Some aspects of the value of Sudan Black B in lipid histochemistry

Summary The lipid dye Sudan Black B, as generally used to demonstrate lipids in the interior of the cell nucleus, was studied with regard to its staining properties for isolated nuclei in relation with its chromatographic characteristics in solution, as well as with a model system consisting of lipid containing polyacrylamide films.Isolated nuclei are stained with Sudan Black B dissolved in ethylalcohol, only when the dye-solution is at least one month old. Extraction with chloroform-methanol (21) before stainig resulted in a decrease of 35% in intensity. Treatment with proteolytic enzymes and DNA-se caused a complete disappearance of the staining capacity. The binding of Sudan Black B with phospholipids enclosed in the form of liposomes in modelfilms when stained with this dye in ethylene glycol obeys the law of Lambert-Beer. Proteins were however, also coloured by the dye.The chromatographic experiments showed that the dye is built up from two main and a number of secondary products. The secondary products which increase by aging of the dye-solution, change the spectrophotometric properties of the total dye and show aspecific binding.The conclusion was reached that on the basis of a positive reaction with Sudan Black B no definite conclusions can be drawn about the presence of lipids in the interior of the cell nucleus.     

Thin layer chrornatography and histochemistry of Sudan Black B

Summary Thin layer chromatography of commercial Sudan Black B on silica gel with chloroform-benzene (11) as the developing solvent reveals two blue main fractions with R_f values of 0.49 and 0.19, SBB-I and SBB-II respectively. Furthermore at least eighteen secondary fractions or impurities have been found. SBB-I and SBB-II were isolated and purified by preparative thin layer chromatography. Commercial Sudan Black B consists of about 20 p.c. SBB-I, 60 p.c. SBB-II and 20 p.c. secondary fractions.From spectrophotometrical and histochemical investigations it appeared that SBB-I stains lipids more pronounced than SBB-II; moreover SBB-I is more specific for neutral lipids than SBB-II, which fraction may also stain some proteins and acid mucopolysaccharides. Contrary to SBB-II the staining with SBB-I is fairly independent of pH. Finally, the colour of SBB-II changes under the influence of light and air, while SBB-I is much more stable.A physico-chemical study of the nature of SBB-I and SBB-II, including spectrophotometry, chromatography, infrared spectroscopy and chemical analysis revealed, that SBB-II is a basic dye, while SBB-I in spite of the structural resemblance behaves as a neutral one, dissolving therefore better in neutral lipids.As yet the chemical composition of SBB-I and SBB-II, and the relation to the scheme of synthesis of Sudan Black B has not been solved. The unspecificity of lipid staining by Sudan Black B is due to the basicity of SBB-II, and to the instability of this dye toward light and air. Moreover the some eighteen impurities may have some influence on the staining properties. The question of solubility or adsorption processes in the case of lipid staining by Sudan dyes is at least partially answered by the proposition of a dissolving fraction SBB-I and an adsorbed fraction SBB-II. The changing absorption spectra by the corresponding solvatochromic and metachromatic effects may give information about the nature of the lipids stained.     

Evaluation of intracellular lipids by standardized staining with a Sudan black B fraction.

The selective staining of neutral lipids in Human Amnion cells in tissue culture was achieved using a particular fraction of the lipid dye, Sudan black B and a standardized staining procedure. The fraction, termed SBB-I, was isolated by thin-layer chromatography. The cytophotometric assessment of intracellular neutral lipids, stained with SBB-I, is described and applied to the study of changes in granulocytic neutral lipids in leukemia.     

The value of simple lipid stains for typing skeletal muscle fibres

Summary Skeletal muscle fibre types can be distinguished rapidly with simple lipid stains. Comparative studies showed that Sudan Black B is superior to Oil Red O for this purpose and that optimum staining is obtained using unfixed sections or sections fixed in calciumglutaraldehyde. Factors that possibly influence the staining reaction, such as freeze-thawing, are considered. The stained lipids were identified by thin layer chromatography.     

Detection of lipids in the plant meristematic cell with the aid of Sudan black staining

As lipids can be a source of artefacts during intracellular localization of enzymes by cytochemical methodsin situ it was the aim of the present work to obtain orientation data on the distribution of lipids in the meristematic plant cells. The different fixation and object embedding methods examined revealed that it is best to fix the material by some formol fixative and without chroming, to embed it in polyethyleneglycol media. An alcoholic solution of Sudan black was found to be most reliable. In the meristematio cells the cytoplasm is usually stained more intensely than the nucleus. The ground cytoplasm is stained weakly while cytoplasmic particles are stained intensely. In some cases an intense black staining of nuclei, particularly in the prolongation zone, can be achieved. The staining intensity of cell components does not decrease on extracting lipids with pyridine. After extracting the dye from stained cell components a browninsh residual coloration remains. Chromatography of Sudan black revealed in all the samples tested slowly moving spots (blue and violet) and rapidly moving ones (red II, yellow, red I, colourless).     

Zur Physikochemie der vitalen Kern- und Plasmafluorochromierung vonAllium cepa-Epidermen

Summary Microfluorimetrically recorded fluorescence bands of vitally stained nuclei and protoplasts from the inner epidermis of yellow onion(Allium cepa) scales are compared with fluorescence bands of Tarions model solutions. The strong fluorescent staining of the nucleus and the weaker hue of the cytoplasm after application of acridine orange is mainly due to the accumulation of monomeric dye cations in polar cytoplasmic lipids. Vital fluorescent staining with neutral red shows similar effects, but in addition an accumulation of the dye base in apolar lipids can be ascertained without doubt. On the other hand, the major accumulation of the acid fluorochrome uranin (sodium fluorescein) does not take place in polar cytoplasmic lipids but, apparently, rather in the form of anions in the water phase of the ground cytoplasm and inner nuclear plasm owing to a mechanism of plasmatic ion trap.The proof that dye ions are present in vitally stained protoplasm suggests the possibility, that the familiar phenomenon of vacuole contraction may depend on a Donnan effect in the case of basic dyes and might be a consequence of the raising of osmotic values by dye anions in the case of acid dyes.Experiments with three phases (dye solution-oil-blood plasma) yield fluorescent stainings of blood plasma with acridine orange, neutral red and uranin, which may be compared to vital staining of nuclei and cytoplasm. The well-known properties of blood plasma-lipids suggest a similar function of cytoplasmic lipids, viz. as a vehicle of lipid transport in a mainly aquatic, molecular-disperse phase. A diagram (Fig. 15) illustrates the cooperation between sheetlike lipoproteid complexes (boundary layers) and lipoproteid complexes of the ground cytoplasm dispersed as particles.     

Propylene and Ethylene Glycol as Solvents for Sudan IV and Sudan Black B

Propylene or ethylene glycol is recommended as a solvent for Sudan IV and Sudan black B to replace the commonly used alcohol-acetone mixtures for general lipid staining in tissue sections. Either glycol is used as a dehydrating agent, dye solvent, and differentiating solution. They offer the advantages of a stable solution, inert with respect to solubilities of lipid material in it, and excellent control of differentiation without loss of dye from lipid particles. Sections remain pliable and are not shrunken by the glycols. Counterstains may be used after staining with Sudan IV but are generally not necessary after staining with Sudan black B. With the use of propylene glycol as a solvent, Sudan IV appears to equal the staining ability of Sudan black B as regards the type of lipid material detected, and the choice of dye to be used would depend on the color contrast desired.     

海水中DHA产生菌的筛选及一株高产菌的鉴定

从海水中筛选产DHA的微生物,共采集海水样品280余份,用苏丹黑染色法得到160株产油脂菌株,在对60株脂肪粒较大的微生物用索氏抽提法提取油脂后,初筛得到油脂含量高于8%的菌株7株。对10株油脂产量较高的菌株进行复筛,编号7-3的菌株油脂含量达到15.9%,DHA在油脂中的含量达到45.2%,选用7-3作为目的菌株。对7-3进行形态特征、培养特征及生理生化特征鉴定,初步判定菌株7-3为酒香酵母属(Brettanomyces sp.)。     

Bromine-Sudan Black: a general stain for lipids including free cholesterol

Synopsis Preliminary bromination (with bromine water) increases the intensity of staining of tissue lipids with Sudan Black and certain other dyes. The mechanism appears to be due to the formation of sudanophilic bromo-derivatives of cholesterol and to the retention of certain other lipids, notably phosphatidyl choline and free fatty acids, during staining. The advantage of the bromine-Sudan Black method is that all tissue lipids are stained, except saturated fatty acids, saturated triglycerides and perhaps saturated cholesterol esters. In practice, such lipids rarely, if ever, occur alone, and normally are admixed with their stainable unsaturated counterparts.     

Romanowsky dyes and the Romanowsky-Giemsa effect. 3. Microspectrophotometric studies of Romanowsky-Giemsa staining. Spectroscopic evidence of a DNA-azure B-eosin Y complex producing the Romanowsky-Giemsa effect

The Romanowsky-Giemsa staining (RG staining) has been studied by means of microspectrophotometry using various staining conditions. As cell material we employed in our model experiments mouse fibroblasts, LM cells. They show a distinct Romanowsky-Giemsa staining pattern. The RG staining was performed with the chemical pure dye stuffs azure B and eosin Y. In addition we stained the cells separately with azure B or eosin Y. Staining parameters were pH value, dye concentration, staining time etc. Besides normal LM cells we also studied cells after RNA or DNA digestion. The spectra of the various cell species were measured with a self constructed microspectrophotometer by photon counting technique. The optical ray pass and the diagramm of electronics are briefly discussed. The nucleus of RG stained LM cells, pH congruent to 7, is purple, the cytoplasm blue. After DNA or RNA digestion the purple respectively blue coloration in the nucleus or the cytoplasm completely disappeares. Therefore DNA and RNA are the preferentially stained biological substrates. In the spectrum of RG stained nuclei, pH congruent to 7, three absorption bands are distinguishable: They are A1 (15400 cm-1, 649 nm), A2 (16800 cm-1, 595 nm) the absorption bands of DNA-bound monomers and dimers of azure B and RB (18100 cm-1, 552 nm) the distinct intense Romanowsky band. Our extensive experimental material shows clearly that RB is produced by a complex of DNA, higher polymers of azure B (degree of association p greater than 2) and eosin Y. The complex is primarily held together by electrostatic interaction: inding of polymer azure B cations to the polyanion DNA generates positively charged binding sites in the DNA-azure B complex which are subsequently occupied by eosin Y anions. It can be spectroscopically shown that the electronic states of the azure B polymers and the attached eosin Y interact. By this interaction the absorption of eosin Y is red shifted and of the azure B polymers blue shifted. The absorption bands of both molecular species overlap and generate the Romanowsky band. Its strong maximum at 18100 cm-1 is due to the eosin Y part of the DNA-azure B-eosin Y complex. The discussed red shift of the eosin Y absorption is the main reason for the purple coloration of RG stained nuclei. Using a special technique it was possible to prepare an artificial DNA-azure B-eosin Y complex with calf thymus DNA as a model nucleic acid and the two dye stuffs azure B and eosin Y.(ABSTRACT TRUNCATED AT 400 WORDS)     

Histochemical lipid studies on Schistosoma mansoni adults maintained in situ and in vitro

Haseeb M. A., Eveland L. K. and Fried B. 1984. Histochemical lipid studies on Schistosoma mansoni adults maintained in situ and in vitro. International Journal for Parasitology14: 83–88. Schistosoma mansoni male and female adults were incubated at 37°C for 0.5 and 1.0 h in Earle's balanced salt solution containing 0.1% glucose and 0.5% lactalbumin hydrolysate, then examined by histochemistry and scanning electron microscopy. Histochemical analysis of cryostat sections stained with Oil Red O showed that males contain neutral lipid mainly in the parenchyma and tubercles, while females contain neutral lipid in the vitellaria. Neutral lipids are released from the tubercles of both paired and unpaired males maintained in vitro. There is evidence of in situ lipid transfer from males to blood vessel walls. Neutral lipid was not seen in females from unisexual infections. Sudan Black B staining fo total lipids is positive in tubercles, parenchyma, and vitellaria. Nile Blue Sulphate stains acidic lipids in male caecal walls. Scanning electron microscopy reveals no tegumental damage.     

Hemocytes of the cochineal insect: ultrastructure

Using transmission electron microscopy, light microscopy (Giemsa May‐Grumwald), and the Periodic Acid‐Schif (PAS) and Sudan Black B staining techniques, hemocytes in the hemolymph of adult female Dactylopius coccus were characterized. The following, in order of abundance, were found: granulocytes, plasmatocytes, prohemocytes, and oenocytoids. Granulocytes varied in size with granulations in the cytoplasm, a large quantity of mitochondria, rugose endoplasmatic reticulum, ribosomes and vesicles, central or exocentric, spherical and occasionally lobulate nucleus. Plasmatocytes were polymorphic with irregularities in the plasma membrane; cytoplasm contained mitochondria, rugose endoplasmatic reticulum and vesicles, and exocentric, spherical, or irregular nucleus. In both types of hemocytes, scant polysaccharides and lipids were found. Prohemocytes were small and spherical with homogeneous cytoplasm and large exocentric nuclei. Oenocytoids were oval or irregular with dense homogeneous cytoplasm and elongated exocentric nuclei. The percentages of granulocytes on different days (d 1 and 10) during the life of the adult female were significantly different, as were those of plasmatocytes on d 30 and 50 and prohemocytes on d 1 and 50. © 2010 Wiley Periodicals, Inc.     

Dynamic of polysaccharide and lipid in the developing microsporangiums of Chinese fir (Cunninghamia lanceolata)

Microsporongium development in Chinese fir (Cunninghamia lanceolata) was investigated using cytochemical methods with a special attention to the fluctuations (in amount and distribution) of polysaccharide and lipid reserves along the development of the microsporangium. Semi-thin sections of microsporangia at different developmental stages were stained with periodic acid–Schiff (PAS) reagent and Sudan Black B to detect insoluble polysaccharides and neutral lipids, respectively. In young microsporangia, microspore mother cells began to accumulate starch grains and lipids, which disappeared during microspore development. Following microspore division, the starch grains present in bicellular pollen disappeared and abundant lipid deposits were accumulated. In mature pollen, only abundant lipids accumulated as storage material. The pollen wall of C. lanceolata is predominantly composed of polysaccharidic intine, and the sporopollenin-containing exine is weakly developed and only forms a thin layer covering the intine.     

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.     

Interaction of negatively charged liposomes with nuclear membranes: adsorption, lipid mixing and lysis of the vesicles

Fluorescence energy transfer studies reveal that negatively charged lipid vesicles interact with nuclei from mouse liver cells. This interaction was observed with charged lipid vesicles composed of PA or PS but not with the uncharged PC or PE:PC vesicles. The vesicles were prepared by bath sonication and contained either a fluorescent marker in the lipid bilayer or in the vesicular interior. The negatively charged vesicles showed an adsorption to the nuclear membrane visible by fluorescence microscopy. The results obtained by resonance energy transfer experiments are interpreted in terms of a mixing of the lipids from the vesicles with the nuclear membrane. Encapsulation studies documented a staining of the nuclei only if the dye molecules of high or low molecular weight were encapsulated inside negatively charged vesicles. As consequence of the vesicle-nuclei interaction morphological changes on the nuclear surface became visible.     

Efficient Lipid Staining in Plant Material with Sudan Red 7B or Fluoral Yellow 088 in Polyethylene Glycol-Glycerol

Polyethylene glycol (400) with 90% glycerol (aqueous) is introduced as an efficient solvent system for lipid stains. Various lipid-soluble dyes were dissolved in this solvent system and tested for their intensity, contrast, and specificity of staining of suberin lamellae in plant tissue. The stability (i.e., lack of precipitation) of the various staining solutions in the presence of fresh tissue was also tested. When dissolved in polyethylene glycol-glycerol, Sudan red 7B (fat red) was the best nonfluorescent stain and fluorol yellow 088 (solvent green 4) was an excellent fluorochrome. These two dyes formed stable staining solutions which efficiently stained lipids in fresh sections without forming precipitates. Estimations of the solubilities of these dyes in the solvent compared with their solubilities in lipids of various chemical types indicated that they should both be effective stains for lipids in general.     

Acetyl Sudan Black: A Nonexistent Reagent?

Acetyl Sudan black (AcSB) has been recommended as a readily prepared reagent which “appears to give less background staining and just as intense lipid staining as the untreated dye,” i.e. as nonacetylated Sudan black B (Lillie and Fullmer 1976).     

Peroxidase and pseudoperoxidase reactions in relation to sudanophilia.

A selective staining of hemoglobin in erythroid cell series was achieved by use of Sudan Black B (modified method of Sheehan and Storey) if optimal amount of hydrogen peroxide was added to the staining mixture. The effect of some inhibitory agents (KCN, wet heat, pH) on this staining as well as on the Lepehne's pseudoperoxidase reaction for hemoglobin was similar. Both reactions were more resistant to these factors than the peroxidase reactions and sudanophilia in granulocytes in which both could be blocked by the pretreatment with absolute methanol. Moreover the effect of some extraction procedures for lipids on both myeloperoxidase reactions and sudanophilia was investigated. The results support the view that the sudanophilia in granulocytes is due to their peroxidase activity and for the staining of hemoglobin by use of Sudan Black B with H2O2 its pseudoperoxidase activity is responsible. In addition the effect of the substitution of phenolphosphate by dihydroxybenzenes on granulocyte sudanophilia is reported.