A Sudan Black B Myelin Stain for Peripheral Nerves

Blocks of fresh issue were fixed 2 or more days in: cobalt sulfate (or nitrate), 1 gm; distilled water, 80 ml; 10% calcium chloride, 10 ml; and formalin, 10 ml. The fixed tissue was washed thoroughly in tap water, embedded in gelatin, frozen sections cut, and mounted on slides with gelatin adhesive. The sections were stained 15-30 min in a saturated, filtered solution of Sudan black B in 70% alcohol, differentiated in 50% alcohol under microscopic observation, and a cover glass applied with glycerol-gelatin. In thick (50-100 μ) sections, myelin stained green to gray-green and this allowed easy differentiation between nerves and other tissue elements.     

STAINING AND ACID PHOSPHATASE REACTIONS OF SPHEROSOMES IN PLANT TISSUE CULTURE CELLS

Spherosomes in plant tissue culture cells from normal sunflower stems and sunflower crown gall tumors reacted similarly to several nonfluorescent and fluorescent lipid dyes. Sudan IV and black B were good selective spherosome stains. The lipid fluorochromes, Nile blue and 3, 4-benzpyrene were excellent selective spherosome stains and visualized the smallest particles more readily than did Sudan IV. Spherosomes could not be seen in tissues stained with Sudan IV or 3,4-benzpyrene after ether-alcohol extraction. Acid phosphatase was detected on the spherosomes in both normal and tumor tissues using the lead sulfide precipitation and the post-incubation coupling procedures.     

Tannic acid, Iron hematoxylin, Alcian blue, and Basic fuchsin for staining islets and reticular fibers of the pancreas

In this technique alpha cells are stained by basic fuchsin, beta cells by iron-hematoxylin, reticular fibers by ferric tannate, and much by alcian blue. Among 6 commonly used fixatives tested, Bouin's fluid fixation (8-12 hr) gave the best staining results. Procedure: paraffin sections to water; 0.5% Li₂CO₃ to remove picric acid; 20% tannic acid, 15 min; wash well; 2-4 sec in 0.5% basic fuchsin containing 10% alcohol; rinse, then differentiate in 1% aniline in 90% alcohol until alpha cells are red and beta cells pink; 1% phosphomolybdic acid, 1 min; 5% hematoxylin in 2% iron alum, 0.5 min; wash well; 1% filtered alcian blue SGX, 15 sec; rinse, dehydrate, clear, and mount in synthtic resin. Results: reticular fibers, black; acinar cells, orange to gray; alpha cells, red; collagenous fibers, red; beta cells, gray granules; ducts, bluish-green. The method was tested on rat, rabbit, dog, hamster, cow and man.     

The Demonstration of a new nerve-cell Organoid

A method is described for the demonstration of a new nerve-cell organoid— the binary spheroid systems (Baker bodies). Zenker-formol or acetic-osmium-bichromate materials are postchromed at 37°C. and embedded in paraffin, sectioned and mounted in the ordinary manner. The sections are colored in a 70% alcohol solution of Sudan black B, rinsed in 70% alcohol, counter-stained in Mayer's carmalum and mounted in Farrant's medium. After examination the cover may be removed and the Sudan black extracted in 96% alcohol. The sections can then be restained by the azan method of Heidenhain, and other cytoplasmic inclusions can be correlated accurately to the functional states of the spheroid systems.     

Staining of depolymerised DNA in mammalian tissues with methyl violet 6B and crystal violet

The paper contains an account of DNA staining with basic dyes; methyl violet 6B and crystal violet in mammalian tissue sections after RNA extraction with cold concentrated phosphoric acid. The study shows that the best staining is obtained at pHs 2.5 and 3.5. Dehydration of stained nuclei is perfect when a mixture of absolute ethanol and n-butanol is used followed by treatment of sections in isoamyl or amyl alcohol. The in situ absorption data of nuclei stained with aqueous solution of methyl violet 6B as well as with crystal violet are also presented. Possible mechanism of staining as well as an explanation for dye-leaching when sections are dehydrated through ethanol are discussed.     

A modification of the tannic acid-phosphomolybdic acid-dye stain for demonstrating myoepithelial cells in formalin fixed tissue

A modified tannic acid-phosphomolybdic acid-dye procedure is used for staining myoepithelial cells in formalin fixed surgical and autopsy material. Paraffin sections are brought to water, mordanted for 1 hr in Bouin's fixative previously heated to 56 C, cooled while still in Bouin's, rinsed in tap water until sections are colorless, rinsed in distilled water, treated with 5% aqueous tannic acid 5-20 min, rinsed in distilled water 30 sec or less, treated with 1% aqueous phosphomolybdic acid 10-15 min, rinsed 30 sec in distilled water, rinsed in methanol, stained 1 hr in a saturated solution of amido black or phloxine B in 9:1 methanol:acetic acid, rinsed in 9:1 methanol:acetic acid, dehydrated, cleared and mounted. Myoepithelial cells of sweat, lacrimal, salivary, bronchial, and mammary glands are blue-green with amido black or pink with phloxine B. Fine processes of myoepithelial cells are well delineated. Background staining is minimal and the procedure is highly reproducible.     

Nuclear stains with soluble metachrome metal mordant dye lakes. The effect of chemical endgroup blocking reactions and the artificial introduction of acid groups into tissues.

Following our study on the effect of deoxyribonucleic acid (DNA) extraction on nuclear staining with soluble metal mordant dye lakes covering 29 dye lakes we chose a series of lakes representing the three groups: (1) readily prevented by DNA removal, (2) weakened by DNA extraction but not prevented, (3) unaffected by DNA removal, for application of other endgroup blockade reactions. The lakes selected were alum and iron hematoxylins, iron alum and ferrous sulfate galleins, Fe2+ gallo blue E, iron alum celestin blue B, iron alum fluorone black and the phenocyanin TC-FeSO4 sequence. Azure A with and without an eosin B neutral stain, was used as a simple cationic (and anionic) dye control. Methylation was less effective than with simple cationic dyes, but did weaken celestin blue, gallo blue E and phenocyanin Fe2+ nuclear stains. These dyes also demonstrate other acid groups: acid mucins, cartilage matrix, mast cells, central nervous corpora amylacea and artificially introduced carboxyl, sulfuric and sulfonic acid groups. Alum hematoxylin stained cartilage weakly and demonstrated sulfation and sulfonation sites. The iron galleins, iron fluorone black and acid iron hematoxylin do not. A pH 4 iron alum hematoxylin gave no staining of these sites; an alum hematoxylin acidified with 1% 12 N HCl gave weaker results. Deamination prevented eosin and orange G counterstains but did not impair nuclear stains with any of the mordant dye lakes. The simple acetylations likewise did not alter mordant dye nuclear staining, the Skraup reagent gave its usual sulfation effect on other tissue elements, but did not alter nuclear stains by mordant dyes. The mordant dyes do not bind to periodic acid engendered aldehyde sites and p-toluidine/acetic acid and borohydride aldehyde blockades did not alter mordant dye lake nuclear staining. Nitration by tetranitromethane, which blocks azo coupling of tyrosine residues, did not alter nuclear staining by the mordant dye lakes. Benzil at pH 13, which prevents the beta-naphthoquinone-4-Na sulfonate (NQS) arginine reaction and the Fullmer reaction of basic nucleoprotein, did not affect iron gallein, iron or alum hematoxylin stains of nuclei or lingual keratohyalin.     

Methods for the Demonstration of Lipid Applied to Compact Bone

Sections of compact bone were cut from the diaphysis of the femur, tibia, and humerus from dogs and monkeys. These sections were either ground thin and decalcified, or decalcified and subjected to frozen sectioning. Decalcification of the sections was effected by immersion in either Decal, 10% formic acid, 10% formic acid-sodium citrate (pH 4.5) or 20% aqueous EDTA. Sections were routinely stained with oil red O, Sudan black B, or Fettrot 7B. In addition, Nile blue A and phosphine 3R were also employed. Sections stained with phosphine were viewed with a fluorescence microscope. Control sections were extracted with lipid solvents prior to application of the staining procedures. The results indicate that lipid is present in compact bone within the osteocytes, lacunae, canaliculi, and organic matrix. The significance of the lipid in these sites, particularly extracellularly, is unknown.     

C. I. acid red 52: a new stain for cells of granulocytic origin

Using the xanthene dye C.I. acid red 52 (C.I. 45100) as a single agent stain applied to coverslip preparations of blood and bone marrow, primary and secondary granules in cells of neutrophilic origin stained brilliant pink. In eosinophils, granules stained dark red. In leukemic myeloblasts that also stained with Sudan black B and demonstrated myeloperoxidase and specific esterase activity, a few bright red staining granules were visualized with acid red 52. In some leukemic promyelocytes, Auer rods stained bright red. In leukemic lymphoblasts, no red granules were seen. Of a wide variety of dyes tested so far, acid red 52 is the most sensitive stain for primary and secondary granules of granulocytes in blood and bone marrow.     

C. I. Acid Red 52: A New Stain for Cells of Granulocytic Origin

Using the xanthene dye C.I. acid red 52 (CI. 45100) as a single agent stain applied to coverslip preparations of blood and bone marrow, primary and secondary granules in cells of neutrophilic origin stained brilliant pink. In eosinophils, granules stained dark red. In leukemic myeloblasts that also stained with Sudan black B and demonstrated myeloperoxidase and specific esterase activity, a few bright red staining granules were visualized with acid red 52- In some leukemic promyelocytes, Auer rods stained bright red. In leukemic lymphoblasts, no red granules were seen. Of a wide variety of dyes tested so far, acid red 52 is the most sensitive stain for primary and secondary granules of granulocytes in blood and bone marrow.     

The application of lipid-soluble stains in plastic-embedded sections

The present study was designed to develop a routine method for direct demonstration and precise localization of lipid substances in tissue sections. A panel of lipid-rich tissues was fixed in 4% buffered formaldehyde, infiltrated, and embedded in the water-soluble plastics Technovit 7100, EFL-67, and JB-4. The use of alcohol containing fluids was avoided. Staining with the lipid-soluble dyes Sudan Black B and Oil Red O revealed excellent preservation of tissue lipids in Technovit 7100 embedded sections when compared with cryostat sections of the same tissue specimens. Lipid preservation in EFL-67 and JB-4 embedded sections was inconsistent, even when infiltration and polymerization procedures were performed at 4 degrees C. Combination of lipid-soluble dyes with the periodic acid Schiff, Jones' methenamine silver, or Gomori' reticulin method allowed for an exact localization of lipids in high-quality Technovit 7100 embedded sections. The procedure herein is easily applicable in routine histopathology practice.     

The Use of Sudan Black B as a Bacterial Fat Stain

Sudan black B was introduced as a specific fat stain for the detection of lipids in tissue sections by L. Lison in 1934. Saturated solutions of Sudan black B in 70% alcohol or in ethylene glycol stain the fat bodies of bacteria a deep blue-black color, and this dye is recommended as superior to the other Sudans.

The method used in staining the bacteria was to suspend a loopful of the cells in a drop of the stain solution and to prepare flat wet mounts. The organisms giving positive fat tests with Sudan black B included Bacillus cereus, Bacillus mycoides, Azotobacter beijerinckii, Rhizobium leguminosarum, Mycobacterium avium, Mycobacterium leprae, Oospora lactis, Bacillus tumescens, water spirilla, and fungi.     

A Modification of the Tannic Acid Phosphomolybdic Acid-Dye Stain for Demonstrating Myoepithelial Cells in Formalin Fixed Tissue

A modified tannic acid-phosphomolybdic acid-dye procedure is used for staining myoepithelial cells in formalin fixed surgical and autopsy material. Paraffin section are brought to water, mordanted for 1 hr in Bouin's fixative previously heated to 56 C, cooled while still in Bouin's, rinsed in tap water until sections are colorless, rinsed in distilled water, treated with 5% aqueous tannic acid 5-20 min, rinsed in distilled water 30 sec or less, treated with 1% aqueous phosphomolybdic acid 10-15 min, rinsed 30 sec in distilled water, rinsed in methanol, stained 1 hr in a saturated solution of amido black or phloxine B in 9:l methanol:acetic acid, rinsed in 9:l methanol:acetic acid, dehydrated, cleared and mounted. Myoepithelial cells of sweat, lacrimal, salivary, bronchial, and mammary glands are blue-green with amido black or pink with phloxine B. Fine processes of myoepithelial cells are well delineated. Background staining is minimal and the procedure is highly reproducible.     

Adaptation of the Millon, Sudan Black B, and Periodic Acid-Schiff Technics for Block Staining of Insect Tissues

Spermatophores and reproductive systems of the beetle, Lytta nuttalli Say, fixed in Bouin's aqueous picroformol or buffered 10% neutral formol were stained in toto by the Millon, Sudan black B and periodic acid-Schiff reactions as follows. Millon: after excess fixative is removed in 70% ethanol, specimens are brought to water, stained in Millon's reagent at 60 C for 1 hr, rinsed in 2% aqueous nitric acid at 40-50 C, dehydrated rapidly, cleared, embedded and sectioned as usual. Sudan black B: specimens are taken to absolute ethanol, stained in a saturated solution of Sudan black B in absolute ethanol at room temperature for 24-48 hr, rinsed and cleared in xylene, embedded and sectioned. PAS: specimens are brought to water, oxidized in 0.5 aqueous HIO₄ at 37 C for 30 min, washed in 2 changes of water, stained in Schiif reagent at room temperature for 1 hr, rinsed in 3 changes of 0.5% aqueous potassium metabisulfite, washed in running water for 10-15 min, dehydrated, cleared, embedded and sectioned. All 3 methods produced their characteristic staining in specimens up to 3 mm thick     

A Differential Staining Method for Elastic Fibers, Collagenic Fibers, and Keratin

A method allowing for the differential presentation of elastic fibers, other connective tissue fibers, epithelial and other types of cytoplasm, and keratin is described. The procedure is based on the affinity of orcein for elastic fibers, of anilin blue for collagenic material, and of orange G for keratin. Bouin-fixed, tissue-mat embedded sections are stained in Pinkus' acid orcein for 1 1/2 hours and rinsed in distilled water. The sections are differentiated in 50% alcohol containing 1% hydrochloric acid, washed in tap and then in distilled water. The sections are next transferred for I to 2 minutes to the anilin blue, orange G, phosphomolybdic acid combination known as solution No. 2 of Mallory's connective tissue stain, diluted 1:1 with distilled water. They are then rinsed in distilled water, quickly passed into 95% alcohol, and dehydrated in absolute alcohol containing some orange G, after which they are cleared and mounted. Within less than two hours sections may be stained and mounted with the following results: elastic fibers — red; collagenic fibers — blue; muscle fibers — yellow; keratin — orange.     

The application of lipid-soluble stains in plastic-embedded sections

Summary The present study was designed to develop a toutine method for direct demonstration and precize localisation of lipid substances in tissue sections. A panel of lipid-rich tissues was fixed in 4% buffered formaldehyde, infiltrated, and embedded in the water-soluble plastics Technovit 7100, EFL-67, and JB-4. The use of alcohol containing fluids was avoided. Staining with the lipid-soluble dyes Sudan Black B and Oil Red O revealed excellent preservation of tissue lipids in Technovit 7100 embedded sections when compared with cryostat sections of the same tissue specimens. Lipid preservation in EFL-67 and JB-4 embedded sections was inconsistent, even when infiltration and polymerization procedures were performed at 4°C. Combination of lipid-soluble dyes with the periodic acid Schiff Jones' methenamine silver, or Gomori' reticulin method allowed for an exact localization of lipids in high-quality Technovit 7100 embedded sections. The procedure herein is casily applicable in routine histopathology practice.     

Simultaneous Demonstration of Lipids and Starch in Plant Tissues

A rapid and easy technique for the simultaneous demonstration of lipids and starch in the same histological section is described. Tissues are prepared by the clamical fixing and Araldite in embedding techniques of election microscopy. Semithin sections are directly stained for 1 hour at 60C with saturated Sudan black B in 70% ethanol without removing the embedding resin. Lipids stain black; stain is shown as white grains contrasting with the blue-grey embedding resin.     

Simultaneous demonstration of lipids and starch in plant tissues.

A rapid and easy technique for the simultaneous demonstration of lipids and starch in the same histological section is described. Tissues are prepared by the classical fixing and Araldite M embedding techniques of electron microscopy. Semithin sections are directly stained for 1 hour at 60C with saturated Sudan black B in 70% ethanol without removing the embedding resin. Lipids stain black; starch is shown as white grains contrasting with the blue-grey embedding resin.     

Taenia taeniaeformis: early inflammatory response around developing metacestodes in the liver of resistant and susceptible mice II. Histochemistry and cytochemistry

Female BALB/cJ (resistant), C3H/HeJ (intermediate resistant), and C3H/HeDub (susceptible) inbred mice, 4-5 wk old, were infected with Taenia taeniaeformis. Liver sections were stained for the enzymes acid phosphatase, beta-glucuronidase, and peroxidase. Eosinophils present around the parasite were identified by the ethanolic Congo red method. Possible gross changes in lipid metabolism in the hepatocytes surrounding the parasite were investigated with the Sudan black B method. The results of observations made by light microscopy were: (1) beta-glucuronidase activity above background levels was observed only in the hepatocytes around the parasite in BALB/cJ mice at 4, 5, and 6 days postinfection (PI); no reaction was observed in the other 2 strains of mice studied; (2) acid phosphatase activity was very strong at 2, 3, and 4 in the 3 strains of mice while this reactivity was weak at 5 and 6 days PI; (3) the cytoplasm of the hepatocytes around the metacestode stained more heavily with Sudan black B than other hepatocytes; and (4) the presence of eosinophils appearing at 3 days PI around the parasite in all 3 strains of mice was demonstrated by staining with Sudan black B, the substrate of peroxidase, and Congo red. Infected C3H/HeJ and BALB/cJ mice had higher numbers of liver eosinophils than infected C3H/HeDub mice throughout the observation time. The present results suggest 2 conclusions: (1) a parasite-liver interaction occurs as is evident by hepatocyte changes in beta-glucuronidase activity and Sudan black B staining, and (2) resistance to the early stages of T. taeniaeformis is associated with the appearance of eosinophils.     

Picrosirius Red Staining of Cardiac Muscle Following Phosphomolybdic Acid Treatment

When the picrosirius red technique was applied to cardiac muscle sections, intense yellow myocyte staining sometimes obscured thin collagenous septa. The picrosirius red technique was modified to include treatment of the sections in 0.2% (w/v) aqueous phosphomolybdic acid prior to staining. With 1-5 min treatment, cytoplasmic staining was eradicated; diminution of collagen staining occurred only with long treatments at much higher concentrations of phosphomolybdic acid. Using this phosphomolybdic acid-picrosirius red technique, collagenous septa as thin as 0.2-0.5 /im and fine collagen fibers making up the septa were clearly discernible. The technique also worked well on sections stained by other techniques and then destained. The phosphomolybdic acid-picrosirius red technique should be useful in experiments designed to investigate the effects of collagen distribution on the electrical and mechanical behavior of cardiac muscle.