrous dimethyl sulfoxide (DMSO) readily induced a strong Schiff reaction in a variety of structures containing polysaccharides, but not glycogen. With the increasing amounts of water added to DMSO, glycogen was also oxidized, while the selective localization of other polysaccharides remained unimpaired. Periodate, solvated in the anhydrous acetic acid-DMSO mixture, rapidly induced concomitant oxidation of nucin and glycogen-containing structures. Sodium bisulfite addition derivatives of carbonyls, induced by periodate oxidation in DMSO, were stained meta- and orthochromatically with toluidine blue at controlled pH. Certain metachromatic tissue components were strongly birefringent in polarized light in contrast to the identical structures oxidized by aqueous periodate. Marked differences in staining reactions elicited in identical structures by periodate in DMSO as compared with aqueous periodate suggest that DMSO-periodate method considerably enhances the range of histochemical oxidations by periodate.
Histochemical demonstration of unsaturated hydrophilic lipids with palladium chloride.
Authors: Kiernan J A
Published: April 2011

Compounds in which olefinic linkages are accessible to aqueous reagents reduce the chloropalladite ion [PdCl4]2-, to metallic palladium. This reaction is used in a histochemical method whereby hydrophilic unsaturated lipids are stained dark brown or black. The specificity of the new method has been confirmed by means of solvent-extraction and chemical blocking procedures and by comparison with other histochemical techniques. Yellow staining of collagen, keratin and cytoplasm is probably due to attachment of the chloropalladite anion to proteins. The yellow background can be largely decolorized by treating the sections with aqueous pyridine, which forms colorless complexes with divalent palladium. A standard technique for staining with palladium is presented and the method is discussed in relation to other histochemical procedures that demonstrate unsaturated lipids.
Cytochemical localization of NADH oxidase in Candida albicans.
Authors: Borgers M , De Nollin S , Thoné F , Van Belle H
Published: April 2011

The application of a recently published technique to localize reduced nicotinamide adenine dinucleotide oxidase activity is described in glutaraldehyde-fixed Candida albicans. The reaction product appears as a finely granular precipitate on the mitochondrial cristae and on the central vacuolar membrane, and, if present, on the vacuolar contents. Fixation should be kept to a minimum and prolonged incubation times up to 2 hr are necessary to show these reactive sites. The reaction appears to be strongly substrate-dependent and not affected by cyanide. Exposure of C. albicans cells to the antimycotic miconazole resulted in a strong increase in reduced nicotinamide, adenine dinucleotide and oxidase activity. The hypothesis is put forward that this enzyme, together with peroxidative and catalatic enzymes, may be implicated in the mechanism by which miconazole exerts its lethal effect on C. albicans.
Altered renal cortical reabsorption of protein and urinary excretion of sodium in relation to vascular leakage induced by horseradish peroxidase.
Authors: Straus W
Published: April 2011

The intravenous injection of horseradish peroxidase (HRP) into rats of the Sprague-Dawley strain caused vascular leakage as detectable by a 20-40% increase in the hematocrit and a 15-20% decrease in plasma protein concentration. These changes did not occur when the same amounts of HRP were injected into rats pretreated with antagonists to histamine and serotonin. After pretreatment with the antagonists, the reabsorption of HRP by the proximal tubule cells (the concentration of HRP in the total particulate fractions) showed a 77% decrease and the urinary excretion of sodium showed more than an 80% increase as compared to the values from rats treated with HRP alone. In addition, the blood clearance rate of HRP was decreased and the urinary excretion of HRP was increased after treatment with the antagonists to histamine and serotonin. Cytochemical observations of formaldehyde vapor-fixed tissue also showed the effects of vascular leakage. After the injection of HRP in physiologic or hypertonic saline, the basal infoldings of the proximal tubule cells were strongly peroxidase-positive. When the same amounts of HRP were injected after pretreatment with antagonists to histamine and serotonin, or with mannitol, the basal infoldings were not stained or were stained faintly. Rats of the Wistar/Furth strain did not show the effects of vascular leakage observed with rats of the Sprague-Dawley strains. The questions are discussed as to whether the marked depression of renal cortical HRP absorption by mannitol and hypertonic saline (J Histochem Cytochem 23:707, 1975) is related to the prevention of vascular leakage, and whether the reabsorption of both sodium and protein is increased during the leakage of serum proteins into the interstitial tissue.
Colloidal gold, a useful marker for transmission and scanning electron microscopy.
Authors: Horisberger M , Rosset J
Published: April 2011

Electron dense markers of a size suitable for transmission electron microscopy and scanning electron microscopy have been prepared with gold granules labeled with a monolayer of specific macromolecules. The optimum conditions for preparing the markers have been ascertained. The method is simple, rapid and seems to be general since gold granules have been labeled with polysaccharides and proteins. As homogeneous populations of gold granules having different sizes can be prepared, the method is also suitable for double marking experiments. The gold technique is illustrated by the localization of polysaccharides and glycoproteins on yeast cell walls and erythrocyte membranes by transmission electron microscopy and on yeast cells and intact erythrocytes by scanning electron microscopy. Good spatial resolution of the marker was achieved in all cases. The method is also suitable for marking thin sections. Spectrophotometric measurements were used to determine the number of gold granules adsorbed per cell.
Immunohistochemical detection of Fc receptor. II. Electron microscopic demonstration of Fc receptor by using soluble immune complexes of ferritin-antiferritin immunoglobulin G.
Authors: Itoh G , Suzuki I
Published: April 2011

The mouse mesenteric lymph node cells were incubated in the soluble immune complexes of ferritin-antiferritin immunoglobulin G at 37 degrees C for 20 min. After being washed, postfixed with OsO4 and dehydrated by degraded ethanol series, the lymph node cells were observed by electron microscope. Apprroximately 15% of the cells (mainly composed of small lymphocytes) bound ferritin particles to the cell surface. The distribution pattern of the binding of ferritin particles (ferritin-antiferritin immunoglobulin G) took the form of discrete patches of irregular distribution interspaced with unlabeled portions. The electron microscopic features of ferritin particles (ferritin-antiferritin immunoglobulin G) attached to the cell surface suggest that a structure of constant conformation (Fc receptor) situated in the cell membrane takes part in the binding of ferritin-antiferritin immunoglobulin G.
Stabilization of cholesterol in myelin with digitonin: observation with polarized light.
Authors: Saland L C , Napolitano L M
Published: April 2011

Cholesterol can be preserved in tissues dehyrated for light microscopy by incorporating digitonin into glutaraldehyde fixation. Fresh and fixed frozen sections of rat sciatic nerve exhibit strong, radially positive birefringence when viewed with polarized optics. However, tissue fixed and dehydrated with standard methods for Epon embedding shows very weak radially positive birefringence. The use of digitonin with a variety of techniques generally permits retention of optical activity in embedded nerves. Such observations suggest a preservation of lipids (presumably cholesterol) in molecular orientations necessary to produce Maltese cross patterns.
Rapid chemical dehydration of biologic samples for scanning electron microscopy using 2,2-dimethoxypropane.
Authors: Maser M D , Trimble J J
Published: April 2011

Acidified 2,2-dimethoxypropane (DMP) has been used as a rapid chemical dehydrating agent en route to plastic embedding and ulthrathin sectioning for conventional electron miscroscopy (J Histochem Cytochem 23:107, 1975). We have used DMP to dehydrate biologic specimens prior to critical point drying and metal coating for scanning electron microscopy. There is no difference in either the gross architecture or the fine surface structure of mouse small intestine and trachea, rat trachea and kidney, and cultured fibroblasts, between samples dehydrated in DMP for 5 min to 30 days and those conventionally dehydrated in ethanol or acetone. DMP dehydration is advantageous in speed, economy and apparent completeness.
Scanning Feulgen-deoxyribonucleic acid cytophotometry of Papanicolaou destained preparations.
Authors: Anthony A , Dolber P C , Romanosky A J
Published: April 2011

Feulgen deoxyribonucleic acid cytophotometry of Papanicolaou destained specimens revealed a differential loss in Feulgen reactivity among human buccal and cervical smears, cultured embryonic lung fibroblasts and invasive cervical carcinoma cells. Loss in Feulgen reactivity in Papanicolaou destained fibroblasts and polyploid nuclei of malignant lesions was observed to result in underestimates of relative Feulgen deoxyribonucleic acid and nuclear area values using scanning integrating microdensitometry. Thus, Papanicolaou stained preparations may not be suitable for deoxyribonucleic acid quantification of high ploidy lesions since distributional absorption error is unpredictably influenced by such factors as ploidy level, nuclear size, chromatin dispersion and differential aldehyde loss during destaining. Feulgen deoxyribonucleic acid cytophotometry of Papanicolaou stained preparations can be useful for differentiating benign from malignant lesions if extent of aneuploidy (as reflected in abnormal deoxyribonucleic acid frequency distribution profile) is used as a diagnostic indicator.
Electron probe x-ray microanalysis of small granulated cells in rat sympathetic ganglia after sequential aldehyde and dichromate treatment.
Authors: Lever J D , Santer R M , Lu K S , Presley R
Published: April 2011

Rat adrenal medulla and celiac-mesenteric sympathetic ganglia were fixed by a glutaraldehyde/formaldehyde-potassium dichromate-osmium treatment sequence and plastic-embedded. Fine sections were examined by electron probe x-ray microanalysis. Comparable peaks for chromium (Kalpha = 5.4 keV) were obtained from cytoplasmic fields containing membrane-bounded inclusion granules in both adrenomedullary noradrenaline cells and a type (type II) of sympathetic small granulated cell whose inclusion granules closely resemble those of the adrenomedullary noradrenaline cell. Chromium was not detected in granules within adrenomedullary adrenaline cells nor in two other sympathetic small granualted cell types. In no material was chromium detected in agranular cytoplasmic or nuclear fields. Since chromium binds to the Schiff monobase formed by glutaraldehyde and noradrenaline during fixation, we infer that noradrenaline is present in the granules of the type II sympathetic small granulated cell, as well as in adrenomedullary noradrenaline cells.
Commercial horseradish peroxidase degrades myelin encephalitogenic protein during coupling for immunohistochemical studies.
Authors: Johnson A B , Cammer W
Published: April 2011

Conjugates of myelin encephalitogenic basic protein (EP) and commercial horseradish peroxidase (HRP) have been used for immunohistochemical demonstrations of anti-EP antibody in animals with experimental allergic encephalomyelitis. We performed gel electrophoresis studies on EP-HRP conjugates prepared with glutaraldehyde and on mixtures of EP and HRP incubated without glutaraldehyde. The results show that under conditions of one-and two-step coupling HRP causes rapid loss of the native EP band, apparently due to EP degradation. The EP-HRP mixtures are not encephalitogenic in rabbits, or encephalitogenic activity is lost during processing. The immunohistochemical reactivity of conjugates, however, signals some preservation of antibody-combining sites. The mechanism of the HRP effect on EP is unknown. The possibilities of a contaminating proteinase or direct peroxidatic attack are suggested. Until this action of HRP can be overcome, the effect of coupling procedures on the biological activities of EP will be difficult to assess, and EP-HRP conjugates cannot be expected to reveal sites that may bind encephalitogenic portions of the EP molecule.
Peroxisomes in pulmonate gastropods.
Authors: Dannen E , Beard M E
Published: April 2011

Organelles with the morphologic characteristics of peroxisomes have been found in the cells of the kidney sac of two terrestrial pulmonate gastropods. Arion ater and Ariolimax columbianus. These peroxisomes appear in profile as circles or ellipses, 0.25 micron in diameter and 0.3-0.8 micron long; They have a finely granular matrix and a single-limiting membrane; the organelles are extensively associated with smooth endoplasmic reticulum. Some Ariolimax peroxisomes contained structures reminiscent of nucleoids while those of Arion did not. The peroxisomes of Arion ater show a strongly-positive staining reaction with the 3,3'-diaminobenzidine technique, which is inhibited in the presence of aminotriazole. Peroxisomes of Ariolimax columbianus did not show a positive reaction, despite a number of variations of the 3,3'-diaminobenzidine protocol. Speculations are made concerning the biochemical reasons for this cytochemical behavior. Peroxisomes in both tissues were negatively stained while lysosomes were positively stained in acid-phosphatase incubations.
Specificity of the glial fibrillary acidic protein for astroglia.
Authors: Bignami A , Dahl D
Published: April 2011

Glial fibrillary acidic protein (GFA) is the main constituent of glial filaments and the close similarity of GFA and neurofilament protein has been recently reported. However, the immunofluorescence staining of peripheral nerve which may be observed with GFA antisera is not due to cross-reaction between GFA and neurofilament protein. Staining of peripheral axons was also observed with control sera obtained by injecting the rabbits with nonimmunogenic GFA preparations isolated with the same procedure. Immune GFA antisera and control sera reacted with sodium dodecyl sulfate extracts of sciatic nerve. However, the precipitin line formed with peripheral nerve crossed the line against GFA protein, thus indicating nonidentity between the two antigens. Buffer extract of sciatic nerves that had been incubated with spinal cord reacted by immunodiffusion with GFA antisera, thus indicating that redistribution of GFA occurred under these conditions.
The dispersal of cells from human gynecologic specimens: chemical agents.
Authors: Escobar G J , Todd P , Sattilaro R F
Published: April 2011

An investigation of nonenzymatic chemical solutions for the dispersal of gynecologic cells in suspension was undertaken using known general information on the composition of cell surfaces and cell adhesive properties. Studies on human cells cultured in vitro showed that thiol reducing agents release microextension-mediated contacts. Dispersing solutions containing lithium diiodosalicylate separated cells, presumably by extracting glycoproteins. Solutions without this agent but containing tetramethylurea and mixed sugars similar to those found on the cell surface separated cells by interacting with hydrogen bonds and other noncovalent polysaccharide interactions thought to hold cells together. Solutions containing ethylenediaminetetraacetate, dithiothreitol, tetramethylurea, mixed sugars, mixed amines and inorganic ions are suggested as mildly-acting dispersal solutions on the basis of evaluationtions by phase contrast microscopy, Papanicolaou staining and particle volume analysis.
Measurement of cytoplasmic fluorescence depolarization of single cells in a flow system.
Authors: Price G B , McCutcheon M J , Taylor W B , Miller R G
Published: April 2011

We have built a flow system in which we can analyze and sort individual viable cells on the basis of their cytoplasmic microviscosity. The average cytoplasmic microviscosity (or cytoplasmic structuredness) of a cell can be quantitated by exciting fluorescein molecules in the cytoplasm of the cell with a polarized light source and measuring the extent of depolarization of the fluorescent signal. Changes in the state of a cell (e.g., the reception of a signal inducing the cell to differentiate) often appear to be associated with changes in cytoplasmic microviscosity, these changes being detectable within hours of the inducing signal. An example of one such change is presented.
Scene segmentation techniques for the analysis of routine bone marrow smears from acute lymphoblastic leukemia patients.
Authors: Brenner J F , Necheles T F , Bonacossa I A , Fristensky R , Weintraub B A , Neurath P W
Published: April 2011

Automated analysis of lymphoblast cell morphology is being evaluated as a basis for predicting the response to therapy of patients with acute lymphoblastic leukemia. A new technique of scene segmentation particularly applicable to the "cluttered" images of cells in routine bone marrow smears is described. Morphologic characteristics of lymphoblasts found in bone marrow smears made at time of diagnosis were measured by an automated, interactive image-processing system using the new scene segmentation technique. These characteristics, on a patient by patient basis, are being compared to remission length and survival data to develop and test new prognostic methods.
An automated method of differential red blood cell classification with application to the diagnosis of anemia.
Authors: Bacus J W , Weens J H
Published: April 2011

A method of automated red cell analysis suitable for the rapid classification of large numbers of red cells from individual blood specimens has been developed, and preliminarily tested on normal bloods and clinically proven cases of anemias and red cell disorders. According to this method digital image processing techniques provide several features relating to shape and internal central pallor configurations of red cells. These features are used with a fully automated decision logic to rapidly provide a quantitative "red cell differential" analysis, a report of the percentage subpopulations of recognized categories of red cells. For each subpopulation, measurements of mean cell area, mean cell hemoglobin content and mean cell hemoglobin density are provided. The nine types of red cell disorders studied with this method were: (a) iron deficiency anemia, (b) the anemia of chronic disease, (c) beta-thalassemia trait, (d) sickle cell anemia, (e) hemoglobin C disease, (f) intravascular hemolysis, (g) hereditary elliptocytosis, (h) hereditary spherocytosis, and (i) megaloblastic anemia due to folic acid deficiency. Preliminary indications are that the red cell differential is useful in distinguishing between these conditions.
A program system for interactive measurements on digitized cell images.
Authors: Holmquist J , Bengtsson E , Eriksson O , Stenkvist B
Published: April 2011

Using a high precision image scanner and a PDP-8/F minicomputer, we have developed a program system for interactive measurements on microscopic images. By giving simple keyboard commands, the operator can run the image scanner and manipulate the digitized images. The interface between the operator and the microscope-computer system is a Tektronix 4010 graphic terminal. The system allows objects to be isolated and parameters to be calculated from each object, e.g., parameters characterizing shape of the object, irregularity in light transmission over the object, area, integrated light transmission, etc. Objects are isolated and parameters are calculated under complete operator control using interactive computer graphics technique. Calculated parameters may be stored in dedicated data records, which are stored in files for later statistical analysis. The system also includes a statistical evaluation part. Technically, the system consists of a command scanner, which translates commands into internal representation, a parser, which checks the syntax of the commands, and an interpreter, which executes the commands. The system is designed so that new commands can be added easily.
A multi-spectral approach for scene analysis of cervical cytology smears.
Authors: Aggarwal R K , Bacus J W
Published: April 2011

A multi-spectral approach for the scene analysis of cervical cytology smears, using multiple images of a scene digitized through suitably chosen color filters matched to the Papanicolaou stain, has been proposed here. This technique involves clustering of two-dimensional data for extracting cytoplasm of the epithelial cells. Its performance on an experimental data set of 233 scenes involving more than 10 types of normal and malignant epithelial cells has been compared with density and gradient thresholding techniques. This resulted in an approximate 83% rate of success compared to approximately 40% for the rest of the other techniques.
Segmentation of cervical cell images.
Authors: Cahn R L , Poulsen R S , Toussaint G
Published: April 2011

A major problem in the automation of cervical cytology screening is the segmentation of cell images. This paper describes various standard segmentation methods plus one which determines a segmentation threshold based on the stability of the perimeter of the cell as the threshold is varied. As well as contour, certain structural information is used to decide upon the threshold which separates cytoplasm from the background. Once the cytoplasm threshold is found, cytoplasm and nucleus are separated by simple clustering into three groups, cytoplasm, folded cytoplasm and nucleus. These techniques have been tested on 1500 cervical cells that belong to one of eight normal classes and five abnormal classes. A minimum Mahalanobis distance classifier was used to compare results. Manually thresholded cells were classified correctly 66.0% of the time for the 13 class problem and 95.2% of the time on the two (normal-abnormal) class problem. The contour tracing technique was 52.9% and 90.0% correct, respectively.
High resolution analysis of cervical cells--a progress report.
Authors: Poulsen R S , Oliver L H , Cahn R L , Louis C , Toussaint G
Published: April 2011

This paper presents preliminary results of research toward the development of a high resolution analysis stage for a dual resolution image processing-based prescreening device for cervical cytology. Experiments using both manual and automatic methods for cell segmentation are described. In both cases, 1500 cervical cells were analyzed and classified as normal or abnormal (dysplastic or malignant) using a minimum Mahalanobis distance classifier with eight subclasses of normal cells, and five subclasses of abnormal cells. With manual segmentation, false positive and false negative error rates of 2.98 and 7.73% were obtained. Similar experiments using automatic cell segmentation methods yielded false positive and false negative error rates of 3.90 and 11.56%, respectively. In both cases, independent training and testing data were used.
The automated classification of mitotic phase for human chromosome spreads.
Authors: Bishop R P , Young I T
Published: April 2011

A system has been developed that automatically recognizes the mitotic phase of human chromosome spreads for karyotyping. Suitable spreads are classified into one of five subphases of mitosis. Classification is performed on the basis of summed chromosome length and most probable chromosome width. Classification requires 100-500 msec. A television camera scans the spread through microscope optics; computer and special purpose electronics process the video signals to generate run length histograms. The histograms are used to determine mitotic phase. Unbanded spreads, 133, were classified with a 4.5% error rate. One hundred banded spreads were classified with a 15% error rate.
Application of Fraunhofer diffraction theory to feature-specific detector design.
Authors: Hardy J A , Wheeless L L
Published: April 2011

Light scatter from epithelial cells in a slit-scan flow system is modeled using the Fraunhofer condition of scalar diffraction theory. Power spectra are calculated for successive positions of model cells in the line focus of a laser beam with a Fourier transform computer program. Using the calculated power spectra, detector configurations are designed to detect specific cell structures of interest. Detector configurations are tested in a static slit-scan scatter apparatus. Data indicating the ability to detect boundaries and cell orientation are discussed.
Experimental findings on gynecologic cell orientation and dynamics for three flow nozzle geometries.
Authors: Kay D B , Wheeless L L
Published: April 2011

Gynecologic cell orientation and dynamics in sheath flow for three flow nozzle geometries are studied and compared. Cell orientations are classified into four categories based upon visual inspection of dark field laser stroboscopic photographs of cells in flow. Results are discussed in the context of slit-scan cytofluorometry.
Two-color immunofluorescence using a fluorescence-activated cell sorter.
Authors: Loken M R , Parks D R , Herzenberg L A
Published: April 2011

A technique for the analysis of fluorescein and rhodamine in a flow system using a single wavelength of excitation is described. Both optics and electronics are used to discriminate the fluorescein and rhodamine signals. This technique has been used to study the relationship between immunoglobulin M and immunoglobulin D on mouse splenic lymphocytes.
Monodispersal and deoxyribonucleic acid analysis of prostatic cell nuclei.
Authors: Goerttler K , Ehemann V , Tschahargane C , Stoehr M
Published: April 2011

We collected prostatic glands from 50 unselected autopsies at the Pathology Institute and compared their histologic sections with cytologic preparations and with results of photometric measurements of isolated prostatic cells and isolated nuclei. The results obtained with single cell photometry and flow-through cytophotometry proved to be comparable with one another and with the results of the cytologic and histologic studies. With these methods of cytophotometry we could differentiate inflammatory conditions, microcarcinomas and frank carcinomas from normal and hyperplastic prostatic tissue. We had difficulties, however, preparing adequate suspensions of cell nuclei from chronic fibrosing prostatitis. Our results indicate that it should be possible for diagnostic purposes to combine the technique of fine needle biopsy of the prostate with that of flow-through cytophotometry and to use the combined techniques for studying diseases of other organs.
Chromomycin A3 as a fluorescent probe for flow cytometry of human gynecologic samples.
Authors: Jensen R H
Published: April 2011

Chemical, physical and optical properties of chromomycin A3 are examined so as to ascertain appropriate staining and analysis procedures for flow cytometry of human gynecologic samples. Fluorescence excitation and emission spectra of chromomycin A3-stained cervical cells are compared with those of chromomycin A3-stained deoxyribonucleic acid. Conditions for deoxyribonucleic acid-specific staining of cervical cells are presented, and staining specificity of cervical cells with chromomycin A3 is compared to that obtained with ethidium bromide, propidium iodide and Hoechst 33258. Also presented is a brief review of two parameter flow cytometry as a prescreening procedure for detection of cervical neoplasia. Results of flow cytometry and cell sorting are interpreted based on the deoxyribonucleic acid-specificity of chromomycin A3 staining.
Computer-assisted monocyte esterase assay by flow-cytophotometry.
Authors: Kaplow L S , Lerner E
Published: April 2011

A Wang model 2200 computer has been interfaced with the Bio/Physics Systems, Inc. model 6300 Cytograf and model 2100 Distribution Analyzer. Using a custom designed software program, in conjunction with an azo-dye technic for staining monocytes for nonspecific esterase activity, it has been possible to obtain rapid and reliable data concerning relative values for intracellular monocyte esterase activity. The method is based on measuring the axial light-loss voltage signal for each of one thousand stained monocytes. Individual stained monocytes were assigned to one of four groups (A, B, C, D), dependent upon the magnitude of the signal and were given different rating values (1, 2, 3, 4) according to their group designation. A "score" was derived for each blood sample by multiplying the percentage of cells (monocytes) in each group category by the appropriate factor and summing these values. The technic permits rapid objective assessment of intracellular nonspecific esterase activity in monocytes suspended in a mixed cell population. Both Gaussian and bi-modal patterns for monocyte esterase were observed. The latter suggests a dual monocyte population.
Multiparameter geometric and densitometric analysis of the G0-G1 transition of WI-38 cells.
Authors: Kendall F M , Wu C T , Giaretti W , Nicolini C A
Published: April 2011

Automated image analyses were performed using Feulgen stained smears of WI-38 cells that were either confluent, or that had received a nutritional stimulus to proliferate 3 hr before collection. These experiments show that it is possible to observe changes in morphometric and densitometric parameters of nuclei that correlate with structural and functional differences in isolated chromatins from quiescent G0 and proliferation G1 cells that have been demonstrated by other means. Scatter plot analyses of the data indicated the presence of nuclear images from the stimulated G1 population that had the same deoxyribonucleic acid content as the confluent G0 cells, but had greater areas, perimeters and horizontal projections and smaller mean free paths, form factors, and average optical densities. Multiparameter cluster analysis permits, even minimally, an objective, model-independent identification of G0 from G1 cells that present an increased nuclear dispersion (i.e., lower average optical density) systematically accompanied by increased nuclear convolution (i.e., lower form factor), both compatible with the reported increase in available binding sites with respect to G0 cells.
Measurement of mammalian sperm deoxyribonucleic acid by flow cytometry. Problems and approaches.
Authors: Van Dilla M A , Gledhill B L , Lake S , Dean P N , Gray J W , Kachel V , Barlogie B , Göhde W
Published: April 2011

Measurement of mammalian sperm deoxyribonucleic acid content is of importance in several areas of biomedical research. When measured in flow systems with orthogonal axes of illumination, flow and detection, an unexpected, distorted distribution consisting of a narrow peak with a lateral extension to the right is observed. Several lines of evidence lead to the conclusion that this effect is an optical-geometric artifact attributable to the flat shape and high index of refraction of mammalian sperm heads. This artifact disappears when an epiillumination flow system is used in which the optic axes for illumination and detection and the flow axis are all coincident. Other approaches also eliminate the artifact. The resulting coefficients of variation observed after acriflavine-Feulgen staining are 4-5%, short of the goal of 1.5% required to distinguish between human sperm bearing X and Y chromosomes and to develop a mutagen test system using mice.
Recognition of cells in mitosis by flow cytofluormetry.
Authors: Darzynkiewicz Z , Traganos F , Sharpless T , Melamed M R
Published: April 2011

Cells in mitosis may be distinguished from interphase cells based on difference in chromatin structure as revealed by two different methods of staining with acridine orange. In the first method, cells are heated and then stained at neutral pH; the difference in stainability between mitotic and interphase cells reflects the difference in the extent of deoxyribonucleic acid denatured by heat in these cells. At a given temperature the deoxyribonucleic acid of the mitotic cell appears to be more extensively denatured than that of the interphase cell. In the second method, cells are treated with buffer at pH 1.5 (1.3 to 1.9) and then stained at pH 2.6 (2.3 to 2.9). The mechanisms involved in the differential stainability of interphase versus mitotic cells at that low pH are currently under investigation. In both methods, in addition to enumerating cells in mitosis, it is possible to quantitate cells in G1, S and G2 phases of the cell cycle.
Fluorometric detection of deoxyribonucleic acid synthesis; possibilities for interfacing bromodeoxyuridine dye techniques with flow fluorometry.
Authors: Latt S A
Published: April 2011

Fluorometric detection of the biosynthetic incorporation of 5-bromodeoxyuridine (BrdU) into deoxyribonucleic acid has permitted cytologic studies of chromosome structure, replication, and repair. Some of these phenomena, previously detected using BrdU-dye techniques on fixed microscopic preparations, should be particularly amenable to analogous experimentation in fluorescence flow systems. Problems involved in interfacing BrdU-dye methodology with flow fluorometry are discussed. The effects of certain chemical modifications of bisbenzimidazole dyes on their spectroscopic properties and potential use for detecting BrdU incorporation into unfixed cells are described. Data on the use and energy transfer characteristics of a pair of deoxyribonucleic acid-binding dyes (33258 Hoechst and ethidium bromide) capable of simultaneously providing information about BrdU substitution and total deoxyribonucleic acid content are presented.
Optimization of the binding of dissociated exfoliated cervico-vaginal cells to glass microscope slides.
Authors: Leif R C , Ingram D , Clay C , Bobbitt D , Gaddis R , Leif S B , Nordqvist S
Published: April 2011

In order to monitor the development of a cell dissociation technique, it was essential to utilize the Centrifugal Cytology rotor to produce glutaraldehyde-fixed even cellular dispersions. The Cytology rotor has been improved to insure rapid alignment with the centrifugal field during both acceleration and deceleration, and the fixative is now delivered to the surface of the slide. The dissociation of the cells results in a loss of their adhesion to glass slides. Three bonding agents were tested: (a) Poly-L-Lysine; (b) Mayer's albumin fixative; (c) positively charging the slides with a silicone coating. The results with 65% albumin-coated slides were clearly superior to the other two. The addition of a postfixation step of 95% ethanol/4% polyethylene glycol did not significantly affect the recovery of the cells, but did eliminate some unevenness in the Centrifugal Cytology preparations, flattened the cells and expedited the procedure.
Flow-microfluorometric analysis of nuclei isolated from various normal and malignant human epithelial tissues. A preliminary report.
Authors: Koss L G , Wolley R C , Schreiber K , Mendecki J
Published: April 2011

In order to obviate some of the technical problems associated with preparation of monocellular cell suspensions required for flow fluorometry, isolation of nuclei from several types of benign and malignant human tissues was undertaken. Satisfactory preparations of nuclei were obtained from epithelia of the uterine cervix and colon and from lung tissue using the citric acid method. The sucrose method was effective with colonic epithelium only. Distribution of deoxyribonucleic acid content in these nuclei was measured based on green fluorescence of acridine orange and red fluorescence of propidium iodide in a Bio-Physics Cytofluorograph. Essentially diploid patterns of deoxyribonucleic acid distribution were observed for all benign samples regardless of tissue origin whereas the malignant samples gave histograms suggestive of abnormal deoxyribonucleic acid distribution. Preliminary observations on distribution of single-stranded nucleic acids using acridine orange red fluorescence showed marked differences between populations of benign and malignant nuclei. Isolated nuclei appear to be suitable for flow-through microfluorometric analysis and offer some significant advantages over intact cells.
Automated classification of blood cell neutrophils.
Authors: Mui J K , Fu K S , Bacus J W
Published: April 2011

The classification of white blood cell neutrophils into band neutrophils (bands) and segmented neutrophils (segs) is a subproblem of the white blood cell differential count. This classification problem is not well defined for at least two reasons: (a) there are no unique quantitative definitions for bands and segs and (b) existing definitions use the shape of the nucleus as the only discriminating criterion. When cells are classified on a slide, decisions are made from the two-dimensional views of these three-dimensional cells. A problem arises because the exact shape of the nucleus becomes indeterminate when the nucleus overlaps so that the filament is hidden. To assess the importance of this problem, this paper quantitates the classification errors due to overlapped nuclei (ON). The results indicate that, using only neutrophils without ON, the classification accuracy is 89%. For neutrophils with ON, the classification accuracy is 65%. This suggests a classification strategy of first classifying neutrophils into three categories: (a) bands without ON, (b) segs without ON and (c) neutrophils with ON. Category III can then be further classified into segs and bands by other stretegies.
The influence of particles on jet breakoff.
Authors: Stovel R T
Published: April 2011

Experimental observations of the breakup of a liquid jet issuing from a vibrated nozzle support the view that particles passing through the orifice tend to create a surface disturbance which, depending on the phase relative to the nozzle vibration, may either add to or subtract from the disturbance created by the nozzle vibration. The result may be either a shortening or lengthening of the jet breakoff length, depending on phase. The implications for sorting large cells with a fluorescence-activated cell sorter are discussed.
Proof without prejudice: use of the Kolmogorov-Smirnov test for the analysis of histograms from flow systems and other sources.
Authors: Young I T
Published: April 2011

In this paper the Kolmogorov-Smirnov statistical test for the analysis of histograms is presented. The test is discussed for both the two-sample case (comparing fn1(X) to fn2 (X)) and the one-sample case (comparing fn1 (X) to f(X)). Presentation of the specific algorithmic steps involved is done through development of an example where the data are from an experiment discussed elsewhere in this issue. It is shown that the two histograms examined come from two different parent populations at the 99.9% confidence level.
A modified technique to obtain uniform precipitation of lanthanum tracer in the extracellular space.
Authors: Shaklai M , Tavassoli M
Published: April 2011

A method for obtaining a uniform, dense precipitate of lanthanum nitrate to delineate extracellular space is described. Improvement of the previous technique is achieved by phosphate precipitation of lanthanum in the tissue carried out at low temperature. This method has been successfully applied to bone marrow.
Strategies for choosing a deoxyribonucleic acid stain for flow cytometry of metaphase chromosomes.
Authors: Jensen R H , Langlois R G , Mayall B H
Published: April 2011

Requirements for flow cytometry of metaphase chromosomes stained with three deoxyribonucleic acid (DNA)-specific fluorescent dyes--Hoechst 33258, Chromomycin A3, and ethidium bromide--are reviewed. Fluorescence properties of these three stains when bound to mitotic cells or to chromosomes in suspension are measured and compared with fluorescence properties when bound to DNA in solution. Conditions are given for high resolution flow cytometry of Chinese hamster chromosomes stained with each of the fluorophors, and histograms are presented that exhibit differences in relative peak position and area. Energy transfer fluorescence between two DNA stains is presented as a potentially useful new parameter for flow cytometry of chromosomes and is illustrated by fluorescence energy transfer from Chromomycin A3 to ethidium bromide when simultaneously bound to hamster mitotic cells.
An improved electronic pantograph.
Authors: Eidelberg E , Davis F
Published: April 2011

A device was developed to permit simultaneous observations of a microscopic field, under any light optic conditions, and permanent recording of the relative X-Y coordinates of the center of the field. A modification is described that allows gray scale values to be assigned to the plots. The system can be readily adapted to digital computer data entry.
Does heparin occur in mucosal mast cells of the rat small intestine?
Authors: Tas J , Berndsen R G
Published: April 2011

Microspectrophotometric analyses combined with model experiments using polyacrylamide films containing different types of purified glycosaminoglycan or glycosaminoglycan and basic protein, have been applied to investigate the chemical characteristics of the glycosaminoglycan moiety present in rat mucosal mast cell granules. The results obtained with these histochemical techniques present evidence of the absence of significant amounts of heparin and the presence of lower sulfated glycosaminoglycan(s) in the granules of these cells.
Variable opacity of glycogen in routine electron micrographs.
Authors: Thornell L E , Sjöström M , Karlsson U , Cedergren E
Published: April 2011

Glycogen in nerve terminals from the reticular zone of frog muscle was identified by transmission electron microscopy of both periodic acid-thiosemicarbazide-silverproteinate treated and UAc-PbCi-stained serial sections. A variable appearance of glycogen in the uranylacetate-lead citrate-stained nerve terminals was seen and is related to the preparative procedure. The study indicates the necessity of cytochemical identification for the assessment of glycogen organization in cells.
An attempt to separate mononuclear cells fused with human red blood cell-ghosts from a cell mixture treated with HVJ (Sendai virus) using a fluorescence activated cell sorter (FACS II).
Authors: Mekada E , Yamaizumi M , Okada Y
Published: April 2011

Nucleated cells (Ehrlich ascites tumor cells or L strain cells) and human red blood cells (RBC)-ghosts were mixed and fused by ultraviolet-inactivated HVJ (Sendai virus). The cell mixture was stained with FITC conjugated anti-RBC ghost antiserum and then applied to FACS II apparatus. The apparatus sorted mononuclear cells fused with RBC-ghosts from the cell mixture on the basis of both the light scattering and fluorescence profiles. When the same procedure was carried out on a mixture containing cells and intact human RBC, the cells sorted by this method were cells into which hemoglobin had been injected. The sorted cells were capable of forming colonies in culture. This sorting method may be useful for collecting cells in which macromolecules have been injected artificially by fusion of RBC-ghosts enclosing macromolecules.