Differential Staining of Yeast for Purified Cell Walls, Broken Cells, And Whole Cells

Intact yeast cells are Gram positive but broken or disrupted cells are Gram negative. A counterstain with methyl green provides differential staining between cell wall and cytoplasm. The cells and cell fragments are dried on a slide and stained by a standard Gram stain. The preparation is then treated for 5 min with 1% phosphomolybdic acid, washed, and stained 0.5 min with 1% aqueous methyl green (unpurified by CHCl₃ extraction). Under these conditions whole, intact cells are dark purple or black, walls of broken cells and purified walls are light green, and the exposed cytoplasm stains light purple. All fractions can be easily differentiated.     

Basic Two-Dye Stains for Epoxy-Embedded 0.3-1 μ Sections

Dyes used in the 3 methods recommended are: I, thionin and acridine orange (T-AO); II, Janus green and Darrow red (JG-DR); III, methyl green and methyl violet (MG-MV). The first 2 methods were two-solution stains, applied in sequence; the third, required only one solution since methyl violet is present in commercial methyl green. Staining solution and timing was as follows: Method I. 0.1% thionin in a 45% ethanolic solution of 0.01 N NaOH, 5 min at 70 C; rinsing in water and followed by 1 min in a 1% aqueous solution of acridine orange made up in 0.02 N NaOH, also at 70 C, then washed, and dried on slides. Method II. 0.5% Janus green in aqueous 0.05 N NaOH, 5 min at 70 C; rinsing in water then into 0.5% Darrow red in 0.05 N NaOH (aq.), 2 min at 70 C., washing, and drying on slides. Method III. 1% methyl green (commercial, unpurified) in 1% aqueous borax for 15-20 min at 20-25 C, washing and attaching to slides. All staining was performed by floating the sections on the staining solutions, all drying at 70 C, and mounting in a resinous medium. T-AO gave blue to violet cytoplasmic structures, darker nuclei which contrasted strongly with yellow connective tissue and the secretion of goblet cells. JG-DR resembled a hematoxylineosin stain, but by shortening the staining time in DR to 0.5-1 min, collagenous and elastic tissue retained more of the green dye. MG-MV gave dark green nuclei in light green cytoplasm, with collagenous and elastic tissues in blue to violet. As with most methods for staining ultrathin sections, thicknesses of less than 1 μ required longer staining times.     

Pyronin Y in the Methyl-Green-Pyronin Histological Stain

Two samples of pyronin Y were found which, with the exception of eosinophilic granules and osteoid, stained only nucleic acids in animal tissues. Good differentiation was obtained. with n-butyl alcohol. It was therefore possible to prepare a differentially staining mixture of either of these pyronins combined with methyl green. This mixture stains polymerized desoxyribose nucleic acid (DNA) clear green, depolymerized DNA and ribonucleic acid red. The red staining of eosinophilic granules and osteoid is readily distinguished by its persistence after ribonuclease or warm-buffer extraction. The staining mixture consists of: (1) pyronin Y (Edward Gurr or G. T. Gurr), CHCl₃ extracted, 2% aq, 12.5 ml; (2) methyl green, CHCl₃ extracted, 2% aq, 7.5 ml; (3) distilled water, 30 ml. The staining procedure is as follows. (1) Immerse slides 6 min in the dye mixture. (2) Blot with filter paper. (3) Immerse in 2 changes of n-butyl alcohol, 5 min each. (4) Xylene, 5 min. (5) Cedar oil, 5 min. (6) Apply Permount and cover.     

Pyronin Y in the Methyl-Green-Pyronin Histological Stain

Two samples of pyronin Y were found which, with the exception of eosinophilic granules and osteoid, stained only nucleic acids in animal tissues. Good differentiation was obtained. with n-butyl alcohol. It was therefore possible to prepare a differentially staining mixture of either of these pyronins combined with methyl green. This mixture stains polymerized desoxyribose nucleic acid (DNA) clear green, depolymerized DNA and ribonucleic acid red. The red staining of eosinophilic granules and osteoid is readily distinguished by its persistence after ribonuclease or warm-buffer extraction. The staining mixture consists of: (1) pyronin Y (Edward Gurr or G. T. Gurr), CHCl₃ extracted, 2% aq, 12.5 ml; (2) methyl green, CHCl₃ extracted, 2% aq, 7.5 ml; (3) distilled water, 30 ml. The staining procedure is as follows. (1) Immerse slides 6 min in the dye mixture. (2) Blot with filter paper. (3) Immerse in 2 changes of n-butyl alcohol, 5 min each. (4) Xylene, 5 min. (5) Cedar oil, 5 min. (6) Apply Permount and cover.     

Use of Gallocyanin as a Myelin Stain for Brain and Spinal Cord

Tissue fixed in 10% formalin, formol saline, CaCO₃ or phosphate buffer neutralized formalin, Baker's formol calcium, Cajal's formol ammonium bromide, formalin-95% ethanol 1:9, formalin-methanol 1:9, Lillie's methanol-chloroform or Salthouse's formol cetyltrimethylammonium bromide was dehydrated and embedded in paraffin. Sections were attached to slides with either albumen or gelatine adhesive and processed throughout at room temperature of 22-25 C. Mordanting 30-60 min in 1% iron alum was followed by a 10 min wash in 4 changes of distilled water. Myelin was stained in a gallocyanin self-differentiating solution for 1-2.5 hr; thick sections requiring the longer time. The staining solution (pH approximately 7.4) consisted of Na₂CO₃, 90 mg; distilled water, 100 ml; gallocyanin, 250 mg; and ethanol, 5 ml. The ethanol was added to this mixture last, and after the other ingredients had been boiled and then cooled to room temperature. After a staining and thorough washing, Nissl granules were stained for 5-10 min in a solution consisting of: 0.1 M acetic acid, 60 ml; 0.1 M sodium acetate, 40 ml; methyl green, 500 mg. Washing, dehydration, clearing and mounting completed the process. Myelin sheaths were stained dark violet; neuronal nuclei, light green with dark granules of chromatin; nucleoli of motor cells and erythrocytes, dark violet; cytoplasm, green with dark green Nissl granules. The simple and reliable method can be adapted easily for use with automatic tissue processors.     

Classification of larval circulating hemocytes of the silkworm,<Emphasis Type="Italic"> Bombyx mori</Emphasis>, by acridine orange and propidium iodide staining

Circulating hemocytes of the silkworm can be classified by fluorescence microscopy following staining with acridine orange and propidium iodide. Based on their fluorescence characteristics, three groups of circulating hemocytes can be distinguished. The first group, granulocytes and spherulocytes, is positive for acridine orange and contain bright green fluorescent granules when observed by fluorescence microscopy. In granulocytes, these green granules are heterogeneous and relatively small. In contrast, in spherulocytes, the green granules appear more homogenous and larger. The second group of hemocytes consists of prohemocytes and plasmatocytes. These cells appear faint green following staining with acridine orange and do not contain any green fluorescent granules in the cytoplasm. Prohemocytes are round, and their nuclei are dark and clear within a background of faint green fluorescence. Inside the nucleus there are one or two small bright green fluorescent bodies. Plasmatocytes are irregularly shaped and their nuclei are invisible. Oenocytoids belong to the third group, and their nuclei are positive for propidium iodide. Therefore, all five types of circulating hemocytes of the silkworm, including many peculiar ones that are difficult to identify by light microscopy, can now be easily classified by fluorescence microscopy following staining with acridine orange and propidium iodide. In addition, we show that hemocytes positive for acridine orange and propidium iodide are in fact living cells based on assays for hemocyte composition, phagocytosis, and mitochondrial enzyme activity.     

Chlorazol Black E as a Stain for Tension Wood

Sections containing gelatinous fibers were cut at 15 μ from material both fixed and stored in formalin-acetic-alcohol, 5:5:90 (of 70%). These sections were stained 5 min in a 1% aqueous solution of lignin pink (G. T. Gurr), differentiated quickly in water, soaked 5 min in 95% ethyl alcohol, dehydrated in absolute ethyl alcohol and counter stained 5 min with a 1% solution of chlorazol black E (G. T. Gurr) in methyl cellosolve, followed by dehydration in absolute ethyl alcohol, clearing in xylene and mounting in Canada balsam. The gelatinous layer was sharply defined as a dense black zone whilst the remainder of the cell wall stained light pink. The specificity of the technique was superior to that of safranin and light green, and was not easily obscured by overstaining. The technique is particularly useful for locating small zones of gelatinous fibres, and for photomicrographical work.     

A Staining Procedure for Use in the Brucella Opsonocytophagic Test

The following staining procedure is recommended for use in the Brucella opsonocytophagic test in order to avoid confusing results obtained with stains of the Hasting or Wright type: Fix spreads for 5 minutes or longer in absolute methyl alcohol. Stain for 10 to 30 minutes in a solution of the following: 0.5 g. NaCl, 0.5 g. phenol, 0.5 g. methylene blue, 0.02 g. Na₂HPO₄+12H₂O, 50 cc. distilled water, 50 cc. methyl alcohol. Wash slides gently in water. Air dry. By this procedure, the bacteria and the nuclei of the leucocytes appear deep blue. The cytoplasm of the leucocytes appears faintly green with the cell outline distinctly visible. Cytoplasmic granules do not stain.     

pH-dependent intraluminal organization of mucin granules in live human mucous/goblet cells

To study the mechanism of gel-forming mucin packaging within mucin granules, we generated human mucous/goblet cells stably expressing a recombinant MUC5AC domain fused to green fluorescent protein (GFP). The fusion protein, named SHGFP-MUC5AC/CK, accumulated in the granules together with native MUC5AC. Inhibition of protein synthesis or disorganization of the Golgi complex did not result in diminished intragranular SHGFP-MUC5AC/CK signals, consistent with long-term storage of the fusion protein. However, SHGFP-MUC5AC/CK was rapidly discharged from the granules upon incubation of the cells with ATP, an established mucin secretagogue. Several criteria indicated that SHGFP-MUC5AC/CK was not covalently linked to endogenous MUC5AC. Analysis of fluorescence recovery after photobleaching suggested that the intragranular SHGFP-MUC5AC/CK mobile fraction and mobility were significantly lower than in the endoplasmic reticulum lumen. Incubation of the cells with bafilomycin A1, a specific inhibitor of the vacuolar H+-ATPase, did not alter the fusion protein mobility, although it significantly increased (approximately 20%) the intragranular SHGFP-MUC5AC/CK mobile fraction. In addition, the granules in bafilomycin-incubated cells typically exhibited a heterogeneous intraluminal distribution of the fluorescent fusion protein. These results are consistent with a model of mucin granule intraluminal organization with two phases: a mobile phase in which secretory proteins diffuse as in the endoplasmic reticulum lumen but at a lower rate and an immobile phase or matrix in which proteins are immobilized by noncovalent pH-dependent interactions. An intraluminal acidic pH, maintained by the vacuolar H+-ATPase, is one of the critical factors for secretory protein binding to the immobile phase and also for its organization.     

Differentiation of Two Types of Basophils In The Adenohypophysis of The Rat and The Mouse

Pituitaries are fixed for 24 hr. in Bouin's fluid containing 0.5% trichloroacetic acid instead of 5% acetic acid, or in a mixture of 9 parts SUSA and 1 part saturated aqueous solution of picric acid. They are embedded in paraffin and horizontal sections are cut at 3-4 μ. The staining method consists of 3 phases: (a) immersion in aldehyde-fuchsin for the selective demonstration of the beta cell granules, (b) staining of the nuclei with Ehrlich's hematoxylin and (c) a rapid one-step counterstain with light green and orange G dissolved in a phosphotungstic-acetic acid mixture for the differentiation of the acidophilic and the delta cell granules.     

A Mixture of Methyl Green and Pyronin for Cell Fractionation Studies

A staining mixture consisting of 0.57% methyl green and 0.1 1 % pyronin B (calculated from the actual dye content) dis- solved in glycerol, 20 ml.; 2% aqueous phenol, 100 ml.; and 95% ethanol, 25 ml., was found to be optimum for differentiating cell components containing desoxypentose and pentose nucleic acids. The stain can be used for either fresh suspensions or unfixed dried smears of tissue homogenates. Nuclei are stained bright blue, and nucleoli and cytoplasmic particles, bright pink.     

Glycoproteins in membranes of secretory granules of the anterior pituitary gland.

Rat anterior pituitary glands were examined by electron microscopy after staining with five different histochemical stains. Histochemical reactions were observed in the cell coat, cell membrane and the membrane surrounding the secretory granules in all anterior pituitary cells following staining with phosphotungstic acid (PTA), chromic acid and PTA, the periodic acid-thiosemicarbazide-silver protein method (PA-TSC-SP) of Thiéry, ruthenium red and concanavalin A. The staining was abolished when the sections were preincubated with pronase, neuraminidase or trypsin and subsequently exposed to PTA, chromic acid and PTA or PA-TSC-SP. The possible functional role of the glycoproteins present in the membrane surrounding the secretory granules is considered.     

EVIDENCE OF DIFFERENCES IN THE DESOXYRIBONUCLEOPROTEIN COMPLEX OF RAPIDLY PROLIFERATING AND NON-DIVIDING CELLS

1. Quantitative cytophotometric analysis of the interphase cells of a rapidly proliferating differentiated tissue such as liver of new born rat, indicates that these cells can be separated into two groups on the basis of their staining characteristics after methanol fixation. 2. These groups are thought to correspond to two stages of interphase. The first, called "autosynthetic interphase," comprises cells which are duplicating chromosomal material in preparation for mitosis, and shows parallel increases in the methyl green and Feulgen staining of DNA and the fast green staining of histone from the diploid (2 C) to double these values (4 C). 3. The second group is designated the "heterosynthetic interphase," during which the cell ceases proliferating and functions in a manner commensurate with its state of differentiation. In this stage Feulgen staining indicates the diploid chromosomal complement, but there is a decreased capacity of the DNA to bind methyl green and of the histone to bind fast green. 4. The difference between the methyl green binding of the heterosynthetic and autosynthetic 2 C cells is due to the presence of a protein in the former which presumably inhibits staining by competing with the dye for binding sites on the DNA. The effect of this inhibition can be removed by extracting the protein, or by blocking the protein basic groups. 5. The decreased fast green staining of histone in the heterosynthetic cells can be minimized by prolonged fixation with formaldehyde. It is thought to stem either from a similar type of inhibition, or from an increased susceptibility of the histone to loss from the cell during this stage. 6. While histone inhibits methyl green staining of DNA in all cells, the differences between the staining properties of the autosynthetic and heterosynthetic interphase cells are believed to be due to another protein, whose properties appear similar to those of the chromosomal "residual protein." It is concluded that a complex of DNA and residual protein existing during the heterosynthetic interphase is dissociated during the autosynthetic interphase.     

颜色对梨小食心虫产卵选择性的影响

梨小食心虫(Grapholitha molesta Busck)是一种重要的果树害虫,早春产卵喜在桃嫩梢叶上,为探明寄主颜色在其产卵选择中的作用,利用彩色卡纸模拟寄主颜色,室内比较了红、粉红、浅粉、橙黄、深黄、浅黄、青绿、深绿、浅绿、蓝、紫、褐色等12种不同颜色基质对其成虫产卵选择性的影响。结果发现:基质颜色对梨小食心虫的产卵选择性有显著影响,其产卵偏嗜浅黄和浅绿色,白色和黑色参比时其产卵选择率分别依次为68.9%、63.8%和64.1%、65.5%,蓝和浅粉色则表现一定的拒避作用,白色和黑色参比时其产卵选择率分别依次为47.7%、40.4%和47.2%、42.7%,且参比色对其产卵选择性影响差异显著。基质颜色对其产卵量亦有显著性影响,无论黑或白色参比,黄、绿颜色上的产卵量均较多,尤其是深黄、深绿和青绿色。基质颜色对1—7日龄梨小食心虫成虫的产卵选择性均有显著影响。白色参比时,2、3日龄蛾对浅绿和浅黄色的产卵选择率显著高于其他颜色;黑色参比时,2日龄时明显偏嗜浅绿色(79.7%),6日龄时明显偏嗜浅黄色(74.8%)。表明寄主颜色在梨小食心虫产卵场所选择中具有重要作用,为其卵期监测和防控中颜色应用乃至进一步揭示其产卵寄主选择机理提供了依据和参考。     

Gross Differential Staining of the Hypophysis

The lobes of the hypophysis of many mammals can be differentiated by staining either the entire gland for 5–8 hr, or gross 1–2 mm slices of the gland for 3–5 min in a mixture of acid fuchsin (National Aniline, certified Andrade indicator) and methylene blue (Fisher certified reagent). For best results, the staining mixture contains 0.8–1.0% acid fuchsin and 0.2–0.4% methylene blue, both made up in 10% formalin adjusted to pH 6.70–6.75 with phosphate buffer. The anterior lobe stains a light blue, the intermediate lobe a dark blue, the posterior lobe a light pink and the capsule a dark pink.     

Glycoproteins in membranes of secretory granules of the anterior pituitary gland

Summary Rat anterior pituitary glands were examined by electron microscopy after staining with five different histochemical stains. Histochemical reactions were observed in the cell coat, cell membrane and the membrane surrounding the secretory granules in all anterior pituitary cells following staining with phosphotungstic acid (PTA), chromic acid and PTA, the periodic acid-thiosemicarbazide-silver protein method (PA-TSC-SP) of Thiéry, ruthenium red and concanavalin A. The staining was abolished when the sections were preincubated with pronase, neuraminidase or trypsin and subsequently exposed to PTA, chromic acid and PTA or PA-TSC-SP. The possible functional role of the glycoproteins present in the membrane surrounding the secretory granules is considered.     

Characterization of mucin isolated from rat tracheal transplants

Subcutaneous rat tracheal grafts yield several milligrams of secretions from which a homogeneous mucin fraction was isolated and purified. Histological evidence demonstrated that a normal mucociliary epithelium and mucous secretion were maintained for the 4-6 weeks of the experiment. The collected secretions were initially characterized by column chromatography on Sepharose CL-6B which separated the excluded high molecular weight mucins (unpurified mucin fraction) from most of the serum-type glycoproteins and proteins, including albumin. A reductive alkylation treatment of the unpurified mucin fraction followed by Sepharose CL-4B chromatography removed contaminating protein and most of the mannose-containing material from the mucin fraction. The void volume material from this column produced a single high molecular weight band upon sodium dodecyl sulfate agarose/acrylamide gel electrophoresis. The purified mucin fraction contained 16.5% protein and primarily galactose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid. This fraction also underwent beta-elimination in the presence of alkaline borohydride, demonstrating the presence of O-glycosidic linkages.     

In Vivo Staining by Dis and Trisazo Dyes, Especially of Bone and Elastic Tissue

The localization and retention of dis and trisazo dyes in connective tissues and bone was studied in rats and rabbits. Chlorazol fast pink, 5% in 0.9% NaCl. was injected intraperitoneally, 25 mg/kg each day for 2 days in newborn, growing, mature and 17-18 day pregnant rats, and up to 5 days in young rabbits. The dye was also injected at different time intervals during the development of strontium-induced rickets in growing rats, and in animals with abscess walls following subcutaneous injection of 0.1 ml turpentine. Animals were killed at-intervals thereafter, and comparison of in vivo staining of 5% solutions of chlorazol fast pink, chlorantine fast red, chlorazol black E, chlorazol sky blue, chlorazol sky pink, chlorazol green, chlorazol violet, pontamine green and pontamine sky blue was made by intraperitoneal injection in rats. Soft and hard tissue specimens were embedded in polyester resin or in paraffin wax and sectioned at 5-7 μ. Chlorazol fast pink stained some connective tissues and growing bones. The main intensity of staining occurred within 24 hr and gradually decreased but was still detectable after 6 mo in elastic tissues. In thin plastic sections, colouration was brilliant, not in osteoid tissue, but at calcifying bone margins and in elastic fibres. Dye localized at calcifying bone margins was incorporated within calcified tissues and then subsequently lost through remodelling. Such staining was not seen in paraffin-embedded material. Dye uptake was greatly reduced in rachitic rats, and wide osteoid seams were coloured faint pink, but where calcification was still occurring, colouration was brilliant. Similarly collagenous tissue in abscess walls was only lightly stained, in contrast to brilliant colouration of elastic tissues and macrophages. Of the 9 dyes tested, only chlorazol fast pink and chlorazol sky blue stained bone and elastic tissue in vivo. This prolonged retention and staining by these 2 dyes, unlike the others, was associated with their presence in the proximal convoluted tubules of the kidney.     

Membrane differentiation markers of airway epithelial secretory cells

We describe here a system for culturing epithelial cells isolated from hamster trachea, which results in a highly enriched population of mucus-secreting cells. The culture system has enabled us to study the process of secretory cell differentiation in vitro. We found that epithelial secretory cells, in vivo and after 5 days in vitro, selectively bind the lectin Helix pomatia agglutinin (HPA) to apical and, to a lesser extent, basolateral surfaces as well as to mucin granules and intracellular secretory organelles. SDS-PAGE gels of detergent extracts of secretory cells cultured for 5 days reveal three HPA-binding glycoproteins with MW of 120 KD, 220 KD, and greater than 400 KD. The high-MW glycoprotein appears identical to mucin, since it is found in secretions from intact trachea and in spent media from 5-day cultures. It does not appear in spent media from 3-day cultures when cells contain few mucous granules and secrete little mucin. The 220 KD HPA-binding glycoprotein is also present in 5-day but not in 3-day cultures. In contrast, the 120 KD glycoprotein is present at both times. HPA-gp120 is a hydrophobic integral membrane protein, whereas HPA-gp220 and mucin are hydrophilic and are membrane associated. These studies define three membrane glycoproteins, one of which is specific for the tracheal epithelial secretory cell regardless of its mucous content, whereas the other two glycoproteins correlate with mucin secretion. They also demonstrate that, in the fully differentiated state, mucin is bound in a non-covalent fashion to the apical plasma membrane of the tracheal epithelial secretory cell.     

Methyl green-pyronin; stoichiometry of reaction with nucleic acids

Study of the stoichiometry of the DNA-methyl green reaction by dialysis, precipitation of stain-nucleic acid mixtures, and the staining of nuclei of known DNA content, indicate that the compound consists of one dye molecule per 10 P. The significance of this result was discussed in the preceding paper (1). Histone and lanthanum (and probably other multivalent cations (3)) compete with the dye for the nucleic acid molecule, indicating a common site of attachment, presumably the phosphoric acid groups. With care in the avoidance of procedures which might depolymerize DNA, and the use of a buffer at about pH 4.1, a quantitative histochemical method for DNA by the use of methyl green is possible. Pyronin staining appears to be of qualitative significance only. Slight differences in degree of polymerization, as between the shad and mammalian DNA appear to have no effect on methyl green staining. It may be that a critical level of polymerization for DNA staining exists. This level must exceed 20 nucleotides to account for the 10 P to 1 dye molecule and the effect on the methyl green absorption spectrum; but it may be considerably greater. Beyond this critical level, whatever it may be, further polymerization probably has no influence on staining.