Hematoxylin as a Pituitary Stain

The accurate picture of the acidophil granules of the anterior pituitary which is provided by iron hematoxylin can be combined with the differential staining of the basophils by either the periodic acid-Schiff (PAS) or combined aldehyde-fuchsin-PAS procedures. To accomplish this the two stages of the iron hematoxylin technique are separated so that mordanting in iron alum precedes and application of hematoxylin follows the basophil procedures.     

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.     

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.     

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.     

Mallory's Connective Tissue Stain Following Hematoxylin

The following combination of hematoxylin with Mallory's connective tissue stain is useful in bringing out nuclei as well as in differentiating tissue:

Slightly overstain in Mayer's hematoxylin (50 g. potassium alum and 0.2 g. sodium iodate added to 1 liter 0.1% aqueous hematoxylin). Wash; and stain 30 seconds to 1 minute in 0.04% aqueous acid fuchsin-Stain 4 minutes in: 0.5 g. anilin blue and 2 g. orange G dissolved hi 100 cc. of 1% aqueous phosphomolybdic acid. Pass thru 95% alcohol to absolute; clear in xylol and mount in balsam.     

Zirconyl Hematoxylin: a New Stain for Acidic Mucins

Most stains for acidic mucins are time-consuming to prepare and have poor stability. Zirconyl hematoxylin is easily prepared and works for a year or more. It is made by adding 5 ml freshly-made 0.1% aqueous sodium iodate, 400 mg zirconyl chloride oc-tahydrate, and 40 ml 25% aqueous glycerol, in that order, to 100 mg of hematoxylin in 5 ml of absolute ethanol and stirring for 5 min. Stain 10 min and do not “blue” the stain. Chlorazole black or kernechtrot and fast green are good counterstains. Zirconyl hematoxylin stains acidic mucins violet or red violet, regardless of how they are fixed. It stains the same mucins as alcian blue in mouse and sheep salivary glands. It shows goblet cells in mouse rectum as well as alcian blue. It stains the same stomach regions in a lizard as alcian blue. Like alcian blue and colloidal iron, zirconyl hematoxylin stains the mucin of cancerous prostate, but not normal prostate.     

Zirconyl Hematoxylin: a New Stain for Acidic Mucins

Most stains for acidic mucins are time-consuming to prepare and have poor stability. Zirconyl hematoxylin is easily prepared and works for a year or more. It is made by adding 5 ml freshly-made 0.1% aqueous sodium iodate, 400 mg zirconyl chloride oc-tahydrate, and 40 ml 25% aqueous glycerol, in that order, to 100 mg of hematoxylin in 5 ml of absolute ethanol and stirring for 5 min. Stain 10 min and do not “blue” the stain. Chlorazole black or kernechtrot and fast green are good counterstains. Zirconyl hematoxylin stains acidic mucins violet or red violet, regardless of how they are fixed. It stains the same mucins as alcian blue in mouse and sheep salivary glands. It shows goblet cells in mouse rectum as well as alcian blue. It stains the same stomach regions in a lizard as alcian blue. Like alcian blue and colloidal iron, zirconyl hematoxylin stains the mucin of cancerous prostate, but not normal prostate.     

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.     

Mordant Blue 3: a Readily Availablx Substitute for Hematoxylin in the Routine Hematoxylin and Eosin Stain

Mordant blue 3 may be used as a suhstitute for hematoxylin in hematoxylin and eosin stains. The staining solution consists of 0.25 g dye, 40 ml of 10% iron dam, 5 ml of cone H₂SO₄, and 955 ml of dirtilled H₂O. Staining the is 5 minutes, followed by differentiation in acid water or acid alcohol. After blueing, the seaions are counterstained with emin. Results closely resemble the hematoxylin and eosin stain.     

Phosphotungstic Acid-Eosin Combined with Hematoxylin as A Stain for Negri Bodies in Paraffin Sections

Experimentation with the Papanicolaou stain in this laboratory led to the discovery that the eosin, combined with phosphotungstic acid, was responsible for differential staining of Negri bodies. Eosin prepared as in EA 36 was used, but without the light green and Bismarck brown. Paraffin sections of hippocampus from brains of animal affected with rabies were fixed in 10% formol or in a mixture of 2 volumes of saturated aqueous HgCl₂ and 1 volume of absolute alcohol. They were stained first with hematoxylin and then with eosin. This procedure gave better results than staining with other types of eosin or by the original EA 36 mixture. The Negri bodies were well stained and their structure easily visible. The best results were obtained from material fixed with the HgCl₂ solution.     

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.     

Phloxine As An Histologic Stain, Especially In Combination With Hematoxylin

A staining schedule employing phloxine as a counter-stain to Erlich's acid hematoxylin is presented. Fixation is best with Zenker's fluid, although formalin can be used. The technic is similar to the standard hematoxylin-eosin formulae but because of the staining advantages of phloxine over eosin, the technic is simpler, and quicker, resulting in clearly differentiated sections which do not fade as soon as do eosin-stained slides. A brief summary of the uses of phloxine as a biological stain is given and its advantages over eosin are discussed.     

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.     

Isohematein as a Biological Stain

The possible use of isohematein as a biological stain is considered. Certain characteristics of the dye are discussed in relation to staining technic. A preliminary series of experiments is described. The stomach of the frog, skeletal muscle of the frog, and spinal cord of the cat were used as representative tissues. The dye has greater tinctorial power than hematoxylin (hematein) but it is not so selective for nuclei. The results at hand indicate that the dye may have some value as a differential stain for nerve cell bodies. Fibrillae in smooth muscle cells and cross striatums in skeletal muscle were also brought out.     

Metallic Lakes of the oxazines (Gallamin Blue, Gallocyanin and Coelestin Blue) as Nuclear Stain Substitutes for Hematoxylin

Becher's investigations upon the soluble metallic lakes of the oxazines have been re-investigated, extended and results described. Gallamin blue, gallocyanin and coelestin blue in combination with ferric ammonium sulfate gave the best results. The dyes are dissolved in a five per cent aqueous solution of ferric ammonium sulfate. The solution is boiled for 2-3 minutes, cooled, filtered and ready for immediate use. The iron lakes of these dyes stain nuclei excellently giving a deep blue or blue black in 3-5 minutes. No differentiation with acid is required. Coelestin blue gives the most stable solution and is recommended as a routine nuclear stain. The protoplasm remains practically colorless and counter-staining with acid dyes such as ethyl-eosin, orange G, or fuchsin gives pictures which cannot be distinguished from a good hematoxylin stain.

Counter-staining with van Gieson solution is also possible. Benda's modification of the van Gieson solution is recommended. Staining of fat with Sudan, scarlet red, etc., does not interfere with nuclear staining by these dyes.

As applied to the central nervous system these dyes are far superior to hematoxylin. Ganglion and glia cells are as excellently stained as with thionin.

The most widely used fixatives, namely formaldehyde, Mueller-formaldehyde, Zenker's and alcohol, give equally as good results. The nature of the staining process is briefly discussed and a prospectus offered.     

Metallic Lakes of the oxazines (Gallamin Blue,Gallocyanin and Coelestin Blue) as Nuclear Stain Substitutes for Hematoxylin

Becher's investigations upon the soluble metallic lakes of the oxazines have been re-investigated, extended and results described. Gallamin blue, gallocyanin and coelestin blue in combination with ferric ammonium sulfate gave the best results. The dyes are dissolved in a five per cent aqueous solution of ferric ammonium sulfate. The solution is boiled for 2–3 minutes, cooled, filtered and ready for immediate use. The iron lakes of these dyes stain nuclei excellently giving a deep blue or blue black in 3–5 minutes. No differentiation with acid is required. Coelestin blue gives the most stable solution and is recommended as a routine nuclear stain. The protoplasm remains practically colorless and counter-staining with acid dyes such as ethyl-eosin, orange G, or fuchsin gives pictures which cannot be distinguished from a good hematoxylin stain.

Counter-staining with van Gieson solution is also possible. Benda's modification of the van Gieson solution is recommended. Staining of fat with Sudan, scarlet red, etc., does not interfere with nuclear staining by these dyes.

As applied to the central nervous system these dyes are far superior to hematoxylin. Ganglion and glia cells are as excellently stained as with thionin.

The most widely used fixatives, namely formaldehyde, Mueller-formaldehyde, Zenker's and alcohol, give equally as good results. The nature of the staining process is briefly discussed and a prospectus offered.     

Trypan Blue as a Stain for Fungi

Trypan blue was tested for use in the study of fungi. It was found that it stained the cell wall of all fungi investigated. Two staining solutions were very satisfactory: (1) A quick-staining solution of 0.1-0.5% dye in 45% acetic acid; (2) A slow-staining solution of 0.1-0.5% dye in lactophenol. In both instances, heating the preparations quickened the action.