A Carbon-Gelatin Injection Mass for Minute Vascular and Respiratory Passages

Coronary arterioles and pulmonary air passages of dogs could be injected completely with a carbon-gelatin mixture heated at the start of the injection to 52-58°C. Formula: chloral hydrate, 40 gm; carbon (Peerless Black, Columbian Carbon Co., 214 44th St., Brooklyn, N. Y.), 20-40 gm; distilled water, 950 ml; mix thoroughly in a Waring blendor, then add a mixture of Tween 80 (Atlas Powder Co., Wilmington, Del.), 20 ml; and water, 30 ml at 40°C, and agitate to a homogeneous suspension. For use, mix equal parts of the suspension and a 16% gelatin solution (Bacto-Gelatin, Difco Laboratories Inc., Detroit, Mich.) at 52-58°C. With this material the precapillary arterioles may be microscopically studied in relation to their myocardial distribution and the finer respiratory passages of lung investigated, providing all the apparatus with which the solution comes in contact is clean and the combined carbon-gelatin mass is injected shortly after mixing.     

Preparation of <Emphasis Type="Italic">in situ</Emphasis> Injectable Chitosan/Gelatin Hydrogel Using an Acid-tolerant Tyrosinase

An in situ injectable chitosan/gelatin hydrogel was formed under slightly acidic conditions (pH 4.0 ~ 4.5) using an acid-tolerant tyrosinase, tyrosinase-CNK. A homogeneous chitosan/tyrosinase-CNK solution was prepared in one part of a dual-barrel syringe, and highly soluble gelatin in distilled water was prepared in the other part of the syringe without any additional crosslinking materials. Chitosan/gelatin hydrogel was formed in situ by simple injection of the solutions at room temperature followed by curing at 37°C. However, conventional mushroom tyrosinase did not catalyze this permanent gel formation. Tyrosinase- CNK-catalyzed glycol chitosan/gelatin hydrogel was similarly formed by this in situ injection approach. The hydrogels exhibited a high swelling ratio of 20-fold their own weight, interconnected micropores with an average diameter of approximately 260 μm and in vitro biodegradability suitable for tissue engineering and drug delivery applications. These results showed that tyrosinase-CNK-mediated chitosan/gelatin hydrogel formation has remarkable potential for the development of novel formulations for in situ injectable gel-forming systems.     

A Colchicine, Hypotonic Citrate, Squash Sequence for Mammalian Chromosomes

The method was developed for bone marrow of mice but is applicable to other tissues and other species of small mammals. Mice are injected intraperitoneally with 0.5 ml of 0.025% colchicine solution and killed 1 hr afterwards. The femurs are dissected out rapidly, the epiphyses are removed, and the marrow is washed out of the shafts by warm hypotonic sodium citrate solution from a hypodermic syringe. Gentle aspiration of the marrow into and out of the syringe converts it into a fine suspension. The suspension is kept in the citrate solution at 37°C for 10 min. Connective tissue and bony spicules are removed by centrifuging through Nylon bolting cloth in a bacterial filtration tube, before fixing in acetic-alcohol (1:3) and staining by the standard Feulgen procedure. The cells are concentrated for each change of reagent by centrifuging slowly. The advantages of colchicine pretreatment and of working with cell suspensions are emphasized.     

The Use of Protein Silver for Staining Protozoa

When commercially prepared silver products suitable for staining protozoa by the Bodian silver technic apparently became unavailable, a substitute for Protargol was prepared as follows: 0.9 g. of gelatin is dissolved by heat in 100 ml. of distilled water; to this 0.1 g. of silver nitrate is added at 60°C; this solution is poured into Columbia staining dishes (10 ml.) in which one or two drops of M/10 sodium hydroxide have been added. Copper is not used in the impregnating bath. Smears fixed in Hollande's or Schaudinn's fixatives are bleached and impregnated for 36 hours or more at 35°C. Impregnated smears are reduced with a mixture of hydroquinone and sodium sulfite, and toned with gold chloride as recommended by Kirby (1945).     

Silver Impregnation of Ciliated Protozoa by the Chatton-Lwoff Technic

The ciliates are manipulated at all stages by means of micropipettes and fine needles, proceeding by the following steps: Fix the material in a small receptacle with Champy's fluid 1-3 minutes following with Da Fano's solution for several hours. Transfer the specimens to a slide, withdraw excess fluid and embed them in warm (35°-45°C.) gelatin containing 0.05% sodium chloride. Refrigerate in a moist chamber until the gelatin has set, and then immerse 10-20 minutes in 3% silver nitrate (aqueous) at 5-10°C. Wash with cold distilled water, submerge the preparation in cold water to a depth of several centimeters and expose to a strong light for 10-30 minutes. Silver is deposited on various pellicular structures which then appear black in the dehydrated and mounted specimens. Neatly revealed are the many longitudinal and transverse fibrils of the “silverline system”, basal granules of the cilia, bases of buccal ciliary organelles, contractile vacuole pores and the cytoproct. None of these structures, which today are considered to be of inestimable value in comparative morphological and taxonomical studies of ciliates in general, is so precisely made evident by any other technic known to the author.     

Organic charge transfer complex based printable biosensor

This paper describes the preparation of an organic charge transfer complex (CTC) based printable enzyme electrode. CTC crystals were prepared by mixing TCNQ powder with TTF solution (in acetonitrile). Glucose oxidase (GOD) was adsorbed at the CTC crystal surface in a monolayer. A printable paste was prepared by mixing GOD-adsorbed crystals with a binder and a solvent. This paste was applied to an electrode cavity and vacuum dried. A thin layer of gelatin was cast on the paste filled dried electrode, and cross-linked with glutaraldehyde in the dry condition. The sensors were fixed in a flow injection system, and continuously polarized at 0·15 V and 37°C, and the samples were automatically injected every 30 min. The developed sensors produced a huge response curren with an extended linear range of detection (0–100 mM) and the response was unaffected by the presence of normal oxygen in the buffer solution. The sensor showed excellent stability. The performance of the sensors was significantly influenced by the binder used.     

Injection of Lymphatics: With Colored Cedar Oil; With Plastic

(1) The oil mass consists of: cedar oil, 1; color in oil (a paint pigment, e.g., Prussian blue), 1; and toluene, 2, parts by volume. To use, add 1 ml of diethyl ether to each 10 ml of mass, mix thoroughly and inject into the fresh organ with a very fine glass or metallic needle. Heat the organ in water at 50-60° C before starting the injection, massage gently after injection, then fix. For macroscopic studies, fix 5 days in 5% formalin, and dissect. For microscopic studies, fix at least 5 days in: formalin, 10 ml; Al₂(SO₄)₃, 2 gm; ZnSO₄, 2 gm; acetic acid, 4 ml; and distilled water, 90 ml. Dehydrate with dioxane, embed in paraffin and section at 10-20 μ. Stain with hematoxylin-eosin or with one of the following modifications of Van Gieson's formula: 1. 1% acid fuchsin, 10; picric acid (sat. aq.), 50; and 5% ZnSO₄, 40 volumes. 2. 1% acid fuchsin, 20; picric acid (sat. aq.), 80; and 5% CoSO₄, 40 volumes.

(2) The plastic mass consists of a 5-10% solution of Rhodopas (a vinyl copolymer) in acetone. Injection is made as with the oil mass except that a plastic squeeze-bottle and glass needle is preferable to a syringe. Indirect injection is used for both procedures, i.e., into the organ substance; not into a cannulated lymphatic vessel. After the plastic has hardened (24 hr), the unfixed tissue is subjected to corrosion by 5-10% NaOH in water.     

Concentrating and Staining Bone Marrow; An On-the-Slide Technique

A 0.5-1 ml sample of bone marrow is aspirated into a syringe containing 3 drops of 15% K₂-EDTA and an additional 1-2 drops of the EDTA solution previously placed on a slide, is then drawn into the syringe. All of the contents are ejected onto this slide, which is carefully tilted 2 or 3 times to an angle of 5-10°, and the edge brought to the center of another slide. The slide with the aspirate is then slowly tilted to 80-90°. Most of the blood and part of the marrow will drain off, leaving spicules of marrow and some blood on the original slide. A small drop of this concentrated marrow is dragged off with the edge of a third slide and deposited about 2 cm from the edge of a fourth slide on which the smear is to be made. The smear is made by bringing a clean (smearing) slide to the slide with the deposited marrow with flat surfaces parallel and the edges at a 90° angle. With gentle pressure, the smearing slide is pushed toward the empty end of the slide upon which the smear is made. This separates the marrow from the circulating blood. Before staining the smear is air dried and heated in an oven at 120-125 C for 2 min; or alternately for satisfactory but less uniform results the smear is heated over a microburner for 10 sec; then the smear is covered with 1 part of undiluted Wright's stain for 30—45 sec which is then diluted with 2 parts of a solution of 0.1-0.2 gm of Na₂S₂O₃ in 1 liter of distilled water and stained for 10-13 min with this diluted stain. Smears made in this manner have 3 concentric zones; the central zone contains the myeloid tissue; the middle, erythropoetic tissue; the outer, a mixture of blood and marrow.     

Effect of transglutaminase and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide on the solubility of fish gelatin–chitosan films

The possibility of decreasing the water solubility of the films made from fish gelatin and chitosan by modification with TGase was investigated. The effectiveness of enzymatic treatment was also compared with chemical crosslinking using EDC. The treatment of the components with TGase in concentration of 0.2 mg/ml of the film-forming solution limited the solubility of the films at 25 °C from 65% to 28% at pH 6 and from 96% to 37% at pH 3. After 15 min of heating at 100 °C, the modified films were soluble in 23% at pH 6 and in 41% at pH 3. Further decrease of the solubility of the fish gelatin–chitosan films was achieved when enzymatic modification was conducted in the presence of 5–10 mM DTT; the solubility was about twice lower than that without DTT at both studied temperatures and pH values. Generally, the composite films modified with EDC in concentration of 30 mM were distinctly less soluble than films made from the components modified with TGase in the presence of DTT.     

Fuchsin staining with naoh clearing for lignified elements of whole plants or plants organs

Three methods that are adapted to the various consistencies of plants are as follows: 1. Samples are placed for 10-14 hr at 60° C in a 1% aqueous solution of basic fuchsin, to which 10 gm of solid NaOH per 100 ml are added. 2. Samples when taken out of 95% alcohol are placed in a 1% solution of basic fuchsin in 95% alcohol for 24 hr; after washing in water, they are placed in a 15% solution of NaOH at 60° C until cleared. 3. Samples are placed in a 15% aqueous solution of NaOH at 60° C until cleared, then for 24 hr at 60° C in 15% NaOH containing basic fuchsin. After being stained and cleared by one of these three methods, the samples are rinsed in water, dehydrated and then passed into a mixture of absolute alcohol and concentrated HC1 (3:1) for 1-15 min, rinsed in absolute alcohol, cleared in xylene and mounted in Canada balsam. The lignified tissues appear red; the others, transparent.     

Staining Mitochondria With Hematoxylin After Formalin-Sublimate Fixation; A Rapid Method

Mitochondria were stained in liver, kidney, pancreas, adrenal and intestinal mucosa of rat and mouse. Tissues 1 mm thick, were fixed in a mixture of saturated aqueous HgCl₂, 90 ml; formalin (37-38% HCHO), 10 ml, at room temperature (25°C) for 1 hr. Deparaffinized sections 3-4μ thick were treated with Lugol's iodine (U.S.P.) followed by Na₂S₂O₃ (5%), rinsed in water and the ribonucleic acid removed by any of the following procedures: 0.2 M McIlavaine's buffer, pH 7.0, 2 hr, or 0.2 M phosphate buffer, pH 7.0, 2 hr at 37°C; 0.1% aqueous ribonuclease, 2 hr at 37°C; 5% aqueous trichloracetic acid overnight at 37°C; or 1% KOH at room temperature for 1 hr. After washing in water, sections were treated with a saturated solution of ferric ammonium alum at 37°C for 8-12 hr and colored by Regaud's ripened hematoxylin for 18 hr. They were then differentiated in 1% ferric ammonium alum solution while under microscopic observation.     

Application of the Nauta-Gygax Technic for Degenerating Axons to Mounted Sections

Frozen sections of formalin-fixed brains containing lesions were mounted on slides that had been coated first with albumen-glycerol (1:1) then 4% gelatin and blotted. The slides were placed in formaldehyde vapor at 56° C for 40-60 min, washed, and stored (optional) in 10% formalin-saline. The staining technic was as follows: after washing, soak 30-40 min in 0.5% phosphomolybdic acid, rinse; put in 0.05% potassium permanganate 9-16 min (usually 12 min); decolorize in a 1:1 mixture of 1% hydroquinone and 1% oxalic acid; wash thoroughly; soak in 1.5% AgNO₃ at about 20° C for 25-35 min; rinse; put into an ammino-silver solution (4.5% AgNO₃, 20 ml; pure ethanol, 10 ml; ammonia, sp. gr. 0.880, 2.4 ml; 2.5% NaOH, 1 ml) for 1-2 min; reduce in acidified formalin (distilled water, 400 ml; pure ethanol, 45 ml; 1 % citric acid, 13.5 ml; 10% formalin, 13.5 ml) for 1-3 min; wash; dehydrate through ascending grades of alcohol, including absolute; coat with 0.5% collodion, allow to dry slightly and harden in absolute alcohol-chloroform (2:1); rehydrate and put into 1% Na₂S₂O₃ for 1 min; dehydrate and cover.     

A preliminary study of a biosensor based on flow injection of the recognition element

A biosensor based on flow injection of the recognition element has been developed. As a model a pH-transducer was used, and urease was chosen as the recognition element. The pH-transducer was immersed in an internal flow-through chamber which was in contact with the sample solution via a semi-permeable membrane. The recognition element, urease, was injected into the buffer solution passing through the biosensor. The enzyme catalysed the hydrolysis of urea and the concomitant increase in pH was recorded. The biosensor response time was about three minutes at a constant flow rate of 0·05 ml/min. The linear range of the calibration curve of the biosensor was 0–5 mM. The observed detection limit was approximately 0.1 mM. The sample throughput was 6–12 per hour. The pH-response of the biosensor, for a sample solution containing urea (3·26 mM), showed a reproducibility (r.s.d) of 28% (n = 5) and a repeatability (r.s.d.) of 8% (n = 5). Operation at elevated temperatures (up to 50°C) was demonstrated. The presence of glucose (28 mM), acetone (6·7 mM), citric acid (0·2 mM) or sodium acetate (0·6 mM) in the sample solution did not interfere with the sensor response. A lowering of the biosensor response which was observed in the presence of copper ions (due to urease inhibition) could be completely eliminated by adding EDTA to the urease solution. Thus, this work demonstrates a new type of biosensors, based on SIRE-technology (Sensors with Injectable Recognition Elements), which show high accuracy and stability, quick response and high sample throughput. These features suggest the suitability of the system for automation. Such sensors should readily be combined with other enzymes or enzyme systems. The enzyme (urease) cost per analysis (injection) for the biosensor was estimated to be approximately US$0·02. This could be substantially reduced by further optimisation and miniaturisation.     

Viability and acrosome integrity of rabbit spermatozoa processed in a gelatin-supplemented extender

The effect of gelatin addition to the semen extender on the viability and acrosome integrity of rabbit spermatozoa was studied. Pooled semen samples were processed in a boar semen extender with or without gelatin addition. Semen samples were stored at 5 °C for 72 h. Viability and acrosome integrity was evaluated by light microscope. Results showed that gelatin addition had a significant positive effect on the quality of the stored semen.     

Cerebral ventricular transport and uptake: Importance for CCK-mediated satiety

Brain cholecystokinin (CCK) peptides have been proposed to be involved in the control of feed intake. We have examined the importance of the cerebral ventricular system in CCK-mediated satiety in sheep. Continuous injection of 0.64 pmol/min CCK-8 into the lateral ventricles (LV) decreased feeding, whereas injection of neither 0.64 nor 2.55 pmol/min CCK-8 into the cisterna magna (CM) significantly affected feeding. Thus, it is likely that the rostral, but not caudal, ventricular compartments and/or adjacent brain areas are involved in CCK-8 mediated satiety. The rate of injection of carrier solution (synthetic cerebrospinal fluid [sCSF]) was found to affect feed intake during a continuous 75 min injection: feed intakes were greater during injection of sCSF at 0.10 ml/min than during either 0.03 ml/min sCSF or no injection (sham). Injection of 0.64 pmol/min CCK-8 in either 0.03 or 0.10 ml/min decreased feeding. The increased feeding during 0.10 ml/min sCSF injection may have been due to dilution of endogenous CCK released into CSF during the meal. To determine the percent recovery from CSF of exogenous CCK-8, CSF samples from CM were collected during 3 hr continuous LV injections of CCK-8 and inulin (for measurement of bulk absorption). Only 20 to 40 percent of administered CCK-8 was recovered in CM CSF. The loss of CCK-8 was probably not due to degradation in the CSF by proteolytic enzymes, since CCK-8 concentrations did not decrease during in vitro incubation at 37°C for up to 24 hr. We propose that CCK-8 is released during feeding into the ventricular system, and subsequently taken up from CSF by specialized ependymal cells for transport to sites of action.     

Method of intravital injection of the internal blood vessels of organs for experimental-morphological research]

Ten minutes before the injection the rat is given heparin (500 units per 1 kg weight) intradermally. To the narcotized animal, the needle with the 1 ml syringe is inserted and 3--5 ml of blood are aspirated (about 2% of the body mass). Then with the same needle another syringe of 10 ml volume containing filtrated and heated up to 40 degrees C undiluted Indian ink is injected. The amount of the Indian ink injected is 8--10 ml per 100 g of the body mass. We can consider the injection oa success if the mucose of the tongue, the skin of the concha auriculae, the sclera of the eye are promptly stained during the injection and the tail vessels are well filled.     

Difference of molecular association in two types of curdlan gel

The molecular association in a curdlan gel formed by neutralizing an alkaline solution of curdlan with carbon dioxide was compared with those in gels obtained by heating aqueous suspensions of curdlan at various temperatures.

The neutralized and 60°C-set preparations were soluble in 0·01 sodium hydroxide, whereas preparations set at above 90°C were soluble only in concentrations of sodium hydroxide above 1 . The absorption of Aniline blue or Congo red to the preparations decreased with an increase in the temperature of heat treatment and the adsorption to a gel heated at 120°C for 4 h was about 30% of that for the unheated neutralized gel. Seventy-three per cent of the heated preparation was resistant to treatment with 32% sulfuric acid at 32°C for 30 days, whereas none of the neutralized gel was resistant. An electron micrograph of the resistant part of the curdlan showed that it had a pseudocrystalline form. X-ray studies showed a much higher crystalline structure in the resistant part than in the preparation without heat treatment. The X-ray patterns were almost the same for preparations treated with 32% sulfuric acid or (1 → 3)-β-glucanase.     

Chitosan and gelatin based edible films: state diagrams, mechanical and permeation properties

Films of chitosan and gelatin were prepared by casting their aqueous solutions (pH≈4.0) at 60°C and evaporating at 22 or 60°C (low- and high-temperature methods, respectively). The physical (thermal, mechanical and gas/water permeation) properties of these composite films, plasticized with water or polyols, were studied. An increase in the total plasticizer content resulted in a considerable decrease of elasticity modulus and tensile strength (up to 50% of the original values when 30% plasticizer was added), whereas the percentage elongation increased (up to 150% compared to the original values). The low-temperature preparation method led to the development of a higher percentage renaturation (crystallinity) of gelatin which resulted in a decrease, by one or two orders of magnitude, of CO₂ and O₂ permeability in the chitosan/gelatin blends. An increase in the total plasticizer content (water, polyols) of these blends was found to be proportional to an increase in their gas permeability.     

A Nuclear Stain for Escherichia Coli And Related Organisms

The dye base of new fuchsin was precipitated by adding potassium hydroxide to the dye solution. The precipitate was filtered out and washed with water. It was then suspended in water, brought into solution and adjusted to a pH of about 5.0 with nitric acid. The staining solution was prepared by adding 0.3 ml. of a 14% aqueous solution of pyrogallol and 0.1 ml. of a 1% aqueous solution of boric acid to 3.0 ml. of the dye solution. Smears of cells were made in water on a slide and allowed to dry before covering with the staining solution which was also permitted to air dry. The smear was then washed in water and mordanted for 5-20 seconds in a 0.1% aqueous solution of mercuric nitrate. After rinsing in water, the smear was air dried. When dry, the slide was placed on a 50° C. warm plate for a few seconds before covering with a very thin film of a 5% aqueous solution of nigrosin which had a pH of about 5.0.     

Differentiated Staining of Osmium Tetroxide-Fixed, Vestopal-w-Embedded Tissue in thin Sections

Tissues were fixed at 20° C for 1 hr in 1% OsO₄, buffered at pH 7.4 with veronal-acetate (Palade's fixative), soaked 5 min in the same buffer without OsO₄, then dehydrated in buffer-acetone mixtures of 30, 50, 75 and 90% acetone content, and finally in anhydrous acetone. Infiltration was accomplished through Vestopal-W-acetone mixtures of 1:3, 1:1, 3:1 to undiluted Vestopal. After polymerisation at 60° C for 24 hr, 1-2 μ sections were cut, dried on slides without adhesive, and stained by any of the following methods. (1) Mayer's acid hemalum: Flood the slides with the staining solution and allow to stand at 20°C for 2-3 hr while the water of the solution evaporates; wash in distilled water, 2 min; differentiate in 1% HCl; rinse 1-2 sec in 10% NH,OH. (2) Iron-trioxyhematein (of Hansen): Apply the staining solution as in method 1; wash 3-5 min in 5% acetic acid; restain for 1-12 hr by flooding with a mixture consisting of staining solution, 2 parts, and 1 part of a 1:1 mixture of 2% acetic acid and 2% H₂SO₄ (observe under microscope for staining intensity); wash 2 min in distilled water and 1 hr in tap water. (3) Iron-hematoxylin (Heidenhain): Mordant 6 hr in 2.5% iron-alum solution; wash 1 min in distilled water; stain in 1% or 0.5% ripened hematoxylin for 3-12 br; differentiate 8 min in 2.5%, and 15 min in 1% iron-alum solution; wash 1 hr in tap water. (4) Aceto-carmine (Schneider): Stain 12-24 hr; wash 0.5-1.0 min in distilled water. (5) Picrofuchsin: Stain 24-48 hr in 1% acid fuchsin dissolved in saturated aqueous picric acid; differentiate for only 1-2 sec in 96% ethanol. (6) Modified Giemsa: Mix 640 ml of a solution of 9.08 gm KH₂PO₄ in 1000 ml of distilled water and 360 ml of a solution of 11.88 gm Na₂HPO₄-2H₂O in 1000 ml of distilled water. Soak sections in this buffer, 12 hr. Dissolve 1.0 gm of azur I in 125 ml of boiling distilled water; add 0.5 gm of methylene blue; filter and add hot distilled water until a volume of 250 ml is reached (solution “AM”). Dissolve 1.5 gm of eosin, yellowish, in 250 ml of hot distilled water; filter (solution “E”). Mix 1.5 ml of “AM” in 100 ml of buffer with 3 ml of “E” in 100 ml of buffer. Stain 12-24 hr. Differentiate 3 sec in 25 ml methyl benzoate in 75 ml dioxane; 3 sec in 35 ml methyl benzoate in 65 ml acetone; 3 sec in 30 ml acetone in 70 ml methyl benzoate; and 3 sec in 5 ml acetone in 95 ml methyl benzoate. Dehydrated sections may be covered in a neutral synthetic resin (Caedax was used).