Changes Of Distilled Water Research Articles

First Report of Corynespora cassiicola Causing Target Spot on Cotton (Gossypium hirsutum) in South India.

Target spot with unusual symptoms was observed in upland cotton at Regional Agricultural Research Station, Lam, Guntur, Andhra Pradesh, India in 2017. Symptoms appeared in about 60-65 days after planting with minute pinhead size light orange to brick red spots that gradually expanded as circular to oval or irregular concentric spots that finally developed as target spot with yellow halo. These symptoms were also observed during 2019-20 and 2020-21 diagnostic field visits at Krishna zone of Andhra Pradesh, India. Frequent showers had intensified disease severity and defoliation of different cultivars (L 1060, RCH 659 BGII, NDLH-1938 and Jaadoo BG II) examined. Leaf bits containing healthy and diseased portions of the specimens were surface sterilized with 0.1% HgCl2 for a minute and rinsed in three changes of distilled water and were placed on PDA at 27±1˚C for pathogen development. Obtained pathogen was purified using single spore isolation technique. Purified colonies exhibited grey colour on top of culture plate and greyish brown on bottom of culture plate with flat, aerial round colonies having fibrous texture with dense mycelium. Conidiophore were indeterminate septate with intermediate branching. Enteroblastic conidiogenous cells produced conidia subhyaline that were solitary or in chains, straight to curved, 83.43±31.73 x 7.61±1.43 µm with 7.27±2.19 pseudospeta. Morphological characteristics of the isolated fungus were consistent with original description of Corynespora cassiicola on cotton (Jones 1961; Fulmer et al., 2012; Conner et al., 2013) which confirmed it as Corynespora morphologically. Pathogenicity test was conducted on popular hybrid Jaadoo BG II using three isolates Lam (CGGL 19001), Koniki (CPIK 19009) and Venkatapuram (CKMB 19011) obtained during 2019-20 survey. After 50 days of sowing conidial suspension containing 1x104 spores per ml was sprayed on healthy plants maintained in pots under protected conditions. Immediately after spraying, the plants were covered with poly propylene covers for 48 h to prevent cross contamination and to ensure humidity for pathogen establishment. Healthy plants sprayed with sterile water served as control. After seven days of incubation, inoculated plants developed brick red spots initially which later developed into target spot. Absence of symptoms in control plants indicated pathogenic nature of the fungus. Pathogen was re-isolated from diseased tissue to confirm its pathogenicity according to Koch's postulates. The identity of the pathogen was further examined using ITS primers. The DNA was isolated from 10 days old culture using CTAB method. Internal transcribed spacer (ITS) region of three isolates were amplified using ITS1 and ITS4 primers and obtained amplicon (550 bp) were used for sequencing. The sequenced data was deposited in NCBI and accession numbers were obtained as MZ314930, MZ314928 and MZ314927. BLAST search in GeneBank revealed 99% homology of CGGL 19001 isolate with sequences of C. cassiicola (accession number of MN393240) while CPIK 19009 and CKMB 19011 isolates showed 100% homology with C. cassiicola in cotton having accession numbers of MN393238, MN228955, MN733135, MN288934 and MN393238. Based on symptoms, fungal morphology, pathogenicity test and ITS sequences obtained, the fungus was identified as C. cassiicola the pathogen of cotton causing target spot disease. Earlier, Sarma and Nayudu (1970) isolated Corynespora from different host plants including cotton but could not prove its pathogenicity on cotton. Later target spot of cotton associated with C. cassiicola had been reported from Central India (Salunkhe et al., 2019) and this is the first report of C. cassiicola in cotton from South India. Hence, this report of C. cassiicola on cotton is the first report from Souh India. Identification of target spot pathogen is very important as it is emerging as a dominant foliar pathogen for which management strategies are to be worked out.

Effect of Maceration Time on Micronutrient Concentrations of Canarium schweinfurthii Pulp Flour

The effect of wet heat-processing on the micronutrient composition of Canarium schweinfurthii (CS) pulps eaten as traditional snack in most parts of Nigeria was investigated. Fresh wholesome seeds of C. schweinfurthii, obtained from Aba (Abia State, Nigeria) were washed in several changes of distilled water and divided into four lots. The first lot was used raw (CS raw ) and the 2nd, 3rd and 4th macerated in water (55oC) for 15, 30 and 45min to obtain CS 15 , CS 30 and CS 45 respectively. CS 30 represents sample traditionally processed to accepted cooking tenderness. The raw and wet-heat treated seed pulps, dried for 48hr in air-circulatory oven (50oC) and milled into flours were evaluated for vitamin and mineral contents using standard methods. The mineral and vitamin contents of the plant food were affected by the processing method. There were progressive increase in the concentrations of Ca, Na, P, Zn, and Pb; and decrease in those of Fe, I, K and Mg as maceration time was increased. The concentrations of Cu, Mn and Se were increased to their peak values and then reduced as the processing time was extended. Highest values were obtained (per 100g sample) for Fe (7.78mg), I (12.0µg), K (11.34mg) and Mg (8.37mg) in CS raw ; Ca (21.41mg), Cu (289.33µg) and Mn (4.76mg) in CS 15 ; Na (23.36mg), Se (8.0µg) and Pb (122.67µg) in CS 30 and; P (21.33µg) and Zn (1.79mg) in CS 45 . Vitamins C (2.37mg/100g) and E (1.97mg/100g) were the most

Key Role for Ceramides in Mediating Insulin Resistance in Human Muscle Cells

Elevated non-esterified fatty acids, triglyceride, diacylglycerol, and ceramide have all been associated with insulin resistance in muscle. We set out to investigate the role of intramyocellular lipid metabolites in the induction of insulin resistance in human primary myoblast cultures. Muscle cells were subjected to adenovirus-mediated expression of perilipin or incubated with fatty acids for 18 h, prior to insulin stimulation and measurement of lipid metabolites and rates of glycogen synthesis. Adenovirus-driven perilipin expression lead to significant accumulation of triacylglycerol in myoblasts, without any detectable effect on insulin sensitivity, as judged by the ability of insulin to stimulate glycogen synthesis. Similarly, incubation of cells with the monounsaturated fatty acid oleate resulted in triacylglycerol accumulation without inhibiting insulin action. By contrast, the saturated fatty acid palmitate induced insulin resistance. Palmitate treatment caused less accumulation of triacylglycerol than did oleate but also induced significant accumulation of both diacylglycerol and ceramide. Insulin resistance was also caused by cell-permeable analogues of ceramide, and palmitate-induced resistance was blocked in the presence of inhibitors of de novo ceramide synthesis. Oleate co-incubation completely prevented the insulin resistance induced by palmitate. Our data are consistent with ceramide being the agent responsible for insulin resistance caused by palmitate exposure. Furthermore, the triacylglycerol derived from oleate was able to exert a protective role in sequestering palmitate, thus preventing its conversion to ceramide.

Phytophthora cinnamomi

<i>Phytophthora cinnamomi</i>

The CryIA(c) Receptor Purified from Manduca sexta Displays Multiple Specificities

The kinetic binding characteristics of four Bacillus thuringiensis CryI insecticidal crystal proteins to a Cry-binding protein, purified from Manduca sexta brush-border vesicles, were analyzed by an optical biosensor. This 120-kilodalton binding protein, previously determined to be aminopeptidase N, was converted to a 115-kilodalton water-soluble form by removing the attached glycosylphosphatidylinositol anchor with phospholipase C. The solubilized form recognized the three major subclasses of CryIA toxins but not CryIC even though all four CryI proteins are toxic to larvae of M. sexta. CryIA(a) and CryIA(b) toxins bound to a single site on the solubilized aminopeptidase N molecule whereas CryIA(c) bound to two distinct sites. Apparent kinetic rate constants were determined for each binding reaction. All three CryIA toxins exhibited moderately fast on rates (approximately 10(-5) M-1 s-1) and a slow reversible off rate (approximately 10(-3) s-1). Although the second CryIA(c)-binding site retained a moderately fast association rate, it was characterized by a rate of dissociation from the amino-peptidase an order of magnitude faster than observed for the other CryIA-binding sites. CryIA(c) binding to both sites was strongly inhibited in the presence of N-acetylgalactosamine (IC50 = 5 mM) but not N-acetylglucosamine, mannose, or glucose. CryIA(a) and CryIA(b) binding were unaffected in the presence of the same sugars. Our results serve to illustrate both the complexity and the diverse nature of toxin interactions with Cry-binding proteins.

Methods for the study of leaf anatomy in palms.

Large size, hardness, combinations of thick-walled fibers and sclereids with thin-walled parenchyma cells, and the occurrence of silica, calcium oxalate, and tannins make anatomical preparations of palm leaves difficult. Samples for anatomical study should encompass one-half a pinna or a comparable portion from palmate and entire leaves including the midrib, all large ribs, and the margin. Similar pieces from herbarium specimens are reconstituted in glycerin alcohol, aerosol OT and distilled water (10:3:90). All samples are fixed in formol-acetic-alcohol (FAA) but stored in glycerin alcohol to minimize hardening. Transverse and longitudinal sections 15 microns thick, epidermal macerations, and pieces for clearing and for scanning electron microscopy are prepared from the FAA fixed material. Samples for electroscanning are gradually changed to 100% acetone, critical point dried with CO2, and coated with 100-300 angstroms of gold. Leaf material for microtomy is treated with hydrofluoric acid, embedded in Paraplast, and sectioned at 15 microns at a temperature of 7.2 C. Paraplast sections are floated directly on a modification of Sass' Adhesive III, mounted unstained or stained in safranin and fast gaee, and observed in polarized light. Epidermal peels are prepared by soaking pieces 5 mm square for 12-24 hours in undiluted bleach. Pieces for observation of transverse veins are cleared by treatment in 5% sodium hydroxide in a 60 C oven, washed rapidly in three changes of distilled water, and placed in one-third strength commercial bleach until clear. The same procedures can be used to prepare reproductive material for anatomical observations, but time schedules must be increased for larger specimens.

Fe II uptake of enterochromaffin Biochemical and histochemical study of the ferrous ion uptake reaction in relation to enterochromaffin cells and mast cells

Summary Formaldehyde precipitates of noradrenaline, DOPAmine, tryptamine and serotonin were prepared by exposing 1 mmol amounts 24 h in 1 M formaldehyde. Other amines yielded no precipitates. The precipitates are washed in 3 changes of distilled water by centrifugation and resuspension. The washed precipitates are then suspended in 10m M ferrouc chloride or sulfate (or ferric chloride in some tests) for 2 h at 60 °C, recentrifuged and rewashed as before in 3 changes of distilled water and then reacted 15 min in 1 % potassium ferricyanide (K 3 Fe(CN) 6 ) in 0.06 N HCl to form Turnbull's blue. Deep blue and blue-green precipitates were obtained with DOPAmine and noradrenaline respectively, yellowish green with serotonin and brownish-yellow with tryptamine. Of the substances tested DOPAmine and noradrenaline gave the best Turnbull's blue reactions, in agreement with the histochemical reaction. Noradrenaline islets of the glutaraldehyde and formaldehyde fixed adrenal differ considerably from enterochromaffin cells (EC) in a number of azo coupling reactions, making DOPAmine the more probable reaetant in enterochromaffin. Further studies on the histochemical reaction confirmed the engendering of Tuksbuli's blue reactions by both ferrous and ferric salts. Rat mast cells produced a definite though weaker Turnbull's blue reaction with freshly prepared FeCl 2 but not with FeCl 3 . The ferric ferricyanide reaction sometimes reported as positive for rat mast cells is considered dubious because the reaction seen in relatively very few of the numerous mast cells seen in controls and because the greenish-blue seen in these was often equaled or surpassed by the similar color seen in some striated muscle fibers in the same section. This reaction is strongly positive in enterochromaffin cells. The findings support the view that enterochromaffin is not serotonin, but more probably dopamine or a similar catecholamine. Ferrous ion uptake reactions are found also in cartilage, colonic mucin, gastric mucin and other locations. These, like the mast cell reaction resist acetylation and oxidation but are prevented by methylation. The reaction of EC, noradrenalin, melanin, neuromelanin are prevented by acetylation and by oxidation but not by methylation. The mast cell reaction is assigned to heparin, not 5-hydroxytryptamine.

Sodium regulation in the larvae of Chironomus dorsalis (Meig.) and Camptochironomus tentans (Fabr.): the effect of salt depletion and some observations on temperature changes

Sodium regulation was studied in fourth instar larvae of Chironomus dorsalis and Camptochironomus tentans. Both maintain a body sodium level well above that of the surrounding medium. The haemolymph contains approximately 90% of total body sodium and approximates to a single compartment freely exchanging sodium with the external medium. The anal papillae play a primary role in sodium regulation, the gut being in secondary importance. Sodium regulation in both species is comparatively insensitive to alterations in acclimatization temperature. C. dorsalis and C. tentans are capable of maintaining sodium balance in media containing 10 mumole Na and 25 mumole Na respectively. When exposed to several changes of distilled water, C. tentansis capable of reducing sodium loss by elaboration of a more dilute urine. This is apparently,supplemented by a reduction in the permeability of the body surface. Activation of sodium uptake in both species is comparatively sluggish, with influx reaching a maximum only after the loss of greater than 30% body sodium.

Methods for Viewing by Scanning Electron Microscopy the Interior Organization of Protoplasts of Plant Cells

The three-dimensional organization of protoplasts of plant cells may be studied by scanning electron microscopy by exposing the cell interiors by freeze-fracturing followed by freeze-drying. Tissue blocks (1 mm3) are double fixed in glutaraldehyde and osmic acid, washed in several changes of distilled water, and positioned in shallow wells drilled into the centers of specimen stubs used for scanning electron microscopy (Kent Cambridge type). Each specimen is frozen into place with a small droplet of water by plunging the specimen holder into liquid nitrogen. While under liquid nitrogen 0. 1-0. 2 mm of tissue left projecting out of the well is cut off level with the specimen holder surface by a single thrust of a pre-cooled razor blade.

Silver staining, featuring rapid reduction, for whole mounts of retina and optic pathways in chick embryos.

Developing and established nerve fibers in the retina and in superficial tracts of the brain can be stained and viewed en bloc. The method was developed on chick embryos of 2 days of incubation to several months post-hatching but could be used on other material provided that the objects of interest were within 35 μ of the surface. Procedure: (1) Place the entire eye or head in 50% pyridine for at least 16 hr. (2) Wash well for 5–7 hr with hourly changes of distilled water or with running tap water for 4–6 hr followed by several changes of distilled water. (3) Transfer to 95% ethanol for 16–48 hr. (4) Impregnate with 1.5% AgNO3 for 2 days at 37 C. (5) Submerge in water and, when staining the retina, remove the vitreous body and apply an aqueous solution of 0.25% pyrogallic acid in 1.25% formalin by directing a narrow stream of this reducer against the retina for 2–5 sec. Wash the eye with distilled water 30–60 sec after applying the reducer. When staining the brain, remove the meninges under water, direct the stream of reducer against the brain for 20–30 sec, and rinse the brain immediately after the nerves have stained. (6) Dissect the specimen and make temporary mounts in glycerol; or, dehydrate and clear for resin mounting. The technique stains both mature and growing axons with their growth cones and sometimes their cell bodies. The fiber patterns show best on the surfaces of the retina and brain. The stain works consistently and is suited to the study of both normal and abnormal development.

Methyl green-pyronin staining: effects of fixation; use in routine pathology.

After formalin fixation and paraffin embedding, plasma cells and other py-roninophilic cells may be demonstrated as follows: The staining solution contains 4 ml of a 2% aqueous solution of methyl green, 6 ml of a 2% aqueous solution of pyronin Y, 2 ml of absolute ethanol, and Walpole's HCl-sodium acetate buffer at pH 3.8 to make a final volume of 100 ml. Staining is for 15–30 sec, differentiation by dipping twice, 1–2 sec each, in 2 changes of distilled water, next by keeping the slides in 2 changes of 96% ethanol for 30 sec each and finally by dipping them 6 times in each of 2 changes of absolute ethanol before clearing in xylene and covering in Canada balsam. Both the methyl green (Merck, C.I. No. 42585) and pyronin Y (National Aniline, C.I. No. 45005, Cert. No. NPy-20) stock solutions should be well extracted with chloroform. Sections of freeze-substituted tissues, frozen sections and smears are stained for 5 min or more in a similar solution made up with 0.1 M sodium acetate-acetic acid at pH 4.8, nex...

High Resolution Stereography of Complementary Freeze-Fracture Replicas Reveals the Presence of Depressions Opposite Membrane Associated Particles of Split Membranes

Complementary replicas have revealed the fact that the two common faces observed in electron micrographs of freeze-fracture and freeze-etch specimens are complementary to each other and are thus the new faces of a split membrane rather than the original inner and outer surfaces (1, 2 and personal observations). The big question raised by published electron micrographs is why do we not see depressions in the complementary face opposite membrane-associated particles? Reports have appeared indicating that some depressions do appear but complementarity on such a fine scale has yet to be shown.Dog cardiac muscle was perfused with glutaraldehyde, washed in distilled water, then transferred to 30% glycerol (material furnished by Dr. Joaquim Sommer, Duke Univ., and VA Hospital, Durham, N.C.). Small strips were freeze-fractured in a Denton Vacuum DFE-2 Freeze-Etch Unit with complementary replica tooling. Replicas were cleaned in chromic acid cleaning solution, then washed in 4 changes of distilled water and mounted on opposite sides of the center wire of a Formvar-coated grid.

Scanning Electron Microscopy of the Choroid Plexus and Ventricular Wall Ependyma in the Rhesus Monkey

The lumen of the lateral ventricles is separated from the brain parenchyma and the circulatory system by a single layer of specialized epithelial cells, the ependyma. This ependyma is divided into choroid plexus ependymal cells, which coat the pia arachnoid invagination in the lateral ventricles, and the wall ependymal cells which separate the lateral ventricle lumen from brain parenchyma. Using transmission electron microscopy, the ultrastructure of these cells has been described with special emphasis on the difference in apical surface membranes. The apical surface of the choroid plexus ependymal cell contains numerous microvilli and an occasional cilia while the wall ependymal cell contains numerous cilia and only a few microvilli. By scanning electron microscope we have demonstrated differences in the apical surface membrane.The specimens were fixed in glutaraldehyde by vascular perfusion or infusion into the ventricles and postfixed in osmium tetroxide. They were rinsed in cacodylate buffer and three changes of distilled water, blotted dry, and freeze dried, using liquid nitrogen. They were coated with gold palladium and examined in a Cambridge Mark II A stereoscan, scanning electron microscope.

Studies on the Ribose-5-Phosphatase of Sugarcane

A study has been made of ribose-5-phosphatase in sugarcane. The enzyme catalyzes the hydrolysis of ribose-5-phosphate to yield free ribose and phosphoric acid. The enzyme was extracted from fresh leaf tissues of 7-month old sugarcane grown in sand culture. Fractionation of cane extracts with ammonium sulfate showed that most of the enzyme was precipitated between 35 to 60 percent saturation. The richest source was spindle tissue. Leaves + 1 and + 2 were employed for studying the enzyme's properties. Dialysis up to 72 hours against several changes of distilled water had no appreciable effect on the phosphatase activity. Optimum pH was 5.5 and the Km was 2.5 X 10-3 moles of ribose-5-phosphate per liter. Good resolution of the enzyme and other protein constituents was obtained by gel filtration on Sephadex G-200. Molybdenum and tungsten inhibited at low concentrations. Molybdenum at 0.1 µmole and tungsten at 1 µmole per ml. severely inhibited the enzyme's action. Inhibition was reversed at higher concentrations. Molybdenum action was specific. Inhibition was lost during prolonged dialysis. It is proposed that ribose-5-phosphatase is a distinctly different enzyme than the other acid phosphatases previously reported for sugarcane. It appears to be functional in the CO2 fixation pathway elucidated by Calvin.

Use of Gallocyanin as a Myelin Stain for Brain and Spinal Cord

Tissue fixed in 10% formalin, formol saline, CaCO3 or phosphate buffer neutralized formalin, Baker's formol calcium, Cajal's formol ammonium bromide, formalin-95% ethanol 1:9, formalin-methanol 1:9, Lillie's methanol-chloroform or Salthouse's formol cetyltrimethylammonium bromide was dehydrated and embedded in paraffin. Sections were attached to slides with either albumen or gelatine adhesive and processed throughout at room temperature of 22–25 C. Mordanting 30–60 min in 1% iron alum was followed by a 10 min wash in 4 changes of distilled water. Myelin was stained in a gallocyanin self-differentiating solution for 1–2.5 hr; thick sections requiring the longer time. The staining solution (pH approximately 7.4) consisted of Na2CO3, 90 mg; distilled water, 100 ml; gallocyanin, 250 mg; and ethanol, 5 ml. The ethanol was added to this mixture last, and after the other ingredients had been boiled and then cooled to room temperature. After a staining and thorough washing, Nissl granules were stained for 5–10 min in a solution consisting of: 0.1 M acetic acid, 60 ml; 0.1 M sodium acetate, 40 ml; methyl green, 500 mg. Washing, dehydration, clearing and mounting completed the process. Myelin sheaths were stained dark violet; neuronal nuclei, light green with dark granules of chromatin; nucleoli of motor cells and erythrocytes, dark violet; cytoplasm, green with dark green Nissl granules. The simple and reliable method can be adapted easily for use with automatic tissue processors.

Selective staining of protein and starch in wheat flour and its products.

The main constituents of wheat flour and many wheat flour products are wheat protein (gluten) and starch granules. The specific staining of the protein present was effected by 10 min in 0.1% aqueous ponceau 2R (C.I. No. 16150) acidified with 3—4 drops of 1 N H2SO4 per 50 ml of staining solution, followed by rinsing in 2 changes of distilled water, dehydrating, clearing and mounting in a resinous medium in the normal way. Staining of starch was as follows: sections or flour smears were brought to water, treated for 10 min in a protein-blocking reagent (Taninol ADR—Imperial Chemical Industries—used in 1% aqueous solution) rinsed, then stained for 3 mins in 0.5% aqueous chlorazol violet R (C.I. No. 32445) or for 10 min in either 0.5% aqueous chlorazol violet N (C.I. No. 22570), or chlorazol black E (C.I. No. 30235). Staining was followed by thorough rinsing, normal dehydration and clearing and mounting in a medium of R.I. about 1.49 to enhance visibility of unstained starch grains. The methods are applicable to flour smears, cryostat and wax sections.

The effect of fixation on the autoradiographic grain count

AMONGST the most popular fixatives of animal tissues for light microscope autoradiography are formalin, ethyl alcohol, ethyl alcohol mixtures (e.g. those of Belanger and Carnoy), and glacial acetic acid mixtures (for example, Bouin’s and its modifications). It is reasonable to expect that the fixative might be important in subsequent autoradiographic studies, and KOPRIWA and LEBLOND (1962) have obtained significantly different counts with different fixatives of pieces of liver from a 3H-cytidine injected mouse. This report outlines a satisfactory technique for light microscopeautoradiography of developing dental tissues and compares the effect of several fixatives on the autoradiographic grain counts from ameloblasts labelled with tritiated proline. Fourteen-day old albino rats (Wistar strain) were injected with 8 PC per g of body weight of jH-proline (Radiochemical Centre, Amersham, England; spec. activ 253 mc/mM). The animals were killed by decapitation at intervals of 20 min, 60 min, and 90 min, after the injection. Each head was quicklydissectedinto fourjawquadrants, which were each placed into one of four fixatives : buffered neutral formalin, Hollande modification of Bouin’s fluid (JONES, 1966), 3 per cent glutaraldehyde in sodium cacodylate buffer, and glutaraldehyde with osmium tetroxide post-Gxation. The first three were processed for light microscopy and the fourth was processed and kept for subsequent electron microscopy. The tissues for light microscopy were fixed for 3 days at room temperature, decalcified in 10 per cent EDTA, washed in several changes of distilled water and embedded in paraffin. Sections 6 p thick were placed on gelatinized slides and prestained with Mayers haematoxylin and eosin. The liquid emulsion technique used for autoradiography came mainly from the methods reported by KOPRIWA and LEBLOND (1962), KOPRIWA (1966) and ROGERS (1967). Ilford K2 emulsion was diluted with an equal volume of 0 5 per cent glycerol in distilled water and all slides were coated in a uniform manner by means of a dipping machine (KOPRIWA, 1966); the time of withdrawalof each slide from the emulsion was 42 sec. The back of each slide was wiped clean and the slide was then placed to cool, face downwards, upon a chilled glass slab. The slides were stacked in Clay-Adams light-tight boxes which were flushed out with COz before sealing, and the sealed boxes of slides were stored at 4°C in a lead-lined container. Each box contained sections from all three fixatives. All slides were developed after 8 days in the following developer: 2 ~2 g sodium sulphite (anhydrous) plus 1 *O g amidol, made up to 620 ml with distilled water and

Use of membrane filters for measurement of 260 mμ absorbing substances from bacterial cells

The repeated use of inexpensive plastic adapters for membrane filtration is described. It was found possible to use plastic adapters marketed as disposable for more than 100 filtrations. Cellulose membranes could be reused up to 10 times after boiling in 3 changes of distilled water. The effect of adapter temperature and pore size was studied and also the release and adsorption of 260 mμ absorbing substances with the unit components. The S.D. expressed as a percentage of the mean for 10 replicate determinations of E 260 was 2.7%

Brazilin-toluidine blue O and hematoxylin-darrow red methods for brain and spinal cord.

Tissue fixed in 10% formalin, formalin-95% ethanol 1:s CaCO2 or phosphate buffer neutralized formalin, or methanol-chloroform 2:1, was dehydrated and embedded in paraffin or double-embedded by infiltration in 1% celloidin followed by a chloroform-paraffin sequence. Sections were attached to slides with either albumen or gelatine adhesive and processed throughout at room temperature of 24–26 C. For either method, mordanting 30–60 min in 1% iron alum was followed by a 10 min wash in 4 changes of distilled water. For brazilin-toluidne blue O, myelin was stained for 20–60 min, depending upon section thickness, in a self-differentiating solution consisting of: 0.15% Li2CO3 75 ml; 6% brazilin in 95% ethanol, 25 ml; and NaIO3 75 mg. After a thorough washing, Nissl material was stained for 3–8 min in a solution consisting of: 0.1 M acetic acid, 90 ml; 0.1 M sodium acetate, 10 ml; and 1% toluidine blue 0, 2.5 ml. For hematoxylin-Darrow red, myelin was stained for 2–6 hr in a self-differentiating solution consisting of: 0.15% Li2,CO3 95 ml; 10% hematoxylin in 95% ethanol, 5 ml; and NaIO3 25 mg. After a thorough washing, Nissl material was stained for 20 min or less in a solution consisting of: 0.1 M acetic acid, 90 ml; 0.1 M sodium acetate, 10 ml; Darrow red, 25 mg. This mixture was first boiled, cooled to room temperature and filtered. In both methods, washing, dehydration, clearing, and mounting completed the process. In the brazilin-toluidine blue technic, myelin sheaths were stained reddish purple; neuronal nuclei light blue with dark granules of chromatin; nucleoli dark blue; and cytoplasm blue with dark blue Nissl granules. In the hematoxylin-Darrow red procedure, myelin sheaths were blue-black; nuclei light red with dark granules of chromatin; nucleoli almost black; and cytoplasm red with bright red Nissl granules.

In Situ Electron-Microprobe Analyses of Tissue Cultures Grown on Glass

The electron microprobe has found several applications in medicine and biology. These were recently reviewed by Tousirnis. He and others have shown it to be useful for the localization of Ca, P, Fe, Ag, Zn, Cr, and Sn in normal and diseased tissues. The majority of these studies were conducted on histological sections, several cells thick, with the cells cut in different planes, and overlapping one another.This paper reports on the analysis of cells grown in a horizontal monolayer. The cells are left intact, and are analyzed on the coverslip on which they are grown. L-929 fibroblasts and KB carcinoma cells were grown on 10 x 50-mm Bellco glass coverslips in Leighton tubes. The culture medium was Hanks' Basal medium of Eagle, supplemented with penicillin, streptomycin, and 10 percent calf serum. The cultures were incubated at 37 C in a horizontal position for periods up to 3 days. They were then fixed with absolute methanol for 10 seconds, washed with several changes of distilled water, and dried. The cultures were prepared for analysis by vacuum deposition of 100 A of elemental carbon.