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.     

Keratin in the Egg Shells of Amphistomes; Histochemical Differentiation from Quinonetanned Protein in Other Trematoda

Various combinations of the oxidation method for demonstrating keratin in shell material of amphistomes were tried. Acidified permanganate worked more efficiently than performic and peracetic acids, and Alcian blue and aldehyde fuchsin excelled other basic dyes for subsequent staining. For the permanganate-Alcian blue reaction, sections of material fixed in Susa or Bouin were oxidized in 0.3% permanganate in 0.3% H₂SO₄ for 5 min., decolourized in 1% oxalic acid, stained in 3% Alcian blue in 2 N H₂SO₄ and counterstained with eosin. The shell globules stained a deep blue. For permanganate aldehyde fuchsin staining, the sections were stained in aldehyde fuchsin for 1 hr, after oxidation with permanganate. The shell globules then stained a deep magenta. The catechol and fast red reactions were negative in amphistomes and the specimens lack the characteristic amber colour due to quinone tanning.     

A Technic for Demonstrating Plasmodesma

Sections of potato tuber and epidermis from veins on the dorsal side of the sunflower leaf have proved most satisfactory in demonstrating plasmodesma. Kill fresh sections 5 minutes in a solution of 0.75 g. KI, 0.50 g. iodine in 100 cc. H₂O. Swell 5 minutes in 10% H₂SO₄. Mordant 5 minutes in a solution of 1.25 g. KI, 1.00 g. iodine in 100 cc. of 5% H₂SO₄. Wash in 5% H₂SO₄ until iodine starts to fade. Stain in a mixture of 0.5% gentian violet solution in 5% H₂SO₄ made up to a deep green color. Wash in a solution of 30 cc. glycerin, 2 g. ZnCl₂, 0.2 g. iodine, and a bit of KI in 60 cc. of water. Brush with a camel's hair brush, wash and mount in the same solution.     

Studies on Polychrome Methylene Blue II. Acid Oxidation Methods of Polychroming

The action of K₂Cr₂O₇, Ag₂O, KMnO₄, HgO and NaIO₃ in polychroming methylene blue is explored. The last two have no action in neutral or acid methylene blue solutions. With the other three reagents the amount of polychroming, as measured by the shift in the absorption spectrum, is roughly proportional to the amount of oxidant used. Various lots of methylene blue produce similar products with similar proportions of K₂Cr₂O₇. With similar quantities of this reagent similar products are produced by polychroming at 100°, 80°, 70° or 60° C. At 100° C. the action of K₂Cr₂O₇ or of Ag₂O appears to be completed in 15 minutes. In K₂Cr₂O₇ polychroming, H₂SO₄ can be substituted for HCl, and subsequent BaCO₃ neutralization removes the salts formed and prevents accidental alkali polychroming. K₂Cr₂O₇ polychroming produces products with narrower absorption bands than alkali polychroming.     

The Destruction of Cytoplasmic Basophilia with Mineral Acids

Cytoplasmic basophilia may be selectively destroyed by the mineral acids, HNO₃, HCl and H₂SO₄. Their specificity is similar to that of ribonuclease. The optimal conditions for their use are 3°C. for 16 hours at 2M concentrations. Removal of cytoplasmic basophilia as with ribonuclease, malt diastase and perchloric acid is most effective on sections prepared from tissues fixed in solutions containing no chromates. Under the conditions herein reported the mineral acids appear to be a satisfactory and economical substitute for ribonuclease or perchloric acid.     

Selective Staining of Neurosecretory Material in Semithin Epoxy Sections by Gomori's Aldehyde Fuchsin

The epoxy resin was removed from semithin (1 μm) sections by immersing them for 30 sec in sodium methoxide (Mayor et al., J. Biophys. Biochem. Cytol., 9: 909-10, 1961) and then processed as follows: (1) left for 1-3 hr at 60 C in a mixture of formalin, 25 ml; glacial acetic acid, 5 ml; CrO₃, 3 gm; and distilled water, 75 ml: (2) oxidized 10 min in a 1:1:6 v/v mixture of 2.5% KMnO₄, 5% H₂SO₄ and distilled water: (3) bleached in 1% oxalic acid, and (4) stained for 15 min in aldehyde fuchsin, 0.125% in 70% alcohol, or in a 1% aqueous solution of toluidine blue. The neurosecretory material is selectively stained.     

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.     

Thermogravimetric study of starch derivatives with amine/ammonium ion-exchanging groups in oxidative environment

The amine/ammonium materials were prepared by cross-linking of starch (S) with epichlorohydrin (E→SE) in the presence of ammonia (A→SEA) or choline (C→SEC–HO⁻) or with 1,3-bis-(3-chloro-2-hydroxypropyl)imidazolium hydrogensulphate (BCHIHS→SHI–HO⁻) and transfered into the acid/salt forms with HCl (SEA–HCl, SEC–Cl⁻, or SHI–Cl⁻), H₂SO₄ (SEA–H₂SO₄, SEC–HSO₄⁻, or SHI–HSO₄⁻), and H₃PO₄ (SEA–H₃PO₄, SEC–H₂PO₄⁻, or SHI–H₂PO₄⁻) and analyzed with thermogravimetry (TG) under dynamic and isothermal conditions in nitrogen or oxygen environment. According to the values of thermooxidation maxima (TM) calculated from the maximal difference of measured residues on the dynamic TG curves run in nitrogen and oxygen environments the order of decreasing thermooxidation resistance is: S>SEA–H₃PO₄>SHI–H₂PO₄⁻>SHI–HSO₄⁻>SEA–H₂SO₄>SEC–HSO₄⁻>SEC–HO⁻>SEA–HCl>HCl–Cl⁻> SEC–Cl⁻>SHI–HO⁻>SEA>SEC–H₂PO₄⁻>SE. The first-order rate constants calculated by the linear regression method (regression coefficient R>0.95) represented the initial rate constants for residue formation (k_r's) and gasification (k_g's). All the derivatives had greater values of rate constants than S and the k_g's were about 1000 times greater than k_r's. The values obtained in nitrogen were smaller than those calculated from runs in oxygen environment with the exception of S. Most of the salt forms had greater values of k_g's in oxygen environment. The activation energies (E's) were usually greater in nitrogen than in oxygen as well as for residue formation than for gasification. The SHI–HO⁻ sample had high k_g's and low E_g's in oxygen environment while for SHI–H₂SO₄⁻ the opposite was true. This we consider as two extremes for labile and resistant samples for gasification.     

Staining Rickettsiae in Yolk-Sac Cultures

Rickettsiae in yolk sacs are not stained well by the Macchiavello technique, and experiments were undertaken to understand the mechanisms involved. It was found that the citric acid destaining step was not effective and that most of the basic fuchsin was lost from the rickettsiae during the application of methylene blue, another basic dye. A staining technique was then evolved with carbol basic fuchsin in pH 7.45 phosphate buffer (0.4% dye, 0.4% phenol, 0.07 M buffer), followed directly by 0.8% aqueous malachite green oxalate. This technique worked well for R. mooseri, R. prowazeki, R. rickettsii, R. akari, and R. buretii, but for R. tsutsugumushi a modification was needed, whereby 4% aqueous Fe(NO₃)₃·9H₂O was used as destaining solution, and 0.5% aqueous fast green as the counter-stain.     

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.     

Stain Technology: A Phenylhydrazine-Leucofuchsin Sequence for Staining Nerves and Nerve Endings in the Integument of Poultry

This sequence for staining cutaneous nerves and nerve endings uses 1% formic acid as a fixative for 1 hr, followed by two treatments of 5 min each in 6% H₂SO₄. The tissue is then submerged in fresh 5% phenylhydrazine hydrochloride for 30 min, washed in running tap water for 10 min, and given a 5 min soak in distilled water. The specimen is placed in Lillie's “cold Schiff” reagent for 4 hr; transferred to 6% H₂SO₄, 4 changes of 5 min each; washed in distilled water, 3 changes of 5 min each; dehydrated in acetone, 4 changes of 10 min each; and cleared in 2 changes of methyl benzoate, the 1st for 1 hr and the 2nd until the tissue clears. Nerve fibers stain pinkish-purple; muscles also take up the stain, yet the nerves are discernible from the muscles. All other tissue remain unstained.     

A Simple Procedure for Crystallization of the Schiff Reagent

Formation of crystals in Schiff reagents prepared from SO₂ gas previously has been reported either soon after preparation, using high dye concentrations and heating, or after long periods of storage at room temperature. With the first type of procedure only a low yield of crystals accompanied by dye precipitation was obtained. Crystallization without dye precipitation took place if the reagent, prepared with pararosaniline base or chloride in a saturated SO₂ solution, was stored for a sufficient time at room temperature in partly filled flasks. These crystals remained colorless if washed with acid alcohol after being separated by filtration. Schiff reagents layered with paraffin oil or supplemented with 0.1 M hydroquinone took much longer to crystallize, suggesting that crystallizaticn is promoted by the partial oxidation of sulfurous acid to sulfuric acid. A high yield of crystals can be obtained at room temperature after as little as 24 hr by adding 0.04 M of H₂SO₄ to a Schiff reagent prepared with 2% pararosaniline chloride in a saturated SO₂ solution. A Schiff reagent prepared with only 0.2% of these crystals gives an intense staining in the Feulgen and in the Periodic acid-Schiff reactions.     

Maillet's Oso4-ZnI2 Fixative and Alcian Blue Staining in the Study of Neurosecretion; Invertebrates

Maillet's OsO₄-ZnI₂ fixation staining can be combined with a subsequent counterstaining by Alcian blue or aldehyde fuchsin to demonstrate neurosecretory cells in addition to cytological details of the nerve tissue. This technic has been applied to various annelids: Eisenia foetida (Oligochaeta), Erpobdella octoculata (Achaeta) and Nereis diversicolor (Polychaeta). The material is fixed in a 1:4 mixture of 2% OsO₄ and 3% ZnI₂ for 15 nr, embedded in paraffin, sectioned at 5 μ and the sections alternatively mounted on two glass slides. One of these is oxidized by a solution of 0.3% KMnO₄ acidified by 0.6% H₂SO₄ and counterstained with 1% Alcian blue, pH 0.2, the other one is mounted in balsam. The two preparations may then be compared to locate the neurosecretory cells among the other neurons shown on a slide treated only by the OsO₄-ZnI₂. Secretory cells are not stained by Maillet's reagent; except for their Golgi bodies and their cellular and nuclear membranes. The zone of grains which is generally strongly stained by the Alcian blue takes a yellowish hue from the OsO₄-ZnI₂ fixation. This method could be successfully applied to the histological controls in regeneration experiments. In these last ones, we must simultaneously observe the regeneration of the nervous fibres and the possibility of intervention of neurosecretory elements.     

Elimination of benzene from a benzyloxy ligand to form a carbonyl group on a tungsten-phosphine center

WH₃(OCH₂C₆H₅) (PMe₃)₄ (1) is formed upon reaction of WH₂(PMe₃)₅ with benzyl alcohol for 12 days at ambient temperatures. Thermolysis of 1 at 80°C in toluene solution gives the carbonyl complex, WH₂(CO)(PMe₃)₄ (2) and benzene. The conversion is slower in the presence of H₂. Reaction of 1 with D₂ leads to H/D exchange in the hydride ligands and in the benzylic and ortho-phenyl positions of the benzyloxide. A mechanism for the thermolysis of 1, based on an H₂ elimination, sequential C-H activations, and CO deinsertion from an acyl ligand, is proposed. Thermolysis of 1 is much faster in the presence of free benzyl alcohol and 2 is not formed. The products under these conditions are toluene, bibenzyl, WH₄(PMe₃)₄, PMe₃ and unidentified material, consistent with the intermediacy of benzyl radicals.     

凋落物去除和氮添加对亚热带阔叶林土壤不同组分碳、氮的影响

人类活动显著增加了氮沉降,对森林生态系统产生了不同程度的影响;凋落物在其分解过程中输入的大量有机碳、氮也会影响土壤碳氮的形成、稳定及转化.本研究选择亚热带常绿阔叶林,对样地进行8年氮添加[对照(0)、低氮(75 kg·hm^-2·a^-1)、高氮(150 kg·hm^-2·a^-1)]和控制凋落物处理(保留凋落物、去除凋落物),之后采集土壤样品,通过K₂SO₄、Na₂B₄O₇、Na₄P₂O₇、NaOH、H₂SO₄、Na₂S₂O₄、HF等化学试剂逐级浸提土壤,测定各浸提液和残渣中的碳、氮含量,研究凋落物及氮添加对土壤矿物结合态碳、氮的影响.结果表明: 整体上,胡敏素(humin,H)组分的土壤碳、氮含量均为最高,分别占土壤全量的33.5%和33.3%.Na₂B₄O₇溶液提取的土壤可溶性碳、氮含量最高,其次是NaOH和Na₄P₂O₇溶液,3种试剂提取的土壤可溶性总碳、可溶性总氮以及可溶性有机氮分别占提取总量的46.2%、47.9%和76.5%.与对照相比,氮添加增加了Na₂S₂O₄和H组分碳、氮含量;与保留凋落物比较,去除凋落物降低了Na₂B₄O₇、H₂SO₄、Na₂S₂O₄和H组分的碳含量,以及NaOH、HF和H组分的氮含量.保留凋落物和氮添加显著增加了K₂SO₄组分氮含量.可见,保留凋落物和外源氮通过影响化学稳定性不同的土壤组分的碳氮变化来改变土壤碳氮过程.     

外源H2O2对盐碱混合胁迫下裸燕麦幼苗生长和抗性生理的影响

为探讨信号分子过氧化氢（H₂O₂）增强裸燕麦盐碱耐性的作用及其生理机制,以裸燕麦品种‘定莜6号’为材料,在日光温室内用珍珠岩培养幼苗至三叶一心期时叶面喷施0.01 mmol·L^-1 H₂O₂的同时根部浇灌75 mmol·L^-1盐碱混合溶液（NaCl：Na₂SO₄：NaHCO₃：Na₂CO₃=12：8：9：1）或添加H₂O₂淬灭剂二甲基硫脲（DMTU）,研究对幼苗生长及叶片光合色素含量、活性氧代谢和渗透调节物质积累的影响。结果表明：喷施H₂O₂能够缓解盐碱混合胁迫对裸燕麦幼苗生长的抑制,提高幼苗根长、株高和植株干重及叶片叶绿素a、叶绿素b、总叶绿素、类胡萝卜素含量和超氧化物歧化酶、过氧化物酶、过氧化氢酶、抗坏血酸过氧化物酶活性,降低超氧阴离子、H₂O₂、丙二醛、抗坏血酸、谷胱甘肽和游离氨基酸含量,促进抗氧化物质类黄酮、总酚和原花青素及渗透调节物质可溶性蛋白质、可溶性糖和脯氨酸积累。添加DMTU部分或完全逆转了H₂O₂的上述作用。采用隶属函数综合评价显示,喷施H₂O₂提高了盐碱混合胁迫下裸燕麦幼苗的综合评价值D,添加DMTU完全逆转了H₂O₂对D值的提升作用。表明外源H₂O₂通过参与活性氧代谢和渗透调节物质积累等生理代谢调控缓解盐碱混合胁迫诱导的氧化伤害和生长抑制,从而提高裸燕麦对盐碱胁迫的适应能力。     

Gross Demonstration of the Mammalian Atrioventricular Bundle by A Periodic Acid-Schiff Procedure

The following procedure stains the atrioventricular conduction system selectively. (1) Wash the fresh heart with physiological saline solution to free it of blood; (2) fix it in 10% formalin containing 0.5% HIO₄ for 1 hr; (3) wash in 3 changes of distilled water for 20 min; (4) keep in 80% alcohol for 12 hr to 2 wk; (5) wash with distilled water; (6) treat with a dilute Schiff's reagent containing 0.1 gm of basic fuchsin per 100 ml for 0.5-2 min; (7) rinse in three changes of 2% Na₂SO₃ in 0.2 N HCI for 3-5 min; (8) wash and examine in 80% alcohol; store in 80% alcohol.     

The Use of a Chrome Alum-Gelatin (Subbing) Solution as a General Adhesive for Paraffin Sections

The adhesion obtained from a chrome alum-gelatin solution has been found far superior to results given by widely used general adhesives (Haupt's gelatin and Mayer's egg albumen) for paraffin sections. The subbing solution, which consists of 5.0 gin gelatin and 0.5 gm chrome alum per liter of water, is easier to apply and gives more consistent results. Sections affixed to subbed slides are resistant to removal by acids and bases: 1.0 and 0.1 N HCl or H₂SO₄, 1 M H₃PO₄, 5% oxalic and trichloroacetic acids, 1% and 10% lactic acid, 1.0 and 0.1 N NaOH or NH₄OH, and other fluids and solutions such as organic solvents, water, hypochlorite, KMnO₄ and thiosulfate. The applied adhesive is virtually unstained by many stains, including hematoxylin, eosin, fast green, safranin, PAS, Sudan IV and Mallory's triple stain. The only treatment yet found to detach affixed section in less than 6 hr is immersion in 5% trichloroacetic acid for 15 min at 100 C. The concentration of gelatin and chrome alum in the solution recommended is much lower than in previously described adhesives, but this does not seem to lessen its ability to affix the sections. If the concentrations of gelatin and chrome alum are decreased from those described, adhesive qualities are also decreased. An increase in the concentration of the ingredients causes the adhesive to become stained. The described solution therefore gives optimum adhesion and “resistance” to staining.     

A Comparison Of Iron Histochemical Methods For Use On Glycol Methacrylate Embedded Tissues

Four histochemical tests for iron and four procedures for its removal were investigated in regard to their suitability for glycol methacrylate embedded tissues. The HCl-ferrocyanide and chlorate hematoxylin methods were easily modified for plastic sections. The latter does not use iron-containing reagents. Desiderization was complete both after a fifteen minute exposure in 1% Na₂S₂O₄ in 0.1 M acetate-HCl buffer (pH 4.5) and, if an acid method is preferred, after twelve hours in 5% oxalic acid. A six hour treatment in 3.7 N H₂SO₄ also removed all histochemical iron but was accompanied by a relatively greater loss of tissue basophilia.     

Acid Fuchsin as a Stain—A Refinement in Manufacture

From the study of 38 samples of acid fuchsin prepared from several types of basic fuchsin and under varying conditions it is found that rosanilin sulfonated between 80° and 85°C. gives the best results in the Van Gieson staining technic. Staining tests also show that a satisfactory acid fuchsin will give the best results when employed with picric acid in the ratio of 1 part of the 1 per cent aqueous acid fuchsin to 20 parts of the aqueous picric acid. Details for the preparation and use of acid fuchsin are given.