The Papanicolaou Stain for Negri Bodies in Paraffin Sections

Paraffin sections ot the hippocampus (Amnion's horn) from brains of dogs and cows, fixed in sublimate-alcohol (HgCl₂, sat. aq., 1 vol.; absolute alcohol, 2 vol.) were stained by Papanicolaou's (1942) method for vaginal smears. Negri bodies were stained a bright rose color, with nucleoli dark blue. Even though the orange G were omitted, good staining of Negri bodies was obtained. Eliminating this dye simplifies the technic without impairing its effectiveness in the diagnosis of rabies, but the complete staining gives a somewhat more colorful and brighter histologic picture.     

A Consistent Phosphotungstic Acid Hematoxylin Stain for Glial Fibers

After deceration, celloidinization and hydration, oxidize 10 micron paraffin sections for 15 min in a solution containing 0.3 g KMnO₄, and 0.1 ml conc. H₂SO₂, per 100 ml distilled water. Wash in water and reduce in 5% oxalic acid until the sections are colorless. Wash thoroughly in water and place in 4% iron alum solution for two hours. Wash briefly in water and stain for two hours in phosphotungstic acid hematoxylin. Rinse briefly in 95% ethanol and dehydrate in n-butyl alcohol or absolute ethanol for 4 min with two changes, clear and mount. Glial fibers, myofibrils, red blood cells, etc. are stained blue while astrocyte cell bodies, collagen, etc. are stained red. This stain has proven highly consistent in a wide variety of astrocytic derangements. Despite the intensity of this PTAH modification, false positive staining was not observed.     

The Combined Gram-Pappenheim Stain as Modified for Films and Paraffin Sections

A combination of the Gram-Pappenheim stains for the examination of gonorrheal pus, cellular exudate and paraffin sections of formalin-fixed tissues has been described elsewhere (Scudder and Lisa, 1931). The crystal violet solution was made stable for the first time by employing phosphate buffers on the acid side of neutrality, and a stable counterstain was prepared for the first time from National Aniline dyes, ethylated methyl green and pyronin yellowish. Original findings were demonstrated by means of color plate I and II (Scudder, 1931) to show gonococci, pneumococci and cells in smears, and formalin-fixed tissue brought down to water in the usual way. A new color plate is published herewith to show the microscopic appearance of cells, Gram-positive and Gram-negative bacteria, higher bacteria, fungi and spermatozoa in the study of genitourinary and gynecological cases. The method has a value in the field of medical jurisprudence. Crystals were well demonstrated, especially those resulting from sulfa drug therapy. The National Aniline methyl green batches numbered NG 10, 11, 13 to 19, and their batches of pyronin numbered NP 5 to 10 were found consistently stable. Earlier dyes were found either too purple or too blue for the technic and the most satisfactory dyes were found to require a ripening time of several days and could be prepared in amounts of from 1 to 4 liters and stored indefinitely without preservatives.     

A Durable Nissl Stain for Frozen and Paraffin Sections

Frozen sections, 25-50 /j. thick, of formalin-fixed nervous tissues are mounted following the Albrecht gelatin technic. Paraffin sections, 15 p., are deparaffinized and transferred to absolute ethanol. The slides are then coated with celloidin. Both frozen and paraffin sections subsequently follow the same steps: absolute ethanol-chloroform (equal parts) for at least 20 min, 95% ethanol, 70% ethanol (1-3 min), then rinsed in distilled water. Sections are stained in Cresylechtviolett (Chroma) 0.5% aqueous solution containing 4 drops of glacial acetic acid per 100 ml, rinsed in distilled water, agitated in 70% ethanol until excess stain leaves the slide, and rinsed in 95% ethanol. Sections are then dehydrated in absolute ethanol, followed by butanol, cleared in xylene, and enclosed in permount.     

A Hematoxylin and Eosin-Like Stain for Glycol Methacrylate Embedded Tissue Sections

A staining procedure is described for use with glycol methacrylate embedded tissue sections which does not stain the plastic embedment or remove the sections from the glass slides. The basic dye is celestine blue B. It is prepared by treating 1 g of the dye with 0.5 ml concentrated sulfuric acid. It is then dissolved with the following solution. Add 14 ml glycerine to 100 ml 2.5 percent ferric ammonium sulfate and warm the solution to 50 C. Finally adjust the pH to 0.8 to 0.9 The acid staining solution consists of 0.075 percent ponceau de xylidine and 0.025 percent acid fuchsin in 10 percent acetic acid. Slides containing the dried plastic sections are immersed in the celestine blue solution for five minutes and in the ponceau-fuchsin solution for ten minutes with an intervening water rinse. After a final wash, the sections are air dried and coverslipped. This staining procedure colors the tissues nearly the same as hematoxylin and eosin procedures.     

A Methylene Blue-Basic Fuchsin Stain for Mast Cells in Paraffin Sections

In paraffin sections of rat tissue it is possible to stain mast cell granules blue in contrast to red nuclei, pale blue cytoplasmic ribonucleic acid, and colorless collagen. This is done by the following mixture: 1% methylene blue (pure, not polychrome), 9 ml; 0.1% basic fuchsin, 9 ml; glacial acetic acid, 2 ml. Stain formol-fixed, paraffin-processed sections for 5 min, wash in water and pass through acetone, 2 changes, 10 sec total, to xylene and a polystyrene mounting medium.     

Application of the Papanicolaou Stain to Paraffin Sections

Routine paraffin sections from tissues fixed either in aqueous formalin, 80% alcohol (with or without 1% trichloracetic acid added), Carnoy's alcohol-chloroform-acetic (6:3:1) and Bouin's fixative were stained as follows: Harris' hematoxylin, 6 min; running water, 2-3 min; ascending grades of alcohol to 95%; orange G, 0.5% and phosphotungstic acid, 0.015% in 95% alcohol, 5 min; 95% alcohol, 2 changes; Papanicolaou's EA36, 2.5 min; dehydration, clearing, and covering in Permount. The results show morphology better than hematoxylin and eosin and the technic is recommended particularly for keratin, which always stains bright orange.     

Acid Thionin Stain for Nissl Bodies on Frozen Sections

Using a buffered acid thionin stain with carbolxylene as a clearing agent, a reliable stain for Nissl bodies may be performed on frozen sections of fresh or old formalin-fixed material in a relatively short period of time. The technic is simple: the buffering of thionin makes regressive differentiation unnecessary.     

Stain Technology: A Simplified Aluminum Stain in Paraffin Sections of Bone from Hemodialysis Patients

A new simplified method has been devised for staining aluminum and has been tested in paraffin sections of bone from 60 patients who have undergone hemodialysis. Iliac crest bone fragments were fixed in 20% phosphate-buffered formalin for less than a day and demineralized at room temperature in 10% phosphate-buffered formalin containing 5% formic acid for only 2 to 3 hr. Four-micron paraffin sections, accompanied by positive controls, were stained with Maloney's aluminum stain, the Berlin blue reaction for iron, dylon or Congo red for amyloid and von Kossa's reaction for calcium. Aluminum and iron were demonstrated particularly at the mineralizing front of bony tissues; aluminum in 52 cases, iron in 45. Dylon staining also gave positive results in 52 cases. It is important in determining whether aluminum deposition is present that the von Kossa reaction remains positive even after demineralization. This method may be more useful for demonstrating aluminum in bony tissues than the complicated and time-consuming resin-embedding method currently used.     

A Vanadate Hematoxylin Stain for Basic Proteins

Ammonium vanadate can act as both an oxidant and a mordant for hematoxylin. Lithium carbonate can remove vanadate hematoxylin from other structures so that only the most basic proteins are stained. Brief diazotization of the tissue sections restricts staining to the histone proteins of the nucleus.     

A Quick-Mixed Aluminum Hematoxylin Stain

Two stock solutions are composed as follows: A) aluminum sulfate, sodium iodate and acetic acid in aqueous propylene glycol and B) hematoxylin in pure propylene glycol. When combined in specified proportions the stock solutions yield aluminum-hematein dissolved in nontoxic propylene glycol. The ready-to-use stain, prepared in small volumes as needed, performs well in paraffin sections of plant tissues.     

Hematoxylin Substitutes Gallein as a Biological Stain

Gallein, hitherto seldom used in histological staining, semes excellently in iron metachrome mixture as a nuclear stain, and with ferrous solfate can give striking detail to muscle axxis striation, at the same time staining erythragtes, myainated nerve fibers and cutaneous and vascular elastin. Metabme mixtures with alnminnm and chromium are relatively ineffective, that with phosphotungstic acid gives stnmg magmta staining withmt the red blue dichroism shown by hematoxyliu. In the Clara reaction iton is well shown; enterochromaffin dog react, but not as well as with hematoxylin; the keratin Ptoreim, react well; the eosinophil leucocyte granule stains moderately gay purple. Young mammalian elastin that colors blue with hematoxylii failed to stain with gallein. If currat difficulties with metachrome iron hemaboxylim continue, gallein can afford an effective replacement.     

Bismuth Hematoxylin Stain for Arginine Residues

Bismuth ions complex with hematoxylin oxidized by sodium iodate to form a dark blue dye that stains structures with high arginine content. In citrate buffer at pH 5.2, staining is confined to cell nuclei and myelin sheaths. Extraction of nucleic acids has little effect on the stain. Blockade of the guanidino groups of arginine completely abolishes staining.     

A Gelatin Fixative for Paraffin Sections

Mayer's albumen fixative, of which the active principle is white of egg, is used almost universally for affixing paraffin ribbons to the slide. About eight years ago the writer's attention was called to a gelatin fixative which has proved to be so superior to albumen that he has used it almost exclusively ever since in the making of a great variety of botanical preparations, and has recommended it to a number of other workers whose experience with it subsequently has been just as satisfactory. The gelatin method was first described by Szombathy¹ and later discussed by Artschwager,² but it does not seem to have received the attention in the literature which its importance deserves. It certainly merits a wide spread use among both botanists and zoologists.     

A Gelatin Fixative for Paraffin Sections

Mayer's albumen fixative, of which the active principle is white of egg, is used almost universally for affixing paraffin ribbons to the slide. About eight years ago the writer's attention was called to a gelatin fixative which has proved to be so superior to albumen that he has used it almost exclusively ever since in the making of a great variety of botanical preparations, and has recommended it to a number of other workers whose experience with it subsequently has been just as satisfactory. The gelatin method was first described by Szombathy¹ and later discussed by Artschwager,² but it does not seem to have received the attention in the literature which its importance deserves. It certainly merits a wide spread use among both botanists and zoologists.     

Blood Serum as an Adhesive for Paraffin Sections

Diluted human and other mammalian blood serum (15 ml. of fresh blood serum diluted with 10 ml. of freshly distilled water, with 6 ml. of 5% formalin solution in distilled water added) can be used as a good adhesive for paraffin sections. It is preferable to Mayer's egg albumin-glycerol mixture because it is easily obtainable, can be quickly prepared, and sections are less subject to loosening after its use.     

Critical Staining of Pancreatic Alpha Granules with Phosphotungstic Acid Hematoxylin

Mammalian pancreatic alpha granules were differentially stained with phosphotungstic acid haematoxylin. Paraffin sections were dewaxed and hydrated, oxidised 5-40 sec in freshly prepared 0.3% KMnO₄ acidified with 0.3% (w/v) H₂SO₄, decolourised in 4% potassium metabisulphite, mordanted 20 min to 2 hr in 4% iron alum, stained in phosphotungstic acid haematoxylin 16-48 hr, rinsed in 95% ethanol until no stain runs from the tissue, dehydrated in absolute ethanol, cleared in xylene, and covered in synthetic resin. Advantages of this procedure are: (1) consistent, reproducible staining; (2) applicability to all the common laboratory mammals and man; (3) wide latitude at each stage, permitting its use as a routine method; and (4) superior visualization of alpha granules, due to suppression of background staining and absence of glare. For fixation, formalin-acetic or Bouin's solution is recommended.     

A One-Solution Tannic-Aced-Iron Stain for Plant Tissue Sections

After completing the bulletin on “Actinomycetes in various parts of the potato and other plants” (Lutman, 1945) the author found the beautiful plates in the atlas to Olivier's monograph (1881) on root structure in which the same intercellular inclusions were shown. Olivier stated that he had stained his sections in “tannate of iron”. Attempts were made by the author to prepare and use such a combination but they were unsuccessful owing to the precipitate that was formed.

The formula used by the U. S. government for ink for official use was tried. This combination is composed of tannic and gallic acids with ferrous sulphate and is acidified with hydrochloric acid. When used double strength, as suggested for special blackness and permanence, the stain was very successful on sections of potato roots and tubers. It stained the Actinomyces hyphae very differentially and was decolorized from all other cell organs. Any other stains used dyed also the pectins and the Actinomyces secretions (melanins) but with this iron tannate combination in one solution, the finest hyphae could be seen and photographed. Since hydrochloric acid was used in this stain, such Actinomyces inclusions must be very tannophylic; much more so than any animal intercellular inclusions so far described.