H. A. Davenport

Golgi'S Dichromate-Silver Method 3. Chromating Fluids

Slices of cat brains that had been fixed in 10% aqueous formalin for various periods from 2 days to over a year were subjected to different types of chromation prior to impregnation with silver nitrate. Acid solutions of Al, Ba, Ca, Cd, Ce, Co, Cu, Fe, K, Ni, Sr and Zn chromates were tested for usefulness as chromating agents. The chromates of Cd, Co, K, Sr and Zn were found to be best; Ca, Ce and Ni gave positive results, but Al, Ba, Cu and Fe were quite unsatisfactory. Acetic acid was somewhat preferable to formic as the acidifying agent. A formula consisting of potassium chromate, 5% aq., 100 ml. and glacial acetic acid 6–8 ml. was found to be similar in action to comparable mixtures that contained the chromate of Cd, Co, Sr or Zn. Brain slices chromated 24–48 hours in these acidified chromates and silvered in 0.75–1.0% silver nitrate for 12–24 hours at 37–40° C. gave at least three times as many good preparations as similar specimens chromated with plain potassium dichromate solution.

Staining Similarity of Foot's and Hio4-Schiff Technics

A striking similarity is noted in the staining pictures of Foots method for reticular tissue and the H104-Schiff technic. This raises the question of the type of chemical bonding involved in the two methods. It suggests that there is a carbohydrate or carbohydrate-type radical involved in the silver staining.

1. Effects of Embedding 2. Experiments with Modifications

Paraffin embedding was found to be satisfactory for brain stained by a modification of the Golgi dichromate-silver method. Nitrocellulose embedding caused fading in a few specimens. Several modifications in which the tissue was impregnated with silver nitrate before treating it with potassium dichromate were investigated. The following one is recommended. Fix pieces of brain 5-6 mm. thick for 2 days in: silver nitrate;0.5%, 90 ml.; formalin, comml. unneutralized (37-40% gas), 10 ml.; pyridine, pure, 0.05-0.1 ml. Mix in the order given and test for pH with brom cresol purple. A pH of 5.5-6.0 is about optimum and the amount of pyridine added can be varied to adjust it. A slight turbidity of the fixing fluid may be disregarded, but precipitation indicates too much alkalinity. Rinse the tissues with distilled water and place them in a mixture of potassium dichromate, 2.5%, 100 ml. and osmic acid, 1%, 1 ml., for 3-5 days. Wash in water, dehydrate with alcohol and embed in soft paraffin for thick sectioning. Gr...

Synthesis of Silver Proteinates for Neurological Staining

Soluble derivatives of the AgNO3 precipitates of various split protein products were prepared by dissolving the precipitate in a 30-40% aqueous solution of pharmaceutical peptone (Cudahy). The split proteins used included pepsin, trypsin and papain digests of albumin, globulin, gelatin, casein, protamine, and tissue proteins from heart, liver and brain; also, an Escherichia coli digest of casein, and commercial products: Amigen (Mead), casein hydrolysate (Squibb) and pharmaceutical peptone (Cudahy). Staining reactions of the silver derivatives were tested on mammalian nervous tissue. The objective of finding a silver-protein compound that stained axis cylinders selectively was attained only with redissolved silver precipitates of pharmaceutical peptone and bacterially digested casein. It was concluded that the manner of degrading a protein prior to combining it with silver was the most important factor in determining the subsequent staining reaction.

The Stainability of Nerve Fibers by Protargol With Various Fixatives And Staining Technics

The influence of the commonly used tissue fixing reagents, individually and in various combinations, on subsequent staining by protargol was studied. The reagents used were formalin, formamide, picric acid, acetic acid, paranitrophenol, pyridine and chloral hydrate. Parraffin sections from intestine and peripheral nerve of cat, dog, monkey and rat were stained with protargol after fixation in various experimental mixtures of the fixing reagents. Satisfactory nerve stains of intestine were not obtained with regularity after any one fixing and staining procedure. (Good fixation and staining appeared to be influenced by properties inherent in the tissue itself and showed marked variations from animal to animal even in the same species.)Stains of nerve fibers in peripheral nerve trunks were much more easily obtained than in the intestine where good stains were sporadic and unpredictable. The use of a mixture of 0.5% protargol and 0.1% fast green FCF, is proposed as a silver-dye staining medium.

Preparation and Test In G Of Silver-Protein Compounds

Silver derivatives of the protargol type were made from 13 different commercial peptones. The peptone samples were purified by precipitating with an ethanol concentration of 75%, reprecipitating once, and discarding any water-insoluble material. A 20–22% solution of the purified material in water, allcalinized with 2 ml. of strong ammonia per 100 ml., was precipitated by adding an equal volume of 25% aqueous AgNO3, and stood in a refrigerator overnight. Thorough washing of the precipitate with distilled water, draining, and then dissolving it in a purified peptone solution (30–35% aqueous), whose volume was 0.6 that of the solution used for silver precipitation, gave the soluble silver derivative. Ammonia was added to facilitate solution and the final pH adjusted to 8.0–8.4. The concentrated solution was dehydrated either with acetone or in a dessicator under reduced pressure and ground to a powder. Staining tests on neurological tissue showed that the Pharmaceutical peptone (Cudahy Packing Co., Omaha, Ne...

Staining sections of peripheral nerves for axis cylinders and for myelin sheaths.

Pieces of mammalian nerves 1 to 2 cm. long were placed under moderate tension and fixed 24–48 hours in: picric acid, saturated aqueous, 90 ml.; formalin, 10 ml.; and trichloracetic acid, 25% aqueous, 2 ml. They were washed in water, cut in two and one end stained with 0.04–0.06% osmic acid solution, while the other was dehydrated, embedded in paraffin, and mounted sections from it stained with protargol. The fixing solution used was selected from a number of combinations of acidified picro-formalin as the one most likely to give satisfactory results when followed by both silver and osmic acid. The use of osmic acid solutions of less than 0.1% concentration avoided the overstaining of myelin sheaths seen frequently when stronger solutions were used with material that had been fixed previously. Protargol, 0.5% solution with fast green FCF added to make 0.05% dye in the final concentration, was used to impregnate sections for axis cylinders. Reduction and toning were done as in Bodians method.

Thiazin Dyes in Supravital Staining of nerve Fibers

Supravital staining by thiazins of segments of small intestine and mesentery of young dogs was studied with reference to specificity for nervous tissue. Attempts to secure a purer form of methylene blue by alumina adsorption and alcohol elution of the commercial, medicinal dye yielded a product which appeared to be structurally different from the original dye. The treated dye had absorption maxima from 620 to 655 mμ in contrast with 665 for the untreated. Small nerve bundles were stained by the treated dye after 2 to 4 hours of immersion, but staining was always incomplete. Staining by untreated methylene blue was compared with that by the leucobase, thionol, methylene green, toluidine blue, new methylene blue and the azures. It was concluded that the specificity for nerve fibers resides mainly in the =N(CH3)2Cl radical, although some specificity appears to be effected by the methyl groups on the trivalent nitrogen, since azure A (dimethyl) and azure C (mono-methyl) stained weakly, but thionin did not. Me...