The Use of Osmium Tetroxide-Potassium Ferrocyanide as an Extracellular Tracer in Electron Microscopy

Secondary treatment of glutaraldehyde fixed liver samples with long term stored solutions of osmium tetroxide and potassium ferrocyanide (Os-Fe) results in tracing of the extracellular spaces of the tissue with an electron opaque substance. This substance does not diffuse across cell membranes, its formation probably being related to the progressive reduction of the O_sO₄ molecule by potassium ferrocyanide. The application of the present method in electron microscopy may be useful in overcoming the artifacts often induced by other tracing techniques such as vascular perfusion with peroxidase or immersion in lanthanum. Although the period of storage of the Os-Fe solution is a clear disadvantage of the method, it seems plausible to anticipate that further studies on the chemical interaction between osmium tetroxide and potassium ferrocyanide will provide us with a Os-Fe mixture having an immediate tracer effect.     

An improved method for perfusion fixation of neural tissues for electron microscopy

A method employing vascular perfusion for improved preservation of biological ultrastructure is described, and its effectiveness demonstrated for mammalian nervous tissues. Following a physiological saline flush into the aorta, hydrogen peroxide and glutaraldehyde in phosphate buffer are perfused. After buffer rinses, tissue blocks are postfixed in osmic acid and potassium ferrocyanide. The success rate is enhanced greatly by close attention to details of perfusion technique. Advantages of the method include more uniform and complete preservation. In particular, superior images of membranous elements, glycogen granules and basal laminar material are achieved. Adjustments in osmolality may render the procedure suitable for non-mammalian forms and other tissues.     

Cytochemical demonstration of phosphatases in the rat liver by a cerium-based method in combination with osmium tetroxide and potassium ferrocyanide postfixation

Acid phosphatase, alkaline phosphatase, glucose-6-phosphatase, Mg-activated adenosine triphosphatase and 5'nucleotidase were demonstrated in the rat liver using a cerium-based method. This method can be applied routinely and yields better results than the lead-based method. The tissue was postfixed in osmium tetroxide and potassium ferrocyanide which considerably enhances the membrane contrast in comparison with solely osmium tetroxide postfixation. This facilitates the precise localization of the reaction product.     

A modified silver method for demonstrating developing nervous tissue in culture

Dissociated explants of 8-day-old embryonic chick cerebrum were cultured for up to 18 days. By the beginning of the 2nd week and thereafter, primary cultures of neurons exhibited characteristic differentiated morphology with an interconnecting neurofibrillary network that became increasingly ramified. Neurons in bipolar, tripolar or multipolar form could be demonstrated positively using a short modified silver impregnation method with potassium ferrocyanide.     

Optimizing medium constituents and fermentation conditions for citric acid production from palmyra jaggery using response surface method

The quantitative effects of pH, temperature, time of fermentation, sugar concentration, nitrogen concentration and potassium ferrocyanide on citric acid production were investigated using a statistical experimental design. It was found that palmyra jaggery (sugar syrup from the palmyra palm) is a suitable substrate for increasing the yield of citric acid using Aspergillus niger MTCC 281 by submerged fermentation. Regression equations were used to model the fermentation in order to determine optimum fermentation conditions. Higher yields were obtained after optimizing media components and conditions of fermentation. Maximum citric acid production was obtained at pH 5.35, 29.76 °C, 5.7 days of fermentation with 221.66 g of substrate/l, 0.479 g of ammonium nitrate/l and 2.33 g of potassium ferrocyanide/l.     

Transport and metabolism of free cyanide and iron cyanide complexes by willow

Cyanide compounds are contaminants of growing importance that could be remediated biologically via phytoremediation, provided the plants possess suitable mechanisms for managing these pollutants without toxicity. The transport and metabolism of two cyanide compounds, potassium cyanide and potassium ferrocyanide, by willow (Salix eriocephala L. var. Michaux) were compared using a hydroponic system that preserved cyanide speciation and solubility. The cyanide compounds were labelled with ¹⁵N to quantify transport while a novel tissue extraction procedure was used to relate tissue ¹⁵N to cyanide content and speciation. These analyses revealed that although little free cyanide was detected in the aerial tissues of plants exposed to either of these two cyanide compounds, significant enrichments in ¹⁵N were observed, suggesting transport and subsequent metabolism of free cyanide as well as ferrocyanide. The results for ferrocyanide are of interest because this molecule is resistant to microbial degradation and if oxidized to ferricyanide is purportedly membrane impermeable. Nevertheless, these results and mass balance calculations for tissue ¹⁵N and solution cyanide confirming 100% recovery for the added ferrocyanide are suggestive of ferrocyanide uptake and metabolism. This study provides new information describing the biological transport and metabolism of these two cyanide compounds in plants. Moreover, the data also suggest that phytoremediation of cyanide may be possible and ecologically safe due to the lack of cyanide bioaccumulation in aerial tissues.     

HALE STAIN FOR SIALIC ACID-CONTAINING MUCINS : Adaptation to Electron Microscopy

The feasibility of using the Hale stain to identify cellular sialic acid-containing mucins by electron microscopy was investigated. Three kinds of mouse ascites tumor cells were fixed in neutral buffered formalin, exposed to fresh colloidal ferric oxide, treated with potassium ferrocyanide, imbedded in Selectron, and sectioned for electron microscopy. Additional staining with uranyl acetate and potassium permanganate was done after sectioning in order to increase contrast. Those cells known to be coated with sialomucin showed deposits of electron-opaque ferric ferrocyanide crystals in the areas where sialomucin concentrations were expected. When these cells were treated with neuraminidase beforehand, these deposits did not appear. It was concluded that, with the precautions and modifications described, the Hale stain can be successfully combined with electron microscopy to identify sialomucin.     

Comparative study of the effect of ferrocyanide and EDTA on the production of ethyl alcohol from molasses by Saccharomyces cerevisiae

The effects of potassium ferrocyanide and EDTA on ethyl alcohol production from molasses by Saccharomyces cerevisiae were investigated on simulated batch pilotplant-scale conditions for alcoholic fermentation of molasses. Ethyl alcohol production was more sensitive to ferrocyanide than to EDTA. When ferrocyanide was introduced into the cultures at the time of inoculation, there was stimulation of ethyl alcohol production, with 261 ppm ferrocyanide producing the maximum effect, which was 3.0% more than in control cultures. When added during the propagation of the yeast, ferrocyanide depressed ethyl alcohol production by 4.0% maximum whereas EDTA stimulated ethyl alcohol production by 2.0%. Addition of ferrocyanide during the fermentation stage produced no significant effect on alcohol production, whereas over a wide range of EDTA concentration there was a steady increase in alcohol yield.     

Binding of Ferric Ions to Nuclei and Other Tissue Sites in Sections Stained for Sulfated Mucosubstances by the High-Iron Diamine Method

Binding of Fe³⁺ occurred in nuclei and several other sites when tissue sections, after a prior staining by the high-iron diamine (HID) method for sulfomucins, were immersed for 1 hr in 0.06 N HCl containing 1% potassium ferrocyanide (Prussian blue reaction). Apparently Fe³⁺, which is derived from FeCl₃ present in the HID dye bath, unites directly with these tissue components, although one cannot exclude the possibility that iron is first bound to colorless diamine complexes and then to tissues. The visualization of Fe³⁺ by ferrocyanide provides a simple way of obtaining a suitable nuclear stain combined with general counters taming for the HID method.     

Effects of nucleophils on kinetic parameters of peroxidase-catalyzed oxidative reactions]

The effect of imidazole on the kinetic parameters of reactions of potassium ferrocyanide and o-dianizidine peroxidation by hydrogen peroxide within a wide range of pH was studied. It was shown that imidazole activates the reaction of o-dianizidine peroxidation at pH greater than or equal to 6.5, but does not affect the oxidation of potassium ferrocyanide. It was also found that imidazole causes a similar increase in the kkat and Km, i. e. non-competitive activation occurs. The data obtained suggest a possible mechanism of the activator effect. Differences in the mechanism of interaction of various substrates uith peroxidase are discussed.     

Double simultaneous staining of B and argyrophil cells of the pancreatic islets: a sequential thiosulphation-aldehyde fuchsin and Grimelius silver procedure

The thiosulphation-aldehyde fuchsin (TAF) method for the insulin-producing B cells can be followed by the Grimelius silver impregnation for the argyrophil cells. This double staining is useful to study, in normal and pathological tissues, the spatial distribution of the two main endocrine cell populations of the pancreatic islets. A treatment with potassium ferrocyanide has been found to enhance the argyrophilia of A cells.     

Flow injection chemiluminescence determination of captopril based on its enhancing effect on the luminol–ferricyanide/ferrocyanide reaction

A new flow injection chemiluminescence method is described for the determination of captopril. It is based on the enhancing effect of captopril on the chemiluminescence reaction of luminol with potassium ferricyanide in alkaline solution in the presence of potassium ferrocyanide. The method allows the determination of captopril over 0.1–40 µg/mL range, with a relative standard deviation (SD) of 1.0% for the determination of 0.5 µg/mL captopril solution in 11 repeated measurements. The method was satisfactorily applied to the determination of captopril in commercial captopril tablets. The possible reaction mechanism is also discussed briefly. Copyright © 2002 John Wiley & Sons, Ltd.     

The suitability of ferric potassium ferrocyanide as a perfusate in cholinesterase histochemistry: ultrastructural and biochemical observations

Synopsis When perfused through an animal immediately after death, ferric potassium ferrocyanide does not pass into or across endothelial cells and does not inhibit tissue cholinesterases. Perfusion of a 2% aqueous solution of the compound offers a convenient and easy method for recognizing the relationship of structures, demonstrated by cholinesterase histochemistry, to injected vessels.     

Nonlethal assay system of β-glucuronidase activity in transgenic tobacco roots

Previously, assay conditions for the GUS enzyme have required lethal and/or destructive conditions which do not allow for the continued observation of living plants. The replacement of sodium phosphate buffer with potassium phosphate buffer and the removal of potassium ferrocyanide and EDTA resulted in an assay for the GUS enzyme that is both nondestructive and nonlethal to tobacco plants and therefore allows observations of tobacco roots through time.     

Activation of dopamine beta-monooxygenase by external and internal electron donors in resealed chromaffin granule ghosts

Membrane ghosts derived from chromaffin vesicles of bovine adrenal medullas have been used to examine the mechanism of reduction of dopamine beta-monooxygenase in its compartmentalized state. The rate of the dopamine beta-monooxygenase-catalyzed conversion of dopamine to norepinephrine is greatly stimulated by the presence of ATP, reflecting substrate hydroxylation on the ghost interior subsequent to the active transport of dopamine. We demonstrate a 2-3-fold increase in the turnover rate for ghosts resealed with 0.2-2 mM potassium ferrocyanide, conditions leading to a slight decrease in the rate of dopamine transport. These data provide the first evidence that an intravesicular pool of reductant can activate dopamine beta-monooxygenase, as required by models in which vesicular ascorbate behaves as enzyme reductant. Although there is sufficient catecholamine (endogenous plus substrate) to keep internal ferrocyanide reduced in these experiments, an additional 2-3-fold increase in turnover occurs in the presence of 0.2-2 mM ascorbate on the ghost exterior. The magnitude of this activation is found to be constant at all concentrations of internal ferrocyanide (both below and above saturation), implying that reductants on opposite sides of the membrane behave independently. Replacement of ascorbate by potassium ferrocyanide as external reductant leads to almost identical results, and we are able to rule out an inward transport of dehydroascorbate as the source of activation by external ascorbate. We conclude that external reductants are capable of reducing membrane-bound dopamine beta-monooxygenase from the exterior face of the vesicle, either by direct reduction or through a membrane-bound mediator. It appears that two viable modes for reduction of dopamine beta-monooxygenase may exist in vivo, involving the reduction of membrane-bound enzyme by cytosolic ascorbate as well as the reduction of soluble enzyme by the pool of intravesicular ascorbate present in chromaffin vesicles.     

FerriDye: colloidal iron binding followed by Perls' reaction for the staining of proteins transferred from sodium dodecyl sulfate gels to nitrocellulose and positively charged nylon membranes

A staining method for proteins on (positively charged) nylon and nitrocellulose membranes is described. The two-step method uses cationic cacodylate iron colloid which is substituted with Tween 20 at an OD460 nm = 0.5, followed by Perls' reaction with acid potassium ferrocyanide. It stains transferred proteins deep blue with low background. The sensitivity is intermediate between that of conventional stains and AuroDye, the colloidal gold stain. This is the first sensitive staining method for proteins transferred on (positively charged) nylon membranes. These membranes have documented advantages in immunoblotting. It will therefore be a useful tool for correlating the position of bands or spots of proteins detected with overlay assays with the complete electropherogram in a duplicate protein blot.     

Ferri- and Ferrocyanide Salts Change the Current/Voltage Relations of Chara corallina: No Correlation with the Transmembrane Redox System

The current/voltage {I/V) relations of the plasma membrane ofChara corallina cells are characteristic when the bathing mediumhas elevated (5-0 mM) concentrations of potassium Addition ofeither 0-5 mM femcyanide or 0-5 mM ferrocyanide usually induceda qualitatively similar increase in the ‘leak’ current,with a concomitant increase in membrane conductance. Both redoxreactants failed for unknown reasons to affect the I/V profileof some of the cells tested In the sensitive cells, femcyanidewas unable to generate extra current over that found upon additionof ferrocyanide Because the feiTocyanide oxidation rate of thecells is only 10% of the femcyanide reduction rate, we concludethat both forms, fern- and ferrocyanide, affect the 'leak' conductanceindependent of the redox state of the reactants, i e ferrocyanidedoes not act indirectly via an oxidation to femcyanide Hence,under the experimental conditions, we were unable to detecta current that could be assigned to the operation of a transmembraneredox system Furthermore, the fern- and ferrocyanide inducedshift in the I/V profile only reversed slowly after withdrawalof the redox reactants. This suggests that the elicited currentis independent of the presence of an extracellular electronacceptor, i.e on the continuous operation of a proposed transmembraneredox system Key words: Current/voltage curves, transmembrane reductase, voltage clamp, femcyanide, ferrocyanide, Chara     

Chemiluminescence inhibition assay for folic acid using flow injection analysis

A new flow injection method for the determination of folic acid is described. A fast oxidation reaction occurred when folic acid was mixed with potassium ferricyanide generating ferrocyanide which then inhibited the chemiluminescent reaction of ferricyanide and luminol in alkaline medium. The decrease of chemiluminescence intensity was correlated with the folic acid concentration in the range 0.1-21 microg/mL; the detection limit for the assay was 0.03 microg/mL (3sigma). A complete analysis of folic acid, including sampling and washing, could be performed within 2 min with a relative standard deviation of less than 4.0%. The proposed method has been applied successfully to the determination of folic acid in pharmaceutical preparations.     

A method for the electron microscopic demonstration of cytochrome oxidase in fresh and formalin-prefixed tissues

Summary Burstone's reaction is adapted for electron microscope purposes. Sections of 300–500 thickness cut by free hand are incubated for sixty minutes in N-phenyl-p-phenylenediamine at room temperature. No coupling agent is used. The dye polymere gained is postchelated with 0.5% copper sulphate dissolved in 4% buffered formalin solution. After thorough washing a second postchelation is performed with potassium ferrocyanide. Double postchelation reduces the otherwise high lipid solubility of the reaction product, so that it withstands embedding into epoxy resins. Postfixation with osmic acid adds further contrast to the reaction product. Cell structure — even in central nervous tissue — could be preserved satisfactorily without major loss of enzyme activity by the use of a brief formalin perfusion prior to incubation. The reaction product appears in the form of droplets localized in the mitochondria.     

Nitric oxide reduces seed dormancy in Arabidopsis

Dormancy is a property of many mature seeds, and experimentation over the past century has identified numerous chemical treatments that will reduce seed dormancy. Nitrogen-containing compounds including nitrate, nitrite, and cyanide break seed dormancy in a range of species. Experiments are described here that were carried out to further our understanding of the mechanism whereby these and other compounds, such as the nitric oxide (NO) donor sodium nitroprusside (SNP), bring about a reduction in seed dormancy of Arabidopsis thaliana. A simple method was devised for applying the products of SNP photolysis through the gas phase. Using this approach it was shown that SNP, as well as potassium ferricyanide (Fe(III)CN) and potassium ferrocyanide (Fe(II)CN), reduced dormancy of Arabidopsis seeds by generating cyanide (CN). The effects of potassium cyanide (KCN) on dormant seeds were tested and it was confirmed that cyanide vapours were sufficient to break Arabidopsis seed dormancy. Nitrate and nitrite also reduced Arabidopsis seed dormancy and resulted in substantial rates of germination. The effects of CN, nitrite, and nitrate on dormancy were prevented by the NO scavenger c-PTIO. It was confirmed that NO plays a role in reducing seed dormancy by using purified NO gas, and a model to explain how nitrogen-containing compounds may break dormancy in Arabidopsis is presented.