The Venetian Turpentine Mounting Medium

As a mounting medium to follow aceto-carmine the following modification of Zirkle's is suggested: Venetian turpentine, 25 ml.; phenol, 50 ml.; propionic acid, 35 ml.; acetic acid, 10 ml.; water, 20 ml. The technic can be employed with either root-tip or pollen-mother smears, and has been used with quite a variety of plants. It is especially valuable where it is desired to make temporary mounts permanent. The method is simple, and with reasonable care no displacement of marked cells occurs.     

Modification of the Marchi Technic

The formula proposed by Swank and Davenport (1935) was modified and applied to human and macaque nervous material. Three groups of experiments were performed and the following observations were made. (1) Diluting the osmic acid component, without altering the relative concentration of the other constituents of the solution resulted in practically no staining of the degenerated fibers. (2) When all constituents of the staining solution were used in much lower concentration than previously suggested, enhancement of staining of the degenerating fibers occurred and the different structures of the normal tissue were more easily identified. (3) At low concentrations of osmic acid and potassium chlorate, the contrast was diminished and artifacts produced by increasing the concentration of acetic acid or formalin or both. The new formula, based on the present results, consists of osmic acid, 0.5%, 11 ml.; potassium chlorate, 1%, 16 ml.; formalin (cone), 3 ml.; acetic acid, 10%, 3 ml.; and distilled water to make 100 ml. (All solutions are aqueous). Good staining after a long period of fixation in formalin, following degeneration of 8-80 days, was obtained and the cost of staining solution greatly reduced.     

A Protargol Method for Staining Nerve Fibers in Frozen or Celloidin Sections

Extensive experimentation with protargol staining of neurons in celloidin and frozen sections of organs has resulted in the following technic: Fix tissue in 10% aqueous formalin. Cut celloidin sections IS to 25 μ, frozen sections 25 to 40 μ. Place sections for 24 hours in 50% alcohol to which 1% by volume of NH₄OH has been added. Transfer the sections directly into a 1% aqueous solution of protargol, containing 0.2 to 0.3 g. of electrolytic copper foil which has been coated with a 0.5% solution of celloidin, and allow to stand for 6 to 8 hours at 37° C. Caution: In this and the succeeding step the sections must not be allowed to come in contact with the copper. From aqueous protargol, place the sections for 24 to 48 hours at 37° C. directly into a pyridinated solution of alcoholic protargol (1.0% aqueous solution protargol, 50 ml.; 95% alcohol, 50 ml.; pyridine, 0.5 to 2.0 ml.), containing 0.2 to 0.3 g. of coated copper. Rinse briefly in 50% alcohol and reduce 10 min. in an alkaline hydroquinone reducer (H₃BO₃, 1.4 g.; Na₂SO₃, anhydrous, 2.0 g.; hydroquinone, 0.3 g.; distilled water, 85 cc; acetone, 15 ml.). Wash thoroly in water and tone for 10 min. in 0.2% aqueous gold chloride, acidified with acetic acid. Wash in distilled water and reduce for 1 to 3 min. in 2% aqueous oxalic acid. Quickly rinse in distilled water and treat the sections 3 to 5 min. with 5% aqueous Na₂S₂O₃+5H₂O. Wash in water and stain overnight in Einarson's gallocyanin. Wash thoroly in water and place in 5% aqueous phosphotungstic acid for 30 min. From phosphotungstic acid transfer directly to a dilution (stock solution, 20 ml.; distilled water, 30 ml.) of the following stock staining solution: anilin blue, 0.01 g.; fast green FCF, 0.5 g.; orange G, 2.0 g.; distilled water, 92.0 ml.; glacial acetic acid, 8 ml.) and stain for 1 hour. Differentiate with 70% and 95% alcohol; pass the sections thru butyl alcohol and cedar oil; mount.     

A Simple Histochemical Reaction for Aldehydes

A study has been made on the possibility of replacing leucofuchsin by colored basic fuchsin for the histochemical demonstration of aldehydes. Several tissues from mammals and various pertinent fixatives were used. Aldehydes were freed from carbohydrates by oxidation and from thymonucleic acid by hydrolysis.

It was found that the colored form and not necessarily the leucoform of basic fuchsin can be used histochemically in demonstrating aldehydes. The technic used is as follows: (1) Treat with 1.0-0.5% H₅IO₆ (or in 1% KIO₄ in M/1 H₂SO₄) for 5 to 10 min. and wash thoroughly. For thymonucleic acid hydrolize with N HCl 5 min. at room temperature, 10 min. at 60°C. and 5 min. at room temperature. (2) Stain for 2-3 min. with 0.05% basic fuchsin in 5% ethanol, 3% phenol. (3). Transfer immediately to 1 or 2 changes of 1% sodium bisulphite or potassium metabisulphite in 0.1-0.2 N H₂SO₄ for a total of 5 min. (4) Rinse with water and treat with M H₂SO₄ in 95% ethanol for 3-5 min. 6. Wash thoroughly in water and dehydrate, clear, and mount. For glycogen and mucin the following counterstaining solution is recommended: orange G, 0.25 g.; light green SFY, 0.10 g.; phosphotungstic acid 0.50 g.; 50% ethanol, 100 ml.; glacial acetic acid, 0.25 ml.     

THE ENUMERATION OF LACTOBACILLI ON GRASS AND IN SILAGE

SUMMARY: For the enumeration of lactobacilli on grass and in silage the following medium has shown promise: peptone, meat extract and glucose, 10 g. each; tomato extract, 200 ml.; yeast autolysate, 50 ml.; Tween 80, 0.5 ml.; agar, 15 g., in a final volume of 1 1. and containing acetic acid/sodium acetate buffer in 0.2M concentration; pH 5–4. The medium was adjusted to pH 5–4 before sterilization and the requisite amount of concentrated pH 5.4 acetate buffer added just before plating. Double laver plates were used.
The only other silage organisms which in this medium formed colonies comparable in size with those of lactobacilli were heterofermentative streptococci and a micrococcus.     

Differential Staining of Degenerating Fascicles in Peripheral Nerves

A study has been made on the possibility of replacing leucofuchsin by colored basic fuchsin for the histochemical demonstration of aldehydes. Several tissues from mammals and various pertinent fixatives were used. Aldehydes were freed from carbohydrates by oxidation and from thymonucleic acid by hydrolysis.

It was found that the colored form and not necessarily the leucoform of basic fuchsin can be used histochemically in demonstrating aldehydes. The technic used is as follows: (1) Treat with 1.0–0.5% H₅IO₆ (or in 1% KIO₄ in M/1 H₂SO₄) for 5 to 10 min. and wash thoroughly. For thymonucleic acid hydrolize with N HCl 5 min. at room temperature, 10 min. at 60°C. and 5 min. at room temperature. (2) Stain for 2–3 min. with 0.05% basic fuchsin in 5% ethanol, 3% phenol. (3). Transfer immediately to 1 or 2 changes of 1% sodium bisulphite or potassium metabisulphite in 0.1–0.2 N H₂SO₄ for a total of 5 min. (4) Rinse with water and treat with M H₂SO₄ in 95% ethanol for 3–5 min. 6. Wash thoroughly in water and dehydrate, clear, and mount. For glycogen and mucin the following counterstaining solution is recommended: orange G, 0.25 g.; light green SFY, 0.10 g.; phosphotungstic acid 0.50 g.; 50% ethanol, 100 ml.; glacial acetic acid, 0.25 ml.     

A Chrome-Alum Preparation for Delicate and Difficult Fixations

A chrome-alum fixative is recommended as a reagent for general use. The basic formula is: C.P. chrome-alum, 3 g.; 40% formaldehyde, 30 ml.; glacial acetic acid, 2 ml.; distilled water, 238 ml. This fixative permits easy sectioning of yolk-rich amphibian embryos. It can be used to make permanent slides of Euglena showing the flagellum. It is a satisfactory fixative for insect larvae and fixes sharply the slime droplets of Planarians. Fixation should not exceed two hours or the material being fixed will swell. Rinses of 70% alcohol or water may follow the fluid. The fluid keeps well, does not harden tissues and gives good cytological detail.     

A Single solution iron-hematoxylin Stain for Intestinal Protozoa

A single solution iron-hematoxylin stain is described for staining fecal smears rapidly and simply. The stain is prepared from the following solutions: Solution A: 1% hematoxylin in 95% alcohol, prepared by diluting a stock solution of 10% hematoxylin in 95% alcohol. Solution B: Ferric ammonium sulfate (violet crystals), 4.0 g.; glacial acetic acid, 1.0 ml.; concentrated sulfuric acid (sp. gr. 1.8),0.12 ml.; distilled water, 100 ml. Mix equal parts of Solution A and Solution B; allow to stand overnight, filter and use. For maximum length of staining life, store in full, air-tight bottles. To stain fecal smears, fix in Schaudinn's, pass through iodine alcohol to 50% alcohol, stain for three minutes, wash in running tap water 5 to 15 minutes, dehydrate and mount.     

A Single solution iron-hematoxylin Stain for Intestinal Protozoa

A single solution iron-hematoxylin stain is described for staining fecal smears rapidly and simply. The stain is prepared from the following solutions: Solution A: 1% hematoxylin in 95% alcohol, prepared by diluting a stock solution of 10% hematoxylin in 95% alcohol. Solution B: Ferric ammonium sulfate (violet crystals), 4.0 g.; glacial acetic acid, 1.0 ml.; concentrated sulfuric acid (sp. gr. 1.8),0.12 ml.; distilled water, 100 ml. Mix equal parts of Solution A and Solution B; allow to stand overnight, filter and use. For maximum length of staining life, store in full, air-tight bottles. To stain fecal smears, fix in Schaudinn's, pass through iodine alcohol to 50% alcohol, stain for three minutes, wash in running tap water 5 to 15 minutes, dehydrate and mount.     

A Mixture of Methyl Green and Pyronin for Cell Fractionation Studies

A staining mixture consisting of 0.57% methyl green and 0.1 1 % pyronin B (calculated from the actual dye content) dis- solved in glycerol, 20 ml.; 2% aqueous phenol, 100 ml.; and 95% ethanol, 25 ml., was found to be optimum for differentiating cell components containing desoxypentose and pentose nucleic acids. The stain can be used for either fresh suspensions or unfixed dried smears of tissue homogenates. Nuclei are stained bright blue, and nucleoli and cytoplasmic particles, bright pink.     

Some Staining Methods for Bacteria and Yeasts

For staining flagella of bacteria use actively motile organisms 20 to 24 hours old, allow to diffuse in sterile water 20 to 30 minutes, transfer droplets of the suspension to clean slides and let evaporate without spreading. Then treat 2 to 4 minutes with the following mordant: tannic acid 10 or 20%, 50 cc.; ferric chloride 5%, 10 to 15 cc.; carbol fuchsin (Ziehl-Nielson), 5 cc.; hydrogen peroxide 3%, 6 to 8 cc. Wash and stain 2 to 3 minutes with a mixture of basic fuchsin, saturated alcoholic, 10 cc.; anilin oil (1 part) and 95% alcohol (3 parts) mixed, 5 cc.; distilled water, 30 cc.; acetic acid, 4%, 1 cc. Wash thoroly with water.     

Staining and Mounting Helminths

The following procedure is recommended: Fix ces-todes and trematodes (while held flat between glass slides) 0.5-2.0 hr. in the following mixture: formalin, 15; acetic acid (gl.), 5; glycerol, 10; 95% ethyl alcohol, 24; distilled H₂O, 46; all proportions by volume. After freeing them from the slides, wash thoroughly in running water and stain immediately thereafter. Stock staining solution: ferric ammonium alum (violet cryst.), 2 g.; distilled H₂O (cold) 100 ml.; after solution, add 2 ml. concentrated H₂SO₄, bring to a boil; add 1 g. coelestin blue B (Nat. Aniline), boil 3-5 min.; cool and add 10 ml. absolute methyl alcohol and 10 ml. glycerol. Dilute 1 vol. with 3 vol. distilled H20 for use. Stain 5-30 min., depending on size of specimens. Wash with 2 changes 0.5 hr. each of distilled H₂O, then 50% isopropyl alcohol 12-16 hr., 50% isopropyl alcohol 2 hr., followed by graded isopropyl alcohol for dehydration. Ether: ethyl alcohol (equal parts), 1 hr., is followed by embedding in celloidin in a sheet just thick enough to cover the specimens. Trim embedded specimens and dehydrate with isopropyl alcohol, 80%, 90% and absolute. Clear in beechwood creosote. Mount in balsam with cover glasses that overlap the edges of the celloidin 1-2 mm. While drying at 37°C, refill edges of mount with fresh balsam as needed. When dry, remove excess balsam and ring the edges with ordinary gloss enamel paint.     

Differential Staining of Gastric Glandular Cells

Fundus of stomach is fixed in 10% formalin (aqueous), Bouin's fluid or 5% trichloracetic acid (aqueous). It is embedded in paraffin, and 7μ sections are cut, mounted, deparaffinized and passed to 70% alcohol and then stained as follows: Mordant 3 min. in saturated Bismarck brown in 70% alcohol. Rinse in 70% alcohol, pass to distilled water, then overstain (2 hr.) in aniline blue, 0.5% solution in 2.5% acetic acid (aqueous). Precipitate the anilin blue with 0.5 ml. of 0.1% methyl violet solution (aqueous) dropped on die slide. Leave on 2 min. or less. Wash and differentiate in 70% alcohol. (Parietal cells dark blue). Stain 30 min. in a mixture of hematein, 0.10g.; A1C13 cryst., 0.05g.; and 70% alcohol 50 ml., prepared just before use and not filtered. Rinse in 70% alcohol and differentiate with an alcoholic extract of saffron (2 g. saffron pistils in 100 ml. 90% alcohol at 60°C. for 6 hr.) while observing the progress of differentiation microscopically. Dehydrate by dropping a 0.1 % solution of acetic acid in absolute alcohol on the section for 30 sec., followed by pure absolute alcohol, xylene, and covering in balsam.     

Differential Staining of Gastric Glandular Cells

Fundus of stomach is fixed in 10% formalin (aqueous), Bouin's fluid or 5% trichloracetic acid (aqueous). It is embedded in paraffin, and 7μ sections are cut, mounted, deparaffinized and passed to 70% alcohol and then stained as follows: Mordant 3 min. in saturated Bismarck brown in 70% alcohol. Rinse in 70% alcohol, pass to distilled water, then overstain (2 hr.) in aniline blue, 0.5% solution in 2.5% acetic acid (aqueous). Precipitate the anilin blue with 0.5 ml. of 0.1% methyl violet solution (aqueous) dropped on die slide. Leave on 2 min. or less. Wash and differentiate in 70% alcohol. (Parietal cells dark blue). Stain 30 min. in a mixture of hematein, 0.10g.; A1C13 cryst., 0.05g.; and 70% alcohol 50 ml., prepared just before use and not filtered. Rinse in 70% alcohol and differentiate with an alcoholic extract of saffron (2 g. saffron pistils in 100 ml. 90% alcohol at 60°C. for 6 hr.) while observing the progress of differentiation microscopically. Dehydrate by dropping a 0.1 % solution of acetic acid in absolute alcohol on the section for 30 sec., followed by pure absolute alcohol, xylene, and covering in balsam.     

Carbol Fuchsin as A Stain for Human Chromosomes

Cells derived from cultures of bone marrow or leucocytes were treated with hypotonic citrate solution, squashed in 45% acetic acid frozen with CO₂ to allow removal of the cover glass without disturbing the smear, and stained by the following schedule: absolute alcohol, 5 min; coat with 0.2% parlodion and air dry; 70% alcohol, 5 min; distilled water, 5 min; stain 2-5 min in a mixture of 45 ml of a 0.3% solution of basic fuchsin in 5% phenol, 6 ml of glacial acetic acid, and 6 ml of 37% formaldehyde. Differentiate and dehydrate in absolute alcohol, clear in xylene and cover. The stain is durable for several weeks if slides are stored in darkness when not in use. Results resemble those obtained by Feulgen or aceto-orcein methods.     

Selective Silver Impregnation of Degenerating Axons In The Central Nervous System

Rat and rabbit brains containing surgical lesions of 5-10 days' duration were fixed in 10% formalin (neutralized with calcium carbonate) for 1 week to 6 months. Frozen sections (15-20 n) were rinsed and then soaked 7 minutes in a 1.7% solution of strong ammonia in distilled water. Subsequent treatment was as follows: rinse; 0.05% aqueous potassium permanganate 5-15 minutes; 0.5% aqueous potassium metabisulfite, 2 changes of 2.5 minutes each; wash thoroughly in 3 changes distilled water; 1.5% aqueous silver nitrate, 0.5-1.0 hr.; 1% citric acid, 5-10 sec.; 2 changes distilled water; 1% sodium thiosulfate, 30 see.; 3 changes distilled water. Each section is then processed separately. Ammoniacal silver solution (450 mg. silver nitrate in 10 ml. distilled water; add 5 ml. ethanol; let cool to room temperature; add 1 ml. strong ammonia water and 0.9 ml. of 2.5% aqueous sodium hydroxide), 0.5-1.0 min. with gentle agitation. Reduction of about 1 minute is accomplished in: distilled water, 45 ml.; ethanol, 5 ml.; 10% formalin, 1.5 ml.; 1% citric acid, 1.5 ml. Rinsing; 1% sodium thiosulfate, 10 sec.; thorough washing followed by dehydration through graded alcohol and 3 changes of xylene or toluene complete the staining process. Normal nerve fibers are slightly stained to unstained, degenerating fibers, black. The treatment in potassium permanganate is critical since too little favors overstaining of normal fibers and too much abolishes staining of degenerating fibers.     

Silver Impregnation of Degenerating Axons in the Central Nervous System: A Modified Technic

Frozen sections of formalin-fixed brains containing surgical lesions, were treated with 15% ethanol for 0.5 hr., soaked in 0.5% phosphomolybdic acid for 0.25-1.0 hr., and subsequently treated with 0.05% potassium permanganate for 4-10 min. (The duration of the latter treatment is critical and individually variable). Subsequent procedure is as follows: decolorize in a mixture of equal parts of 1% hydroquinone and 1% oxalic acid; wash thoroughly and soak sections in 1.5% silver nitrate for 20-30 min.; ammoniacal silver nitrate (silver nitrate 0.9 g., distilled water 20 ml., pure ethanol 10 ml., strong ammonia 1.8 ml., 2.5% sodium hydroxide 1.5 ml.) 0.5-1.0 min.; reduce in acidified formalin (distilled water 400 ml., pure ethanol 45 ml., 1% citric acid 13.5 ml., 10% formalin 13.5 ml.) 1 min.; wash, and pass section through 1 % sodium thiosulf ate (0.5-1.0 min.); wash thoroughly and pass sections through graded alcohols and xylene (3 changes); cover in neutral synthetic resin.     

Anaerobic Phenol Degradation by Microorganisms of Swine Manure

Swine manure contains diverse groups of aerobic and anaerobic bacteria. An anaerobic bacterial consortium containing sulfate-reducing bacteria (SRB) and acetate-utilizing methanogenic bacteria was isolated from swine manure. This consortium used phenol as its sole source of carbon and converted it to methane and CO₂. The sulfate-reducing bacterial members of the consortium are the incomplete oxidizers, unable to carry out the terminal oxidation of organic substrates, leaving acetic acid as the end product. The methanogenic bacteria of the consortium converted the acetic acid to methane. When a methanogen inhibitor was used in the culture medium, phenol was converted to acetic acid by the SRB, but the acetic acid did not undergo further metabolism. On the other hand, when the growth of SRB in the consortium was suppressed with a specific SRB inhibitor, namely, molybdenum tetroxide, the phenol was not degraded. Thus, the metabolic activities of both the sulfate-reducing bacteria and the methanogenic bacteria were essential for complete degradation of phenol. Received: 31 January 1997 / Accepted: 7 March 1997     

Enhanced Differentiation of Zaprionus Chromosomes by Prestaining Acid Hydrolysis

Two variations of orcein staining have been adapted to salivary gland chromosomes of Zaprionus. Method I: after dissection, glands are transferred to 1 N HCl at 60° C for 5 min, stained with 2.5% orcein in 60% acetic add for 15-20 min, and squashed in 60% acetic acid. Method II: after dissection, glands are transferred to 1 N HCl at 60° C for 5 min, transferred to a saturated solution of carmine in 45% acetic acid for 1 min, then to a mixture of 50 ml of 1% orcein in concentrated lactic acid and 50 ml of 30% acetic add for 5 min. They are squashed in the same mixture. The unproved differentiation of chromosomes from cytoplasm is attributed to the removal of cytoplasmic ribonucleic add by add hydrolysis.     

Staining and Mounting Helminths

The following procedure is recommended: Fix ces-todes and trematodes (while held flat between glass slides) 0.5–2.0 hr. in the following mixture: formalin, 15; acetic acid (gl.), 5; glycerol, 10; 95% ethyl alcohol, 24; distilled H₂O, 46; all proportions by volume. After freeing them from the slides, wash thoroughly in running water and stain immediately thereafter. Stock staining solution: ferric ammonium alum (violet cryst.), 2 g.; distilled H₂O (cold) 100 ml.; after solution, add 2 ml. concentrated H₂SO₄, bring to a boil; add 1 g. coelestin blue B (Nat. Aniline), boil 3–5 min.; cool and add 10 ml. absolute methyl alcohol and 10 ml. glycerol. Dilute 1 vol. with 3 vol. distilled H20 for use. Stain 5–30 min., depending on size of specimens. Wash with 2 changes 0.5 hr. each of distilled H₂O, then 50% isopropyl alcohol 12–16 hr., 50% isopropyl alcohol 2 hr., followed by graded isopropyl alcohol for dehydration. Ether: ethyl alcohol (equal parts), 1 hr., is followed by embedding in celloidin in a sheet just thick enough to cover the specimens. Trim embedded specimens and dehydrate with isopropyl alcohol, 80%, 90% and absolute. Clear in beechwood creosote. Mount in balsam with cover glasses that overlap the edges of the celloidin 1–2 mm. While drying at 37°C, refill edges of mount with fresh balsam as needed. When dry, remove excess balsam and ring the edges with ordinary gloss enamel paint.