Showing papers in "Biotechnic & Histochemistry in 1953"

A Quick-Freeze Method for Making Smear Slides Permanent

Abstract: Many types of smear slides can be made permanent rapidly and effectively by substituting for the usual dehydration series a single-step process of freezing the slide on a block of dry ice, placing it immediately in 95% or absolute alcohol, and then mounting it. Advantages of the technic are its speed, the ease of separation of cover slip from slide with a minimum loss of cells, and the superiority of the resulting permanent slides.

Silver Impregnation of Ciliated Protozoa by the Chatton-Lwoff Technic

Abstract: 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 tod...

A staining combination for phloem and contiguous tissues.

Abstract: A technic is described for producing critically stained preparations of phloem tissue. The preparations promise to be relatively stable. Sections of fixed unembedded or of embedded (paraffin or celloidin) phloem, cambium, and xylem are (1) stained in Foster's tannic acid-ferric chloride combination; (2) treated with 1% NaHCOg in 25% or 50% ethyl alcohol for 30 minutes; (3) stained in a saturated solution of lacmoid (made alkaline by adding a few ml. of 1% NaHCO3 in 25% alcohol) for 12 to 18 hours; (4) dehydrated and cleared in a series composed of 1% solution of NaHCOs in 50% ethyl alcohol, 80%, 95%, and absolute alcohol, equal proportions of absolute alcohol, clove oil, and xylene, and finally pure xylene; and (5) mounted in a neutral resin. Callose and lignified secondary walls are blue or blue-green in color, cellulose walls and stainable protoplasmic contents are generally light brown. The technic has been successful with sections from 5 to 40μ in thickness, and the staining has been satisfactory for ...

Gomori's hematoxylin as a chromosome stain.

Abstract: The chromic hematoxylin of Gomori (1941) can be used as an excellent chromosome stain after hydrolysis of the tissue in warm 1–N hydrochloric acid. The hydrolysis must be accurately timed for different material as in the case of the Feulgen reaction. The staining of sections can be performed at room temperature and requires about 15 minutes. For pieces of tissue and whole preparations, it is recommended to stain at 60°C. for 40 minutes. Sections stained at room temperature can be differentiated in 1% hydrochloric acid alcohol for one minute and can be counterstained with phloxine according to Gomori's formula. Whole preparations or sections stained at 60°C. must be differentiated in 45% acetic acid for half an hour or more. Tissue pieces may, after staining, be squashed and examined in the acetic acid, but the preparations can also be made permanent. The blue-black stain is very selective and has the advantage of giving high contrast, and it is nonfading, and insoluble in water and other common reagents. ...

Histochemical demonstration of carbonic anhydrase activity.

Abstract: Fresh tissue slices fixed in chilled acetone for 1 hour and washed in distilled water for 10–30 minutes were incubated for 30–45 minutes at 37°C. in the freshly prepared incubating mixture: filtrate of a mixture of 8% sodium bicarbonate, 100 ml., and MnCl2·4H2O, 1 g. After washing in distilled water for 1 hour, they were dehydrated and embedded in paraffin. Sections were cut 15–20μU, deparaffinized, rinsed in absolute alcohol and placed in a 0.1% solution of potassium periodate for 48 hours at 37°C. The mounted sections were counterstained (if desired), dehydrated in alcohol, cleared in xylene (not carbol-xylene) and mounted in balsam. Many brown granules were produced on the sites of enzyme activity by this procedure. The results obtained seem to be in good agreement with previous findings by biochemical determinations.

Nile blue histochemical method for phospholipids.

Abstract: The technic recommended is: Fix 6–12 hr. in 10% formalin containing 1% CaCl2. Cut frozen sections without embedding or after gelatin or carbowax. Stain 90 min. at 60°C. in saturated aqueous Nile bl...

A one-solution stain for spermatozoa.

Abstract: Fresh semen is allowed to liquefy 30–60 minutes and thin, even smears of it made on clean slides or cover glasses. The smears are fixed 3 minutes with an equal-parts mixture of alcohol and ether, then air dried. They are stained 5–7 minutes in an aqueous solution made by mixing 2 volumes of 5% aniline blue (water soluble), 1 volume of 5% eosin B and 1 volume of 1% phenol. Staining at 40–60°C. is recommended. After staining, the smears are washed with distilled water, air dried and mounted in balsam or synthetic resin. The method was used on over 2000 samples of dog semen and some human specimens. Good preservation and differentiation of cytological structures was obtained uniformly, but tests were not made with other species.

Histological, chromatographic and spectrophotometric studies of toluidine blue.

Abstract: Chromatographic analysis of commercial batches of toluidine blue shows these to be dye mixtures. Histologically, some samples were found to be poor metachromatic dyes. These unsatisfactory stains contained blue dyes with little or no metachromatic properties as well as a metachromatic fraction. On the other hand, contaminating dyes in histologically satisfactory samples had poor staining qualities and hence did not interfere with the color produced by the metachromatic fraction.Chromatographic fractionation of different commercial batches of toluidine blue yielded identical, homogeneous metachromatic dyes. These purified dyes had a peak absorption at 615 mμ in contrast to that of purified azure A whose peak absorption was at 622.5 mμ.

Coumarin in chromosome analysis.

Abstract: Root tips of monocotyledons were soaked 2.5-3.0 hours at 25–27° C. in saturated aqueous coumarin solution and stained in a mixture of N HC 1 and 2% aceto-orcein (1:9 by volume) 3-4 seconds over a flame. They were then squashed in 1% orcein under a cover glass, the excess stain blotted and the cover sealed. Preparations could be kept about one week. Good chromosome morphology was secured.

Culture of pollen tubes for chromosomal analysis at the pollen tube division.

Abstract: It is possible to grow pollen tubes routinely for cytological analysis of nuclei at the pollen tube division. Pollen has been grown successfully after temperature, pressure, gas, moisture, and radiation treatments. The technic for growing Tradescantia pollen is described, but any method is satisfactory which ensures that: (a) the pollen is kept dry before sowing, (b) a minimum of time elapses between sowing pollen on culture medium and placing in a moist growing box, and (c) the growing pollen tubes are not allowed to dry out. Pollen of other species can be grown by the same methods.

Ultra-thin sectioning for electron microscopy.

Abstract: A technic is described for obtaining thin sections of animal tissue suitable for electron microscopy. Fixation is accomplished by perfusion of the whole animal with neutral formalin or alcohol formalin followed by immersion of pieces to be examined in neutralized osmium tetroxide. The embedding medium is a mixture of equal parts of n-butyl and ethyl methacrylate polymerized by ultra-violet light. Sectioning is done by means of a glass knife on an International ultra-thin sectioning microtome set at 0.1 μ. The sections are floated on warm water to spread, then placed on Formvar-coated grids, dried, and put into toluene to dissolve the plastic. The technic produces routinely usable, thin sections that show a minimum of damage owing to fixation, embedding, and sectioning.

A mordanting fixation for intense staining of small chromosomes.

Abstract: A combination iron-mordant fixative in which propionic acid is substituted for acetic acid has been found useful in preparing small plant chromosomes for carmine stained squashes. Propionic acid is better than acetic acid because it holds more iron in stable solution. The fixative is a 3:1 mixture of 95% alcohol and pure propionic acid which contains 400 mg. of Fe(OH)3 per 100 ml. of propionic acid. The latter is previously prepared by dissolving the dry freshly prepared Fe(OH)3 in it. To each 10 ml. vial of fixative is added a few drops of carmine stain. Standard aceto-carmine squashes of material fixed in this mixture show quick intense staining and are especially useful for differentiated chromosomes at mitotic prophase.

Softening paraffin-embedded plant tissues.

Abstract: Soaking paraffin-embedded plant specimens 2–3 days at 37°C. in a mixture of glycerol, 10 ml., Dreft, 1 g., and water 90 ml. is an effective means of softening them prior to sectioning. One side of the paraffin block must be pared away to expose the tissue before immersion in the softening solution.

Simple methods for mammalian chromosomes.

Abstract: The ordinary Feulgen-squash technic, after acetic-alcohol fixation, provides a simple and reliable method of preparing many mammalian tissues for chromosome counts. Tissues best suited to the technic were the seminiferous tubules. Small pieces of tissue about 3-6 mm. long and 1-2 mm. wide were immersed in a freshly made fixative (composed of 3 parts absolute ethyl alcohol and 1 part glacial acetic acid) immediately after removal by biopsy or from a freshly killed animal. After fixation for 3-12 hours the tissue was stained by the standard Feulgen procedure after hydrolysis for 12 minutes in normal HCl at 60 °C A 1-2 mm. piece was then teased apart on a slide in 45% acetic acid with a pair of mounted needles, and the teased tissue was squashed between the slide and the cover slip. During squashing the pressure was applied by hand and was regulated so as to avoid any excessive scattering of the chromosomes. The preparations were made permanent by dehydrating and mounting in Euparal.

Preparation and use of aldehyde fuchsin stain in the dry form.

Abstract: A three-day old aldehyde fuchsin staining solution (Gomori, 1950) was precipitated in a separatory funnel by adding 50 ml. of chloroform and 200 ml. of distilled water to each 100 ml. of the staining solution. The mixture was shaken, allowed to settle and the precipitate-bearing layer filtered through paper. The precipitate was dried at 50°C, scraped from the paper and stored in a stoppered vial. To use, 0.5 g. of the dry stain was dissolved in 100 ml. of 70% ethanol containing 1 ml. of concentrated hydrochloric acid. In the dry form, the dye has retained its property of staining thyrotroph cells and neurosecretory substance in the hypophysis of the rat for several months.

Organic chelating agents for decalcification of bones and teeth.

Abstract: Lower jaws of adult guinea pigs were fixed in 10% neutral formalin 24 hours following intra-arterial and intra-man-dibular injection of the same fluid. They were then decalcified 3 weeks by 3 changes of Versene or Sequestrene (ethylene diamine tetracetic acid, disodium salt). Examination of the sections revealed complete decalcification, excellent fixation and selective staining of bone, cartilage, tooth and other tissues. Comparison with acid-decalcified preparations leads us to conclude that chelate-decalcification is at least as good if not superior to acid-decalcification in the preparation of such hard tissues.

Quantitative determination of dye uptake by bacterial cells.

Abstract: A colorimetric method is presented for the quantitative determination of dye uptake by bacterial cells. Experiments showed that the dye to cell ratio was of major importance in controlling the amount of dye taken up per weight of bacterial cells. Approximate dye saturation of cells could be obtained (at pH 6.1 to 6.3) although the dye uptake curves did not absolutely level off.

Sectioning insects with sclerotized cuticle.

Abstract: Adult insects of different orders including beetles were fixed in a mixture of a saturated solution of picric acid in 90% alcohol, 75 parts; formalin, 25 parts; nitric acid (cone), 8 parts, 4–6 days and even up to 10 days depending upon the hardness of the cuticle (addition of 5% mercuric chloride to this mixture is recommended when prolonged immersion is required), or in Carnoy and Lebruns' fluid 24–48 hours and then transferred to a solution of 3–6 parts of nitric acid in 100 parts of 90% alcohol (3–6 days). After dehydration in different grades of alcohol, the insects were double embedded in celloidin and paraffin, either by (1) clove oil for 1 day, then to a saturated solution of celloidin in clove oil matured for at least 2 months for 20–40 days, or (2) the conventional ether-alcohol-celloidin mixture for 7 days; followed by hardening in chloroform. The difficulty in the proper infiltration of paraffin into celloidin hardened by chloroform around the insect is avoided by keeping the block overnight i...

Feulgen hydrolysis with phosphoric acid.

Abstract: Sections from rat tissues fixed in a 10% solution of formalin in 90% alcohol were treated with phosphoric acid in concentrations varying from 30 to 85% at room temperature (28°), and subsequently stained with the Schiff reagent. Intense staining of the nuclear material was obtained in 5 to 15 minutes when 40 to 75% phosphoric acid was used. The intense staining after the higher concentrations of phosphoric acid may be due to the low concentration of water present, thus minimizing diffusion. The nucleoli in the rat-liver cells were well stained, especially the peripheral portion. The nucleoli of nerve cells, however, were only faintly stained and the Nissl substance was completely negative. The accompanying plate of photomicrographs shows the nuclei stained by this method in the Graafian follicle, the liver, intestinal villi of the rat and a metastatic carcinoma in the human pituitary.

An ammoniated silver carbonate technic for nervous structures in mounted paraffin sections.

Abstract: Specimens of both vertebrate and invertebrate nerve-containing tissues were fixed 2-3 days in Bouin's fluid, soaked 2 days in alcohol containing 2% strong ammonia water, dehydrated and embedded in paraffin. The sections were mounted with gelatin adhesive according to Masson's procedure, dewaxed, passed through graded alcohols to water, then back to 2% ammoniated 80% alcohol for 12-24 hours. The slides were rinsed 3-5 seconds in distilled water, impregnated about one and a half hours in 40% AgNO3 at increasing temperature up to 45°C. The slides were flooded with 62.5% formalin and this solution allowed to remain 3-5 minutes; they were then blotted with filter paper. A second impregnation in ammoniated silver carbonate, controlled under the microscope, was followed by a 10-minute treatment with 10% aqueous acetic acid, toning with gold chloride, then thiosulfate and finally washing. Counterstaining with ponceau red or acid fuchsin, eventually followed by aniline blue or fast green, dehydration and covering,...

An improved lead-tetraacetate: Schiff procedure.

Abstract: Sections from tissues fixed in a 10% solution of formalin in 90% alcohol were treated with lead tetraacetate (PbAc4) in different solvents: glacial acetic acid, dilute acetic acid, methanol, ethanol, toluene and benzene. The excess reagent was removed with a 2% solution of ethylene diamine tetraacetate (EDTA) at pH 8. The sections were then stained with Schiff's reagent for 20 minutes. The chemical stability of PbAc4 in different solvents, the effect of its concentration and the time of exposure on the intensity of the Schiff reaction, were studied. A 0.023 N solution of PbAc4 in benzene with a reaction time of 5 minutes is recommended. The stability of PbAc4 in benzene permitted such solutions to be used for 3–4 days. Satisfactory results were obtained with mammalian tissues, algae, bacteria, fungi and protozoa. Some of the preparations obtained by using the technics described are illustrated in an accompanying plate of photomicrographs.

Effects of oxidation on neurofibrillar argyrophilia.

Abstract: The effect of oxidation on neurofibrillar argyrophilia was studied by subjecting nervous tissues containing both normal and degenerating fibers to the action of potassium permanganate, periodic acid, chromic acid, lead tetraacetate, and sodium bismuthate prior to silver impregnation. The argyrophilic response of normal fibers to such treatment was studied with the Nonidez silver nitrate block technic, the double impregnation method of Bielschowsky on both blocks and sections, and a silver proteinate procedure. The response of degenerating fibers was studied by the Cajal formula 6 block technic and the modified Bielschowsky procedure of Nauta and Ryan for sections. The experimental data indicated that such oxidation did not produce any differential staining effects between normal or degenerating fibers.

Embedding stained mammary glands in plastic.

Abstract: A simplified method of embedding stained mammary spreads of small mammals in plastic (Selection) is presented. Two standard 50 × 75 × 1 mm. glass slides, separated by 2 narrow glass strips of similar thickness, are used to form the embedding chamber. The glands are stained in toto in alum-carmine, dehydrated, defatted, infiltrated with uncatalyzed plastic and embedded in catalyzed plastic. After baking and cooling, the glass chamber separates readily and provides a thin square slide of plastic suitable for low-power microscopic examination, projection, and filing.

A Bacterial Cell Wall Stain1

Abstract: A new method is given to stain bacterial cell walls, especially of Escherichia coli and Bacillus cereus. The cells are smeared in water on a slide and, as soon as air-dry, are stained 3-4 minutes w...

An Arginine Histochemical Method Using Sakaguchi's New Reagent

Abstract: A mounted paraffin section of material fixed in Bouin's, Carnoy's or 10% formalin is allowed to stand 15 minutes at room temperature in a 0.3% solution of 8-hydroxyquinoline in 30% ethanol. The slide, with adhering solution, is placed in 0.15 N hypochlorite (with enough KOH added to make the solution 0.015 N KOH) for 60 seconds, then (without draining) into a solution containing: 10 ml. of 0.15 N KOH; 15 g. of urea; 70 ml. of tertiary butyl alcohol, and water to make 100 ml. Here it is gently agitated for 10 sec. and then kept in a second change of the same solution for 2 min. Two changes of pure tertiary butyl alcohol, 10 sec. and 4 min.; one in aniline, 3 min.; and one of 10 sec. in xylene, complete the procedure. Permount containing 0.02% aniline is used as a mounting medium.

The destruction of cytoplasmic basophilia with mineral acids.

Abstract: Cytoplasmic basophilia may be selectively destroyed by the mineral acids, HNO3, HCl and H2SO4. Their specificity is similar to that of ribonuclease. The optimal conditions for their use are 3°C. for 16 hours at 2M concentrations. Removal of cytoplasmic basophilia as with ribonuclease, malt diastase and perchloric acid is most effective on sections prepared from tissues fixed in solutions containing no chromates. Under the conditions herein reported the mineral acids appear to be a satisfactory and economical substitute for ribonuclease or perchloric acid.

Periodic-acid-foot stain for connective tissue

Abstract: A marked increase in reticular argyrophilia may be obtained in the Foot ammoniated silver carbonate technic by interposing a strong periodic acid oxidation, 4% aqueous for 2 hours at 25–27°C., prior to silvering. Sections so oxidized before the silver bath show a histological picture of connective tissue that is stronger than that given by the original technic. Stroma of lymphoid tissues (but not other types) is further intensified by brief (5–10 sec.) passage through aqueous 1.5% uranium nitrate after oxidation but before silver impregnation. The specific action of periodic acid (cleavage of the 1,2-glycol linkage to produce aldehyde radicals) strengthens the premise that the carbonyl radical plays an important part in the phenomenon of connective tissue argyrophilia.

Golgi impregnation after formalin fixation.

Abstract: Formalin fixed (10% aqueous) brain from cat, rabbit and man cut to blocks 3-4 mm thick was placed in a mixture of potassium bichromate, 5 g; chloral hydrate, 3 g and water 90 ml for 24 hours The specimens were rinsed through 3 changes of water, and transferred through 3 changes of 1% silver nitrate, 1-3 minutes each, then placed for 24 hours in 15% silver nitrate Frozen sections, 40-50 μ were dehydrated and mounted with a cover glass, using Permount No deterioration of the stain was seen after 5 months Some brains had been in formalin for 9 months; others only 7 days

Stains for the primary wall of the cotton fiber.

Abstract: Several methods are described for distinguishing between the primary wall of the cotton fiber and other fiber components, such as the lumen and the secondary wall. The primary wall, a membrane less than 0.5 μ thick covering the entire fiber, has been stained while still attached to the fiber as well as after it has been mechanically stripped from the fiber. The stains include aqueous or alcoholic solutions of ruthenium red, methylene blue chloride, Nile blue sulfate, oil red, Sudan black B, iodine, and Simons' stain. Various concentrations of sodium hydroxide, cupri-ethylenediamine hydroxide, or sulfuric acid have been used to enhance color changes and to cause cellulosic swelling. Fibers that have been stained with Simons' stain and then swelled with dilute cupri-ethylenediamine hydroxide have shown the greatest color differences between the primary wall and the lumen.