Modified Whipf's Polychrome: A Connective Tissue Stain with Special Application for Demonstrating Leishmania

A polychrome stain procedure was developed to demonstrate amastigotes of the protozoan parasite Leishmania braziliensis as well as cytoplasmic and other tissue components in cutaneous lesions of infected animals. The procedure is as follows: stain nuclei for 10 minutes with an iron hematoxylin containing 0.5% hematoxylin and 0.75% ferric ammonium sulfate dissolved in 1:1 0.6 N H₂SO₄:95% ethanol; rinse 4 minutes in distilled water. Cytoplasmic staining is achieved by exposing tissues for 10 minutes to a solution containing 0.25% Biebrich scarlet, 0.45% orange G, 0.5% phosphomolybdic acid and 0.5% phosphotungstic acid in 1% aqueous acetic acid. These first two solutions are modified from Whipf's polychrome stain. Sections are differentiated for 10 seconds in 50% ethanol, rinsed in water, stained 3 minutes in 0.1% aniline blue WS in saturated aqueous picric acid, rinsed in water and differentiated for 1 minute in absolute ethanol containing 0.05% acetic acid. Mordanting overnight in 6% picric acid in 95% ethanol produced optimal results.

This procedure was applied to sectioned material from experimental animals with various protozoa. Trypanosoma cruzi, Besnoitia Jellisoni, Toxoplasma gondii and especially Leishmania braziliensis were well demonstrated. Combining cytoplasmic dyes and phosphomolybdic-phosphotungstic acids into one solution afforded differential staining of tissues by Biebrich scarlet and orange G; connective tissues were stained by this solution. Substantially improved definition of connective tissues resulted after counterstaining. This procedure differs from the Massou sequence in which connective tissues are first stained by cytoplasmic dyes along with other tissues and then destained prior to specific counter-staining. in comparing dyes structurally related to Biebrich scarlet, it was found that Crocein scarlet MOO, but not Poncenu S, was an acceptable substitute. Sirius supra blue GL and Sirius red FSBA were not useful as replacements for aniline blue WS in this procedure.     

A Simplified Stain for Hemoglobin in Tissues Or Smears Using Patent Blue

The following technic, based on the patent blue V hemoglobin reaction, is useful for identifying hemoglobin in tissue fixed in neutral formaldehyde solution and embedded in paraffin:

Stain the deparaffinized, hydrated sections 3 to 5 minutes in the working reagent, prepared by adding 2 ml. of glacial acetic acid and 1 ml. of 3% hydrogen peroxide to 10 ml. of the filtered stock solution (1 g. patent blue, 10 g. zinc powder, and 2 ml. glacial acetic acid). Counterstain 30 to 60 seconds in 1:1000 safranin solution in 1% acetic acid, rinse, dehydrate with alcohols, clear in xylene and mount in clarite. Total time required, 37 minutes.

Blood and tissue and smears may be stained, following fixation in methyl alcohol, by applying the working reagent as above.     

Methylene Violet (Bernthsen), by Zinc-Alkali-Chlorate Hydrolysis of Methylene Blue

Though Bernthsen's methylene violet (MV) is a common constituent of polychrome methylene blue, the hydrolytic oxydation of methylene blue to yield azure-free MV has been considered a difficult chemical reaction since the time of Bernthsen, who used Ag₂O in the hydrolysis. MV is qualitatively distinguished from azures by Bernthsen's criteria and the author's new tests: (1) light-excited isomeric change, (2) reactivity to acidity, (3) reaction with KCIO, and (4) reaction with Na₂SO₃ of azures in CHCI₃, while MV gives none. But MV shows (5) indicator properties at pH 4, while azures do not. For practical hydrolysis, treat methylene blue (10 parts by weight) and KCIO₃ (1 part) with 1-2 N NaOH to convert methylene blue to a mixture of MV and azures. Then dilute the solution, add a Zn salt and NaHCO₃ in excess of the amount needed to convert the NaOH to Na₂CO₃. Boil the solution gently for 1-2 hr. The end point of the reaction is found by pipetting a drop of reactant into 3% acetic acid in a test tube, adding CHC1₃ and extracting. The acetic layer should then be almost colorless while the CHC1₃ is colored intensely cherry red. After cooling, the precipitated dye is filtered and dried. This procedure gives good yields of a dye which meets the criteria given by Bernhsen. The peak of the absorption curve in solution, pH 4-11, is at 624 mμ (Bernthsen 625 mμ) and in acid solution, pH 0-4, 588 mμ (Biological Stains, 1953; 580 μ). The dye contains so little azures, that purification of the MV fraction obtained from the reaction mixture is unnecessary when it is used in the Wright-type Romanowsky stain. The remarkable staining effect of MV is its power to bring out red azurophil granules of monocytes and lymphocytes when used with eosinated thiazins in Wright's stain.     

Initial and Persisting Staining power of Solutions of Iron-Hematoxylin Lake

A study was made of factors affecting the initial staining power and the stability of iron-hematoxylin lake solutions. The findings were applied to the preparation of a superior hematoxylin staining solution. This is made up as follows: in 50 ml. water dissolve, in order, 1.0 g. ferric ammonium sulfate [FeNE₄ (SO₄)₂⋅ 12H₂O], 0.8 ml. sulfuric acid, 50 ml. 95% ethyl alcohol, 0.5 g. hematoxylin. Filter the solution to remove the insoluble, white crust of the ferric ammonium sulfate. The solution stains well ten minutes after it has been made. Peak performance is attained within 5 hours, and is maintained for 4 to 8 weeks. Staining time is 3 to 30 minutes. Excess stain can be rinsed off the slide and section by immersion in water, after which destaining, if necessary, can be accomplished with a solution of 50 ml. water, 50 ml. 95% ethyl alcohol, 0.18 ml. sulfuric acid. The slides may or may not be placed next in a neutralizing solution of 50 ml. water, 50 ml. 95% ethyl alcohol, 0.5 g. sodium bicarbonate. They may then be passed through 50 ml. water, 50 ml. 95% ethyl alcohol on the way to alcoholic counterstaining solutions, or through water leading to aqueous counterstains.

The nuclear stain produced is black, intense and very sharp and has proved to be consistently excellent on a variety of animal and human tissues following a number of different fixatives.     

Histochemical 1,2-Glycol Cleavage with Acetates of Manganese

Both manganic triacetate and tetraacetate will act to split the 1,2-glycol linkage to produce aldehyde radicals which may be demonstrated with either leucofushsin or hydrazine compounds. Thirty to sixty minutes oxidation in a saturated glacial acetic acid solution of either acetate will usually be sufficient to produce satisfactory histochemical localization of the 1,2-glycol linkage. The tetraacetate is the more reliable of the two compounds, giving good localizations more constantly than the triacetate. Present studies indicate that the histological picture after such staining is identical with the localizations given by lead tetraacetate. The principle value of these reagents in glycol cleavage is essentially academic although, since their mechanism of cleavage is believed to differ from the established periodic acid and lead tetraacetate technics, there is the probability that they may be of value in comparisons between the two types of reaction.     

Histochemical 1,2-Glycol Cleavage with Acetates of Manganese

Both manganic triacetate and tetraacetate will act to split the 1,2-glycol linkage to produce aldehyde radicals which may be demonstrated with either leucofushsin or hydrazine compounds. Thirty to sixty minutes oxidation in a saturated glacial acetic acid solution of either acetate will usually be sufficient to produce satisfactory histochemical localization of the 1,2-glycol linkage. The tetraacetate is the more reliable of the two compounds, giving good localizations more constantly than the triacetate. Present studies indicate that the histological picture after such staining is identical with the localizations given by lead tetraacetate. The principle value of these reagents in glycol cleavage is essentially academic although, since their mechanism of cleavage is believed to differ from the established periodic acid and lead tetraacetate technics, there is the probability that they may be of value in comparisons between the two types of reaction.     

Concurrent Demonstration of Desoxyribose Nucleic Acid and 1,2-Glycols

Sections from rat tissue were fixed in 10% formalin in 90% alcohol and placed in a 1.0% suspension of sodium bismuthate (NaBiO₃) in 40% phosphoric acid for 40 minutes at room temperature. Bismuth phosphate crystals were removed with 2N HCl. The sections were next placed in the Schiff reagent for 20 minutes. By this method the DNA was hydrolyzed by the phosphoric acid and the 1,2-glycols were oxidized by the NaBiO₃. In both cases aldehyde groups were released and subsequently stained by the Schiff reagent. A photomicrograph is included demonstrating the nuclei, goblet cells, striated border and basement membrane stained by this combined method.     

The Fixing and Staining of Liriodendron Tulepifera Root Tips and their Mycorrehizal Fungus

Root tips of Liriodendron Tulipifera forming with a fungus of the Mycelium Radicis group an endotrophic mycorrhiza, were subjected to several different fixations in which the action of cationic chromium and anionic chromium were compared. Anionic chromium in the form of chromic acid was combined with several substituted benzene compounds while cationic chromium in the form of chromic sulfate (Cr₂(SO₄)₃·15 H₂O) in 4% formaldehyde (HCHO) was used with the same ring compounds. In addition, several fixatives not containing chromium were tested.

Five percent chromic sulfate (Cr₂(SO₄)₂·15 H₂O) in 4% formaldehyde (HCHO) or in 1% osmic acid with saturated aqueous salicylic and/or picric acid preserved the histological and cytological details of the mycorrhiza, as was clearly demonstrated when followed by staining in 3% acetic acid saturated with orseillin BB and counterstained with 1% crystal violet in clove oil.

Two percent ferric chloride (Fe Cl₃) in 4% formaldehyde showed the relationship of the tannin contents of the cells to the invading hyphae when followed by suitable staining.

Cationic chromium appeared to be superior to anionic chromium in preserving cell walls as well as the general histologic features of the material investigated.     

The Use Of Lead Tetraacetate,Benzidine, O-Dianisidine and A “Film Test” In Investigating The Periodic-Acid-Schiff Technic

In order to obtain a better understanding of the “periodic-acid-Schiff” reaction, also known as the “periodic-acid fuchsin-sulfurous-acid” reaction, three types of investigations were carried out

1) The Schiff reagent was replaced by other aldehyde reagents: benzidine or o-dianisidine. There was no significant change in the histological distribution and intensity of the reactions occurring after periodic acid oxidation.

2) Periodic acid was replaced by another oxidizing agent: lead tetraacetate (dissolved in acetic acid). There was no significant change in the histological distribution of the reactions with the Schiff reagent, but some change in their intensity. It was concluded that 1,2-glycols and a-amino alcohols play the main role in the reactions with both oxidants. The presence of α-hydroxy acids in some types of mucous cells is suggested by the results with lead tetraacetate.

Incidently, glycogen and starch are not sufficiently oxidized by lead tetraacetate (in acetic acid) at room temperature to give positive reactions with the Schiff reagent, while cellulose and other periodic-acid-Schiff reactive substances are.

3) The staining of films of presumed reactive substances with the periodic-acid-Schiff technic C O the intense reactivity of many polysaccharides, mucopolysaccharides and mucoproteins, but not of ordinary proteins. (Hyaluronic and chondroitin sulfuric acid are, however, not reactive in vitro).

In conclusion, the periodic-acid-Schiff technic consists of an oxidation of 1,2-glycols and a-amino alcohols to produce aldehyde groups, which are then stained by the Schiff reagent. The “film test” reveals that these radicals are present in certain polysaccharides, mucopolysaccharides and mucoproteins.     

Staining Nerve Fibers After Sublimate-Acetic and After Bouin'S Fluid

A silver staining method for paraffin sections of material fixed in HgCl₂, sat. aq., with 5% acetic acid is as follows. Process the sections through the usual sequence of reagents, and including I-KI in 70% alcohol, thiosulfate (5% aq.), washing and back to 70% alcohol containing 5% of NH₄OH (conc. aq.). After 3 minutes in the ammoniated alcohol, wash through tap water and 2 changes of distilled water and silver 5-10 minutes at 25°C. in 15% AgNO₃ aq. to which 0.02 ml. of pyridine per 100 ml. has been added. Blot the slide, but not the section and do not rinse. Reduce at 45°C. in 0.1% pyrogallol in 55% alcohol, then rinse in 55% alcohol and wash in water. The remainder of the process consists of gold toning, intensifying in oxalic acid, fixing in 5% Na₂S₂O₃, washing, dehydrating, clearing and covering. When the specimen contains much smooth muscle, the I-KI solution is acidified before use by adding 2 ml. of 1N nitric acid per 100 ml., and the sections treated for 3 minutes instead of the usual 2 minutes. Formalin should not be added to sublimate-acetic, but specimens that do not contain strongly argyrophilic nonneural tissue may be fixed in formalin or, preferably, Bouin's fluid. Sections of tissue after the latter type of fixation will not require the I-KI and thiosulfate but can go from 95% alcohol to the ammoniated alcohol. The advantages of fixing in HgCl₂-acetic acid are suppression of the staining of connective tissue and intensifying the staining of nerve fibers.     

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.     

淋洗法修复化工厂遗留地重金属污染土壤的可行性

通过室内模拟试验,采用振荡淋洗的方法研究了蒸馏水、HCl、H₃PO₄、草酸、CaCl₂等淋洗剂对化工厂遗留地污染土壤中重金属的淋洗效果,探讨了淋洗剂浓度、淋洗时间、淋洗次数等对淋洗效果的影响,并研究了HCl处理前后土壤中重金属形态的变化.结果表明：蒸馏水、H₃PO₄、CaCl₂等对Cr、Pb、Zn、Cu、Cd的去除率较低,大多数条件下重金属去除率均在1%以下,最大去除率仅为3.58%.2 mol·L^-1 HCl在土水比为1∶3、反应时间为1 h、2次淋洗的条件下可以达到最佳淋洗效果,Cr、Pb、Zn、Cu、Cd的去除率分别达到80.75％、88.69％、98.00％、79.33％和95.52％.形态分析结果表明,HCl能有效去除土壤中各种结合形态的重金属.     

Histochemical Use of Sodium Bismuthate

Histochemical 1,2-glycoI cleavage, similar to that obtained with periodic acid and lead tetraacetate, may be obtained with sodium bismuthate. Routinely prepared slide sections, from tissues fixed in 10% formalin, are run down through xylene and graded alcohols to water and then oxidized for three minutes in a 1% sodium bismuthate 20% aqueous phosphoric acid solution. The oxidizing solution must be freshly prepared and used immediately. Following oxidation, sections are rinsed 15 sec. in IN HC1 to remove bismuth pentoxide precipitate, a by-product of the reaction. The sections are then washed in distilled water and placed in leuco-fushsin for 10 min., or in a saturated 30%) alcoholic solution of p-nitrophenylhydrazine for 5 min. or 2,4-dinitrophenylhydrazine for 30 minutes. After staining, the sections are rinsed in 30% alcohol if the nitrophenylhydrazines were used, or in the standard dilute sulfite bath followed by running tap water for 5 min. if leucofuchsin were used. Sections are routinely dehydrated, cleared, and covered. On examination, the sites of 1,2-glycol linkages will be stained violet by leucofushsin or yellow by the nitrophenylhydrazines.     

Histochemical Use of Sodium Bismuthate

Histochemical 1,2-glycoI cleavage, similar to that obtained with periodic acid and lead tetraacetate, may be obtained with sodium bismuthate. Routinely prepared slide sections, from tissues fixed in 10% formalin, are run down through xylene and graded alcohols to water and then oxidized for three minutes in a 1% sodium bismuthate 20% aqueous phosphoric acid solution. The oxidizing solution must be freshly prepared and used immediately. Following oxidation, sections are rinsed 15 sec. in IN HC1 to remove bismuth pentoxide precipitate, a by-product of the reaction. The sections are then washed in distilled water and placed in leuco-fushsin for 10 min., or in a saturated 30%) alcoholic solution of p-nitrophenylhydrazine for 5 min. or 2,4-dinitrophenylhydrazine for 30 minutes. After staining, the sections are rinsed in 30% alcohol if the nitrophenylhydrazines were used, or in the standard dilute sulfite bath followed by running tap water for 5 min. if leucofuchsin were used. Sections are routinely dehydrated, cleared, and covered. On examination, the sites of 1,2-glycol linkages will be stained violet by leucofushsin or yellow by the nitrophenylhydrazines.     

Histochemical Use Of Lead Tetra-Acetate I. Cleavage Of 1-2 Glycols

Lead tetra-acetate acts specifically to split the carbon-carbon single bond of the 1,2-glycol linkage to produce aldehyde radicals which may then be demonstrated by means of leucofuchsin, 2,4-dinitrophenlyhydrazine, or p-nitrophenylhydrazine. Routinely prepared slide sections from tissues fixed in 10% formalin are run down to 95% alcohol, rinsed in glacial acetic acid and then treated for 2 minutes in a saturated solution of lead tetra-acetate in glacial acetic acid with 5 g. of potassium acetate added for each 100 ml. of reagent. The sections are then washed in distilled water and placed in leucofuchsin for 10 minutes, or in a saturated 30% alcoholic solution of p-nitrophenylhydrazine for 5 minutes or 2,4-dini-trophenylhydrazine for 30 minutes. After staining, the sections are rinsed in 30% alcohol if the nitrophenylhydrazines were used, or in the standard dilute sulfite bath followed by running tap water for 5 minutes if leucofuchsin were used. Sections are routinely dehydrated, cleared, and covered. On examination, the sites of 1,2-glycol linkages will be stained violet by leucofuchsin or yellow by the nitrophenylhydrazines.     

The Feulgen Reaction after Glutaraldehyde Fixation

A technique is described for performing the Feulgen reaction for DNA on cells and tissues fixed in glutaraldehyde. Blockade free aldehydes by reducing them with fresh 0.5% NaBH₄ in 1% NaH₂PO₄ for 1 hr at room temperature, then rinse in water. Follow by a Feulgen reaction (hydrolysis at room temperature in 6 N HCI for 20 min, Schiff's reagent for 60 min.). Controls assure the completeness and irreversibility of the borohydride blockade. Cytophotometry shows that the DNA content per nucleus is unaffected by the blockade procedure.     

Bromphenol Blue as A Stain for Muscle Striations

Sections from formalin-fixed muscle are stained 4-24 hr with a 0.05% solution of bromphenol blue in 2% acetic acid, rinsed with water and placed in 0.5% acetic acid for 5-10 min. They are then treated 30 sec with tap water substitute (KHCO₃, 0.2 gm; MgSO₄, 2 gm; distilled water, 100 ml), rinsed, dehydrated in alcohol, cleared in xylene and covered in a polystyrene mountant Striatums of both cardiac and skeletal muscle fibers are fully resolved under oil immersion, against the blue background of the other parts of the fibers.     

Staining Tissue of the Central Nervous System with Luxol Fast Blue and Neutral Red

The tissue is fixed in 10% neutral saline formalin for 1 day to 3 wk depending on the size of the block, dehydrated and embedded in paraffin. The sections are stained at 57° C for 2 hr, then at 22° C for 30 min, in a 0.0125% solution of Luxol fast blue in 95% alcohol acidified by 0.1% acetic acid. They are differentiated in a solution consisting of: Li₂CO₃, 5.0 gm; LiOH-H₂O, 0.01 gm; and distilled water, 1 liter at 0-1° C, followed by 70% alcohol, and then treated with 0.2% NaHSO₃. They are soaked 1 min in an acetic acid-sodium acetate buffer 0.1 N, pH 5.6, then stained with 0.03% buffered aqueous neutral red. Sections are washed in distilled water, 1 sec, then treated with the following solution: CuSO₄·5H₂O, 0.5 gm; CrK(SO₄)₂·12H₂O, 0.5 gm; 10% acetic acid, 3 ml; and distilled water, 250 ml. Dehydration, clearing and covering complete the process. Myelin sheaths are stained bright blue; meninges and the adventitia of blood vessels are blue; red blood cells are green. Nissl material is stained brilliant red; axon hillocks, axis cylinders, ependyma, nuclei and some cytoplasm of neuroglia, media and endothelium of blood vessels are pink.     

Staining Nuclei and Chromosomes in Protozoa

Technics for free-living forms such as Paramecium and for parasitic forms such as the opalinid ciliates are described.

Paramecium: Fix paramecia in hot Schaudinn's fluid containing 5% of glacial acetic acid for 5-15 minutes. (A hot water bath for maintaining the proper temperature of the fixative is described.) Dehydrate up to 83% alcohol. Mount the specimens on albuminized cover glasses. (A table for mounting animals on cover glasses is described.) Apply a thin layer of collodion to the cover glass to prevent the loss of the specimens during the subsequent handling. Pass through descending grades of alcohol to water. Mordant in 4% iron alum for 24 hours. Stain in 0.5% hematoxylin for 24 hours. Destain in saturated aqueous picric acid. Rinse in tap water, expose to ammonia vapor for a second, and then rinse again in tap water. Wash in running water for 1 hour. Dehydrate. Clear, then mount in damar.

Opalinid Ciliates: Make smears on cover glasses and fix them while wet. If the opalinids are to be subsequently stained in hematoxylin, fix in hot Schaudinn's fluid (containing 5% of glacial acetic acid) for 5-15 minutes. Pass through descending grades of alcohol to water. Mordant in iron alum for 24 hours. Stain in hematoxylin for 24 hours. Destain in saturated aqueous picric acid. For Feulgen reaction, fix in a modified weak Flemming's fluid for 1 hour. Wash in running water for 30 minutes. Hydrolyze. Leave 3 hours in fuchsin decolorized with H₂SO₃ (Feulgen formula). Wash in H₂SO₃, then in running water for 15 minutes. Dehydrate up to 95% alcohol. Counterstain with fast green FCF for 2 minutes. Dehydrate in absolute alcohol. Clear, then mount in damar.     

Determination of 17-oxosteroid glucuronides and sulfates in urine and serum by fluorescence high-performance liquid chromatography using dansyl hydrazine as a pre-labeling reagent

A fluorescence high-performance liquid chromatographic method is described for the direct determination of conjugated 17-oxosteroids in biological fluids without hydrolysis. Conjugated 17-oxosteroids are extracted with Sep-Pak C₁₈ cartridge, labeled with dansyl hydrazine in trichloroacetic acid—benzene solution and then separated by high-performance liquid chromatography on reversed-phase μBondapak C₁₈ column using 0.01M sodium acetate in methanol—water—acetic acid (65:35:1, v/v) as the mobile phase. The eluate is monitored by a fluorophotometer at 365 nm (excitation) and 520 nm (emission). Linearities of fluorescence intensities (peak heights) with the amounts of various conjugated 17-oxosteroids were obtained between 10 pmol and 100 pmol. This method is sensitive, reliable and useful for the simultaneous determination of conjugated 17-oxosteroids in urine and serum.