Veronal Acetate Buffer Research Articles

Improved lipid visualization with a modified osmium tetroxide method using ultrasonic treatment and intensification with imidazole or triazole.

Formalin fixed autopsy tissue containing lipids were cut into 1-5 mm thick blocks, washed well, then postfixed in 2% OsO4 in 0.03 M veronal acetate buffer for 30, 60, 90, 120, or 180 min with or without ultrasonic treatment. Tissues exposed to ultrasound for 90 min showed superior penetration of OsO4 and well preserved histological architecture. Tissues also were immersed for 1 hr in veronal acetate buffer (pH 7.4) containing 0.5% imidazole or triazole and compared with untreated controls. Paraffin sections, 4 microns thick, were examined under a light microscope with an image analyzer. Both intensity and percentage area of osmium blackening were significantly higher in samples immersed in imidazole or triazole than in untreated controls. No difference was observed between imidazole- and triazole-immersed samples. The OsO4 method, modified by ultrasound treatment and imidazole- or triazole-immersion, can be applied to routine formalin fixed autopsy materials for improved lipid visualization.

A radioautographic study of the incorporation of iron 55 by the ameloblasts in the zone of maturation of rat incisors.

This study was designed to study the time course of the incorporation of 55Fe into the ameloblasts of maturation in rat incisors. Male Sherman rats (100 +/- 5 gm) were injected intravenously with 0.9 mCi of 55Fe and sacrificed in pairs by perfusion at various time intervals from 5 min to 7 days after injection. The incisors were demineralized in 4.13% disodium EDTA, postfixed in 1% osmium tetroxide in veronal acetate buffer, and embedded in Epon. Incisors from control rats injected with only physiological saline were treated in the same way. Sections from blocks of tissue in the zone of maturation were prepared for light microscope radioautographic observations. The greatest incorporation of iron occurred at 9 mm within the zone; at this site the ameloblasts contained few pigment granules. About 5 mm deeper into the zone the activity fell off to zero, as observed at 2.5 hr after injection of 55Fe. Between 1 day and 7 days after injection the 55Fe labeling was found over the cells containing many pigment granules, while the initial labeling over the cells within 9 mm of the zone had diminished. These data have shown that at any given time, from 30 min to 4 hr, the iron enters the maturation ameloblasts over a wide extent of the zone, reaching a maximum at about 9 mm from the onset of maturation. However, at longer times (1 day to 7 days) the labeling curve shifts and shows the greatest activity beyond 9 mm within the zone.

A simple method of staining juxtaglomerular granules in the rat kidney for high resolution light microscopy.

A simple method for the demonstration of juxtaglomerular granules in Epon embedded semithin (0.5-1 micrometer) sections has been developed as follows: sections are prepared as for routine electron microscopy except that before dehydration, the tissues are immersed in 0.5% uranyl acetate in Veronal acetate buffer (pH 5.0) overnight at room temperature. After sectioning on an ultramicrotome, the semithin sections are briefly stained with toluidine blue-pyronin Y. After staining, the section is rinsed in running tap water and then air dried. Under a light microscope with a 40 X or a 100 X objective, the juxtaglomerular granules appear as deep purple particles and are thus easily separated from the bluish cytoplasm of the juxtaglomerular cells. Cellular organelles in other cells of the kidney were also clearly stained and their fine structure distinguishable.

リサイクルによる木材パルプ繊維の変質 ＩＩＩ リサイクルによるパルプ繊維細胞壁の形態的変化

After being beaten to about 350 ml C.s.f. by a Tappi Standard beater, commercial hardwood and softwood bleached pulps were dewatered by centrifuge in a cotton bag, shredded by hand and dried in a forced air circulation oven at 80°C for 6 days. Pulp fibres recycled 0, 1, 3 and 5 times by a sequence of wetting, defibrating, dewatering and drying, were wetted and dewatered by stepwise replacements with 30, 50, 70, 80, 90, 95 and finally 100% ethanol, succeeded by iso-amyl alcohol, which was flushed with liquid carbon dioxide in a critical point drying apparatus. Surfaces of these fibres were observed by a scanning electron microscope JSM-25. At the same time, fibre width was measured from SEM micrographs taken at the inclination angle of 0°. Also recycled fibres were fixed by Luft solution (1.2% KMnO4 & Veronal acetate buffer), followed by a stepwise dewatering by ethanol from 30 to 100%, a mixture of ethanol and propyleneoxide (1 : 1) and finally propyleneoxide. These fibres were embedded in a mixture of Epon 812, DDSA, MNA and hardner DMP 30. Embedded blocks were cut into a specimens thinner than 2, 000 Å by a ultra-microtome. Ultra thin specimens were electron stained with the Reynolds' lead staining method and observed under a transmittance electron microscope JEM-100 C. Handsheets of recycled fibres were dried on P2O5 and applied to a mercury intrusion porosimeter Shimazu 903-1 to measure the void volume.The following results have been obtained from the above experiments.(1) Parts of outer layer, Si, which were fibrillated by beating, were gradually removed from the surface of the fibre with recycling. This was supposedly taken place by separation of Si and S2 layers due to their difference in shrinkage rates during drying.(2) From the measurement of diameter of recycled fibres by SEM micrographs, a peak of the distribution curve for fibre diameter was shown to shift to the thinner side with recycling. (3) Separation of S1 layer from S2 layer and delamination of S2 layer were observed after 1st recycle from TEM micrographs as well. The more parts of S1 layer were removed from the fibre surface, the more S2 layer exposed. Fissures or cracks in the radial direction of the fibre wall appeared after 3rd recycle. After 5th recycle, S2layer was more exposed on the fibre surface, more delaminated and more split in the radial direction.(4) The mercury intrusion curves showed that the void volume of recycled fibre sheets increased with recycling at the pore diameter of severalμm, which correspond to the inter-fibre clearances and also of several tenths Am, correspond to pores in the cell wall. The latter seemed to be cracks appeared in the radial direction after 3 rd recycle on TEM micrographs. Finally, it can be concluded that fibre walls of recycled pulp fibres are irreversiblly changed by recycling so as to loose fibrillated S1 layer and to cause delamination and radial split in S2 layer.

The influence of different fixatives and osmolality on the ultrastructure of the cochlear neuroepithelium

To investigate which fixative is most suitable for the preservation of cochlear hair cells three main groups of fixatives were used: 1. 3% glutar aldehyde in various buffers (osmolality altered). 2. Glutar aldehyde/paraform aldehyde combination (ad modum Karnovsky). 3. Osmic acid solutions (OsO4) in veronal acetate buffer according to Rhodin (1954). The 2% osmic acid fixative (350--370mosm/kg) resulted in an excellent ultrastructural preservation; an acceptable result was obtained with a 3% glutar aldehyde fixative in 0.1 M sodium phosphate buffer, 600--700 mosm/kg and the Karnovsky's fixative.

Electron Microscopical Observations on the Curative Effect of Ursodesoxycholic Acid in Alloxan-Induced Pancreatic Islet Cell Injury

The preventive effect of ursodesoxycholic acid on pancreatic injury by alloxan (alloxan diabetes) has been reported by Watari, et al. (1976). In the following experiment, to pursue the findings further, ursodesoxycholic acid was used curatively for alloxan diabetes. A first group of animals (5 mice) were injected with alloxan (4 mg) twice at the fifth and tenth day. The second group (5 mice) was injected with ursodesoxycholic acid (0.2 mg each) for 14 days during the experiment in addition to the same alloxan dosage/frequency as the first group. A third group of animals (5 mice) served as the control. The animals were sacrificed on the 15th day and the blood sugar levels were examined, using commercial test paper. The pancreatic tissues were fixed in a mixture of 2.5% glutaraldehyde and 2% osmid acid solution, which was adjusted at pH 7.4 with a veronal acetate buffer; the osmotic pressure was also regulated by adding sucrose of 0.045 g/ml. Following dehydration using a series of alcohol concentrations, the tissues were embedded in Epon 812. Thin sections were cut with a Porter-Blum MT-2B ultramicrotome, stained with both uranyl acetate and a lead mixture, and then observed by electron microscopy. The results were as follows: The pancreatic islet cells, especially of B-cells in the first group of animals injected with alloxan only, were seriously damaged and contained myelinated mitochondria. Golgi apparatus, and an increasing number of autophagic vacuoles. Some B-cells revealed hydropic degeneration. Some B-granules changed into vacuoles after diacrine secretion. Pancreatic A-cells were increased in number and showed no cell injuries. On the other hand, the pancreatic B-cells of mice treated with both alloxan and ursodesoxycholic acid maintained almost normal fine structures. In summary, ursodesoxycholic acid has a curative effect on alloxan-induced pancreatic B-cell injury.

Palpal Sensilla of Selected Anopheline Mosquitoes

Palps of both sexes of Anopheles stephensi, A. albimanus, A. quadrimaculatus, and female A. gambiae were examined and found to be equipped to perceive both mechanical and olfactory stimuli. Present on the palps are sensilla chaetica, tactile and/or air current receptors, campaniform sensilla, proprioceptors, and thin-walled capitate pegs, olfactory receptors probably sensitive to carbon dioxide. The fine structure of each type of sensillum is described. Mosquitoes have various types of antennal and palpal sensilla which are responsible for the reception of external stimuli, including those involved in host finding and selection. Several investigators have reported on the detailed structure (Elizarov and Chaika, 1972; McIver, 1971, 1972; McIver and Charlton, 1970; McIver and Hudson, 1972) and function (Bassler, 1958; Kellogg, 1970) of palpal sensilla of culicine mosquitoes, but information about those on anopheline mosquitoes has been lacking. This work was conducted to (a) determine the types of palpal sensilla on both sexes of Anopheles stephensi Liston, Anopheles albimanus Wiedemann, and Anopheles quadrimaculatus Say, and female Anopheles gambiae Giles and (b) describe the fine structure of the sensilla on female A. stephensi. MATERIALS AND METHODS Specimens of A. stephensi, A. gambiae, A. albimanus, and A. quadrimaculatus were obtained from laboratory colonies maintained, respectively, at the Department of Parasitology, Department of Botany, University of Toronto, Central American Malaria Research Station, San Salvador, and the Center for Disease Control, Atlanta, Georgia. For transmission electron microscopy, palps were removed from mosquitoes immersed in Karnovsky's fixative (Karnovsky, 1965) at pH 7 and 4 C and left in the fixative for 18 hr. After rinsing in 0.05 M Sorensen's 10% sucrose buffer, pH 7, the tissue was postfixed in 1% Os04 in veronal acetate buffer at pH 5 and at room temperature for 2 hr. Dehydration through ethanol was followed by embedding in Spurr's low viscosity epoxy medium (Spurr, 1969). For scanning electron microscopy specimens were fixed in 5% formalin and dehydrated through a graded series of ethanols to xylene (Slifer, 1972), attached to the stubs with silver conductive paint, Received for publication 9 December 1974. coated with gold during spin rotation, and examined in a Cambridge Stereoscan microscope. For counting the sensilla, double coverslip mounts of heads were prepared as described by McIver (1969). All numerical results are means of at least 10 counts or measurements. Statistical significance was determined using Student's t test at the 5% level and adjustment for multiple comparison effect was made. Permeable areas in the cuticle were demonstrated by staining whole specimens with crystal violet (Slifer, 1960) from 30 min to 2 hr.

Zur Pathomorphologie der Corticoidmyopathie. Ultrastrukturelle Veränderungen des Plasmalemms der Skelett- und Herzmuskelzelle im Vergleich zur intrazellulären Reaktion

Zur Pathomorphologie der Corticoidmyopathie. Ultrastrukturelle Veränderungen des Plasmalemms der Skelett- und Herzmuskelzelle im Vergleich zur intrazellulären Reaktion

Ultrastructure of the Metacercarial Cyst of Posthodiplostomum minimum (Maccallum, 1921)

The 2-layered cyst consists of an outer fibrous cellular capsule composed of endothelial cells and an inner noncellular primary cyst wall of homogeneous and moderately electron-dense texture. An osmiophilic interface zone separates these layers. Adjacent to the primary cyst wall is a cyst cavity filled with flocculent material and membrane-bound vesicles. These vesicles are found in close proximity to the cyst wall and resemble similar vesicles in teguminal extrusions of the metacercaria, suggesting that cystogenous materials are formed by the parasite, released into the cyst cavity, and eventually deposited onto the cyst wall. The endothelial layers of the outer fibrous capsule contain numerous intracellular microfilaments that may function in providing additional tensile strength, protecting the metacercaria during ingestion by the definitive host. Micropinocytotic vesicles and extracellular deposits of collagen are characteristic of loose fibroblasts which surround the periphery of the cyst. Necrotic areas are designated the interface zone. It is suggested that they may be attributed to mechanical compression or isolation from oxygen and important nutrients. The metacercariae of the trematode Posthodiplostomum minimum utilize numerous species of freshwater fish as second intermediate hosts, particularly sunfish of the family Centrarchidae. The cercariae penetrate epidermal tissues and enter the general systemic circulation. The larval trematode forms a thin cyst wall which appears as a frail baglike structure loosely covering the metacercariae after reaching those visceral organs which are usually associated with a plentiful blood supply, e.g., liver, heart, or kidney. The process of encystation has been studied by electron microscopy in certain families of digenetic trematodes in order to elucidate the structure, function, host response, and relative contributions of the host and parasite during elaboration of the cyst wall. Electron microscopy of Fasciola hepatica metacercariae which openly encyst in an external environment has revealed that cyst components form a fourlayer wall which is solely contributed by cystogenous gland secretions from the parasite. The quinone-tanned, keratinlike proteinaceous layers are rapidly deposited, providing protection against desiccation and fungal and bacterial injury (Dixon and Mercer, 1964, 1967; Mercer and Dixon, 1967). The cyst of Notocotylus attenuatus has been described by Southgate (1971) as three-layered, originating in a similar manner from parenchymal gland Received for publication 1 May 1973. 67 cell secretions. Cyst wall ultrastructure within a second intermediate host has been described in one psilostomatid (Macy et al., 1968) and in two microphallid species (Strong and Cable, 1972; Stanier et al., 1968). These cysts, protected internally by their hosts from the environment, complete differentiation after periods of up to 4 weeks in which as many as four layers may be present. There is evidence in various heterophyid species of Ascocotyle that the cyst wall is derived from tegumental extrusions which form one or more layers. Host involvement is limited to peripheral infiltration of fibroblasts with extracellular reticular collagen (Lumsden, 1968; Stein and Lumsden, 1971, 1972). This paper reports on the ultrastructure of Posthodiplostomum minimum metacercarial cyst wall organization as an initial step in describing the process of encystation in strigeids. MATERIALS AND METHODS Metacercarial cysts were obtained from naturally infected sunfish (Lepomis sp.) which were seined from ponds in the vicinity of Marion, Ohio, and the Bonnet Carre' Spillway, Norco, Louisiana. Fish were maintained in aquaria and fed a diet of commercial fish food, mealworms, and earthworms. Cysts were excised from liver parenchyma and processed for electron microscopy. Cysts were fixed for 6 hr (1 to 4 C) in 3% glutaraldehyde buffered at pH 7.3 in 0.12 mI monobasic sodium phosphate-NaOH, containing 1% CaCl2 and 3% sucrose. These were washed overnight in cold phosphate buffer, postfixed for 1/, hr with 1% phosphate-buffered OsO4, and This content downloaded from 207.46.13.15 on Fri, 26 Aug 2016 05:32:13 UTC All use subject to http://about.jstor.org/terms 68 THE JOURNAL OF PARASITOLOGY, VOL. 60, NO. 1, FEBRUARY 1974 washed in several rinses of veronal acetate buffer at pH 5.3. Tissue was stained en bloc in 1% uranyl acetate (in veronal acetate buffer, pH 5.3) for 1 hr, and dehydrated with ethanol before gradual embedment in low viscosity epoxy resin mixture (Spurr, 1969). Sections were cut on glass knives with a Sorvall MT-2 ultramicrotome, collected on uncoated copper grids, and stained with 1% aqueous uranyl acetate and with lead citrate. Electron micrographs were made with a Siemens Elmiskop IA operating at an accelerating voltage of 80 kv. Cysts were cut open during fixation allowing access of the fixatives and embedding media into both inner and outer surfaces of the cyst wall.

Lysis of photographic emulsions by mammalian and chicken spermatozoa.

Gaddum & Blandau (1970) studied the proteolytic activity of individual mammalian spermatozoa by directly observing the dissolution of pure gelatin membranes which had been blackened by the inclusion of India ink and fixed with glutaraldehyde. Their observations showed that lytic enzymes were released from the acrosomal region of the sperm head, but not from other parts of the cell. Gaddum & Blandau's technique has two important practical disadvantages. Firstly, the `home-made' gelatin membranes used are tedious to prepare and variable in thickness, and, secondly, the incorporation of India ink obscures the images of the spermatozoa. To counter the second of these disadvantages, Ben\l=i'\tez-Bribiesca& Ve\l=a'\zques-Meza(1972) used Ponceau stain instead of India ink, and Gaddum-Rosse & Blandau (1972) used clear unstained membranes. To eliminate both kinds of disadvantage, we have explored the use of commercial photographic emulsions as a source ofgelatin substrate, a technique which has been used for many years to study the enzymatic activity of tissue slices (Gordon, Levin & Whitehead, 1952; Adams, Fernand & Schneiden, 1958; Adams & Tuquan, 1961; Kirchner, 1972). Our first observations were made with blackened photographic plates, such as Kodak P300. After exposure to light, these plates were processed in the usual way and, after washing, were fixed in 0-05 % glutaraldehyde in veronal-acetate buffer (pH 8-2) for 2 min and then rinsed in buffer and distilled water. Washed spermatozoa, suspended in a mixture of Hanks' BSS medium (B.D.H.) and veronal acetate buffer, were spread onto these plates and incubated at 37° C in a humid atmosphere. The results obtained were essentially the same as those described by Gaddum & Blandau (1970) but with less variability. The areas of depolymerization were very clear, but details of the reacting spermatozoa remained obscured by the blackened emulsions. Very much better images were then obtained by using colour film, such as Kodachrome II, exposed and developed to produce a light red or light blue background. These films are, however, relatively expensive and celluloid film is much less convenient to use than glass plate, which can be cut to the size of a microscope slide and remains solid and flat without additional mounting. We therefore returned to the use of black and white photographic plates but fixed them with Kodafix without exposure either to light or chemical development. These plates are easy to prepare and have given excellent results. Their gelatin coats are transparent,

Formation of protoplasts in Azotobacter vinelandii.

Protoplasts of Azotobacter vinelandii were formed by incubating whole cells in lysozyme and EDTA in Tris-HCl buffer (0.05 M, pH 8.0) supplemented with sucrose (15% w/v). This appeared to be related to the special chelating ability of EDTA and Tris-HCl since substitution of the former by nitrilotriacetic acid or by trisodium citrate and the latter by veronal-acetate buffer or tris-maleate buffer over a pH range of 5.2 to 8.6 yielded only spheroplasts. Of nine strains of Azotobacter studied, only A. vinelandii strain 12837 and strain 0 formed protoplasts.

Localization of Cadmium in the Mouse Liver Cells Induced by Acute Cadmium Poisoning

Acute Cadmium-poisoning of a mouse was induced by two intraperitoneal injections of U00 ppm Cadmium chloride solution. 2 hrs after the second injection, the mouse liver was cut into small blocks in the following chilled prefixative. The same volume of 8 % paraformaldehyde and 1/7M veronal acetate buffer solution was mixed and adjusted to pH 7-0, then Oxine was saturated in this fixative. Oxine makes insoluble precipitate with Cadmium(Cd) sensitively(0.1𝛄) Prefixation was proceeded by 2 hrs in ice. The blocks were postfixed with 1 % OsO4, followed by dehydration and embedded in Epon. G-6-Pase(Gomori) and acid phosphatase(Karnovsky) were also examined. Hitachi HU-11E was used for ordinary observations and selected area electron diffraction, and JEOL-IOO-B fitted with nuclear diode Silicon-detector for energy dispersive analysis of X-rays from an Epon embedded section(EDAX), and transmission type scanning electron image system(TSEl).

Cytochemical demonstration of proteolytic activity of human and rat spermatozoa.

Summary. Proteolytic activity was demonstrated around the acrosomes of intact sperm cells with a simple cytochemical method using a gelatin substrate. The proteolytic activity was localized to the acrosomal region of both rat and human spermatozoa. Not all sperm cells showed the same digestive capacity in each sample studied. Rabbit sperm acrosomes have a denuding effect on the zona pellucida, as Srivastava, Adams & Hartree (1965a, b) have demonstrated. This finding suggested that penetration of a spermatozoon through the zona pellucida is mediated, at least in part, by the presence of hydrolytic enzymes. Recently, Stambaugh & Buckley (1969) found hyaluronidase and a 'trypsin-like' enzyme in acrosomal extracts of rabbit spermatozoa; apparently the proteolytic enzyme is mainly responsible for the ability to penetrate the zona pellucida in this species. The localization of proteolytic activity in intact sperm cells was accomplished by Gaddum & Blandau (1970), using an india-ink gelatin substrate method. The technique promised to be simple enough to allow further studies and even semiquantitative estimations of protease activity; however, in trying to repeat the method, we found that it gave inconstant results and obscured a great deal of the cytological details of the cells under study, nor was it possible to obtain clear-cut 'digestion halos' around the acrosomes, as illustrated in Gaddum & Blandau's original publication. We therefore attempted to develop a method that could clearly show proteolytic activity against gelatin, without significantly obscuring cytological detail. Human ejaculates and rat epididymal spermatozoa were used. The sperma¬ tozoa were washed twice in Hanks' solution with sodium bicarbonate 0-35 g/1 at a final pH of 7-4. The gelatin solution (5%) was prepared in a Veronalacetate buffer at a different pH ranging from 7-4 to 9. Washed spermatozoa were spread evenly on a glass slide; after 2 min at room temperature, smears were immersed once in the gelatin solution kept at about 28 to 30° C; the slides were set in a vertical position for a few minutes to obtain a thin gelatin film. After blotting the bottom edge, the slides were allowed to gel in a horizontal position in a humid chamber at room temperature from 12 to 48 hr. After incubation,

Ultrastructure of the Actinomycele Ceodermatophilus Obscurus

Geodermatophilus, a new generic taxon of the Dermatophylaceae (Actinomycetales), was first described by Luedemann and shown to display close similarities to Dermatophilus congolensis, previously described in our laboratory.Although Geodermatophilus has been studied by electron microscopy, the present investigation was undertaken to clarify further some ultrastructural aspects of this organism in relation to those of D. congolensis. Standard procedures for chemical fixation, as well as recently developed methods for physical fixation, were chosen but the present report is concerned only with results of thin sections. Cells from 16 hr. to 15-day-old cultures, in the medium described by Luedemann, were fixed with cacodylate buffered glutaraldehyde followed by osmium tetroxide in veronal acetate buffer, or according to Kellenberger et al. Dehydration was in ethanol and embedding in Epon 812.

Electron Microscopy of Mycoplasma Pneumoniae

Information concerning the morphology and replication of organism of the family Mycoplasmataceae remains, despite over 70 years of study, highly controversial. Due to their small size observations by light microscopy have not been rewarding. Furthermore, not only are these organisms extremely pleomorphic but their morphology also changes according to growth phase. This study deals with the morphological aspects of M. pneumoniae strain 3546 in relation to growth, interaction with HeLa cells and possible mechanisms of replication.The organisms were grown aerobically at 37°C in a soy peptone yeast dialysate medium supplemented with 12% gamma-globulin free horse serum. The medium was buffered at pH 7.3 with TES [N-tris (hyroxymethyl) methyl-2-aminoethane sulfonic acid] at 10mM concentration. The inoculum, an actively growing culture, was filtered through a 0.5 μm polycarbonate “nuclepore” filter to prevent transfer of all but the smallest aggregates. Growth was assessed at specific periods by colony counts and 800 ml samples of organisms were fixed in situ with 2.5% glutaraldehyde for 3 hrs. at 4°C. Washed cells for sectioning were post-fixed in 0.8% OSO4 in veronal-acetate buffer pH 6.1 for 1 hr. at 21°C. HeLa cells were infected with a filtered inoculum of M. pneumoniae and incubated for 9 days in Leighton tubes with coverslips. The cells were then removed and processed for electron microscopy.

Dynamics of Pusmalemmal Invagination in the Synchronized Cells of Saccharomyces Cerevisiae

Plasmalemma of Saccharomyces cerevisiae invaginates inside the cytoplasm. A double aldehyde fixation technique gave a superior preservation of membrane material compared to that obtained by other procedures.Stationary phase equal size single cells of Saccharomyces cerevisiae strain 1829 were separated by gravity sedimentation at 4°C on a 0.5, 1.0, 2.0 M discontinuous sorbitol density gradient. The separated cells were washed free of sorbitol and were grown in 1% yeast extract and 1% glucose. Counting under phase microscope showed that the cells were synchronized for 2-3 generations with a generation time of approximately 2 hr. At different time intervals of growth, cells were washed and fixed in a mixture of paraformaldehyde (2%) and gluteraldehyde (2.5%) in 0.1 M phosphate buffer pH 7.0 for 30 min. These cells were fixed in 1% OSO4 in veronal-acetate buffer pH 6.1 for 15 hr and treated with 0.5% uranyl acetate for 2 hr and further embedded in Epon after dehydration. Thin sections were stained in lead citrate for 30 min.

Cytochemistry of the Pellicle of Bloodstream Forms of Trypanosoma (Trypanozoon) brucei

The cell coat of Trypanosoma (Trypanozoon) brucei has been found to vary in thickness and in differentiation into layers according to preparative techniques for electron microscopy. Measurements of coat and cell membrane are given. Cytochemical staining techniques indicate the presence of a prominent inner, and less prominent outer, layer of carbohydrates within the cell coat. Localization of the carbohydrates in the coat material is shown by trypsin digestion of living trypanosomes. Carbohydrates and protein may be the sole components of the cell coat. Negatively charged groups could not be demonstrated in the cell coat or membrane. An extracellular layer has recently been demonstrated as the outermost component of the pellicle of bloodstream forms of several species of trypanosomes (Fuge, 1968; Vickerman, 1969a, b; Godfrey and Taylor, 1969; Wright et al., 1970). Such an external cell coat may be suspected of playing an important role in the antigen-antibody interactions with the host, in providing structural support to the cell surface, and in having a modifying effect on the passage of metabolites from the environment to the cell membrane. The chemistry of the cell coat is therefore of some interest. This report extends the examination of the trypanosome cell coat to the cytochemical level by application of techniques to localize carbohydrates and by trypsin digestion of the coat. MATERIALS AND METHODS Bloodstream forms of Trypanosoma brucei were obtained from 3 to 5-day-old infections in white mice produced from the same stabilates as those used in an earlier study of filopodium formation by these animals (Wright et al., 1970). Trypanosomes were fixed both directly after hematocrit centrifugation and after resuspension in Hanks' salt solution. The basic morphology of the pellicle was determined from trypanosomes fixed by 2 procedures: (1) 20 to 30-min fixation at room temperature in 2.5% glutaraldehyde in Sorensen's 0.1 M phosphate buffer pH 7.4, followed by washing in phosphate-buffered 4% sucrose, and 1 hr postfixation in 1% osmium tetroxide in veronal acetate (0.05 M barbital) buffer; (2) 20 to 30min fixation at room temperature in 5% glutaraldehyde in 0.066 M cacodylate pH 7.4, followed by washing in cacodylate-buffered 5% sucrose, and 1 hr postfixation in 1% osmium in 0.066 M cacodylate. Some pellets were treated with 0.5% Received for publication 26 August 1969. * Assistance of National Research Council of Canada grant A3757 is gratefully acknowledged. uranyl acetate prior to dehydration (Terzakis, 1968). All were embedded in an Epon-Araldite mixture. Sections were stained either with saturated methanolic uranyl acetate followed by lead citrate, or with 2% potassium permangenate followed by lead citrate. For cytochemical examination of the cell coat, only the 5% glutaraldehyde-cacodylate fixation procedure was used. Cytochemical techniques for carbohydrates Sections to be stained by the periodic acid-thiosemicarbazide-silver proteinate technique and the periodic acid-silver methenamine technique were collected on gold grids and stained as recommended by Thiery (1967). Control sections were treated similar to these experimental sections except for the omission of periodic acid oxidation in some, and blockading of aldehyde groups with dimedone in others. Blocking of aldehydes with dimedone was done before, in in place of, or after periodic acid oxidation. The sections were floated on a 2% dimedone solution in 1% acetic acid for 1 hr at 60 C (Swift and Saxton, 1967) and were subsequently well rinsed before completing the staining technique. Pellets of trypanosomes fixed in glutaraldehyde were stained 1.5 hr with positively charged colloidal iron at pH 1.8 prepared according to Gasic et al. (1968). All blocks were postfixed in osmium. Ruthenium red staining was carried out as recommended by Luft (1964); some pellets were fixed in glutaraldehyde-ruthenium for 30 min and postfixed in osmium-ruthenium for hr, while some were fixed in glutaraldehyde-ruthenium for 24 hr and postfixed in osmium-ruthenium for 3 hr.

Preservation of fine structures in yeast by fixation in a dimethyl sulfoxide-acrolein-glutaraldehyde solution.

The primary fixative containing 2% acrolein, 2% glutaraldehyde in 50% aqueous dimethyl sulfoxide (DMSO) buffered at pH 7.4, was applied for 7 hr in the cold. After a short wash in 0.02 M s-collidine buffer, pH 7.4, containing 0.2 M sucrose and 0.001 M CaCl2, the yeast cells were postfixed in 3% OsO4 at pH 4.0 (veronal acetate buffer). This method preserves many cytoplasmic features such as lipid deposits and ribosomes which are usually destroyed by permanganate fixation. DMSO apparently acts as a permeating agent allowing maximum penetration of the cell wall by the fixative without disrupting cellular fine structure

Incomplete Maturation of Iridal Pigment in Heterochromia Iridum

Heterochromia is an abnormality of iridal pigmentation characterized by marked differences in the color of the two irides or in segments of one iris. The affected areas are pale blue to grayish white. The condition is known to be present in a variety of species including man, dog, mouse, rabbit, cat, pig, and horse. In some species heterochromia has been transmitted as an autosomal dominant trait. It is also a dominant characteristic in Waardenberg's oculo-dermato-auditive syndrome. In several breeds of dogs heterochromia is associated with merle (or harlequin or dappled) coat color which is controlled by an autosomal dominant gene. Other factors are involved in Fuch's heterochromic cyclitis.The present study resulted from the spontaneous occurrence of 5 cases of heterochromia in a large colony of beagles produced to study the long-term effects of low doses of radiation administered at various ages. When ten weeks old (3 or 4 weeks after the dog eye has attained adult characteristics), the affected dogs were anesthetized with pentobarbital sodium. The eyes were extirpated and quickly hemisected dorsoventrally. One calotte was fixed in cold glutaraldehyde-acrolein for 90 minutes. Representative blocks through the iris were cut and stored in phosphate buffer (pH 7. 3) overnight, washed in veronal-acetate buffer, pH 7.3, and post-fixed in 2% OsO4 in veronal-acetate buffer. Further processing was by standard techniques. The sections were stained with uranyl acetate and lead citrate. The other calotte was fixed for 24 hours in 6.25% glutaraldehyde for light microscopy. The calotte was rinsed in phosphate buffer, pH 7.3., embedded in paraffin, sectioned at 6μ and stained with hema-toxylin and eosin.

Ferrous iron oxidation by Ferrobacillus ferrooxidans. Purification and properties of Fe++-cytochrome c reductase.

The electron transport chain and Fe++-cytochrome c reductase of Ferrobacillus ferrooxidans were studied to elucidate the mechanism of iron oxidation by this autotrophic bacterium.The iron oxidation involved the cytochrome c and a type cytochrome of F. ferrooxidans in the electron transport system. The initial enzyme of the oxidation system was found to be Fe++-cytochrome c reductase. The iron oxidase system was labile to freezing or sonication; either treatment disrupted some link between the cellular cytochromes c and a. Fe++-cytochrome c reductase and cytochrome oxidase retained their individual activities after either treatment.Fe++-cytochrome c reductase was purified 60-fold to 70-fold. The enzyme was judged to be approximately 90% pure by disc electrophoresis, sedimentation, and DEAE-cellulose chromatography. A suitable assay system with a veronal–acetate buffer was developed for the determination of enzyme activity. The effects of inhibitors and potential activators were studied. No specific inhibitor or cofactor was found, although the enzyme was inhibited by various ionic compounds.Fe++-cytochrome c reductase was dissociated into two subunits, one protein and the other ribonucleic acid (RNA). Neither of the subunits had enzymatic activity and efforts to reconstitute the holoenzyme from the two subunits were unsuccessful. The molecular weights of the holoenzyme, protein subunit, and RNA subunit were determined as 100,000–110,000, 27,000–30,000, and 315,000–330,000, respectively. The protein subunit contained one non-heme iron atom per protein molecule. It was concluded that RNA is an essential component of the enzyme and the failure to recover the activity from subunits is due to the aggregation of RNA after dissociation.