Improved Stability of Methanolic Wright's Stain Solution with Dual Additives

Degradation of methanolic Wright's stain solutions was greatly diminished with the addition of diethylamine hydrochloride and dimcthylamine hydrochloride as costabilizers. Precipitation problems were eliminated by the dual additives. The stabilized stain solutions demonstrated good staining performance on blood smears. Methods for predicting the shelf life using calculated analytical parameters are described. Using these methods, the shelf life of a control stain solution was predicted to be 0.7 years; predicted shelf life was more than tripled with the addition of diethylamine hydrochloride and was increased approximately 27 times with the addition of both diethylamine hydrochloride and dimethylamine hydrochloride.     

Improved stability of methanolic Wright's stain solution with dual additives

Degradation of methanolic Wright's stain solutions was greatly diminished with the addition of diethylamine hydrochloride and dimethylamine hydrochloride as costabilizers. Precipitation problems were eliminated by the dual additives. The stabilized stain solutions demonstrated good staining performance on blood smears. Methods for predicting the shelf life using calculated analytical parameters are described. Using these methods, the shelf life of a control stain solution was predicted to be 0.7 years; predicted shelf life was more than tripled with the addition of diethylamine hydrochloride and was increased approximately 27 times with the addition of both diethylamine hydrochloride and dimethylamine hydrochloride.     

The analysis of some commercial dyes and Romamowsky stains by high-performance liquid chromatography.

High-performance liquid chromatography has been used to quantitate batch variations in commericial samples of thiazine dyes, thiazine eosinates, and Romanowsky-type blood stains. It has been observed that all the dyes and eosinates examined, only methylene blue chloride and thionin were reasonably free of their methylated, demethylated, or oxidized homologs. Large variations in composition were observed between most of the samples of each type examined. In several instances the labeled compound was a minority species. In one instance a dye was apparently mislabeled. Large compositional variation was found between various batches of Wright and Giemsa stains, whereas significant differences between the thiazine composition of these two stain types were minor. Very little compositional variation was observed between the lots of LARC stain examined. The thiazine composition of Ames stain was similar for the three lots examined. Ames stain, however, was found to contain several components of unknown composition which have been linked to degradation products formed when stains are aged in methanolic solution.     

The degradation of Romanowsky-type blood stains in methanol.

The oxidative demethylation of Romanowsky-type stains in methanol has been examined quantitatively with respect to its effect upon the staining of blood smears. Spectral changes in bound dye, observed through two color filters, have been measured for the nuclei and cytoplasm of segmented neutrophils and monocytes utilizing the LARC automated differential analyzer. Stain decomposition in methanol results in a large loss in staining intensity with little change in color. The loss in intensity has been correlated with the observed spectral changes in the degraded stain. High-performance liquid chromatographic analysis of degraded stain samples has shown the products of methanolic degradation to be different from those obtained in aqueous polychroming reactions. To maintain a stain of defined thiazine dye composition and thus defined staining properties, refrigeration is recommended.     

The Degradation of Romanowsky-Type Blood Stains in Methanol

The oxidative demethylation of Romanowsky-type stains in methanol has been examined quantitatively with respect to its effect upon the staining of blood smears. Spectral changes in hound dye, observed through two color filters, have been measured for the nuclei and cytoplasm of segmented neutrophils and monocytes utilizing the LARC™ automated differential analyzer. Stain decomposition in methanol results in a large loss in staining intensity with little change in color. The loss in intensity has been correlated with the observed spectral changes in the degraded stain. High-performance liquid chromatographic analysis of degraded stain samples has shown the products of methanolic degradation to be different from those obtained in aqueous polychroming reactions. To maintain a stain of defined thiazine dye composition and thus defined staining properties, refrigeration is recommended.     

Elicitation of thiomolybdates from the iron-molybdenum cofactor of nitrogenase. Comparison with synthetic Fe-Mo-S complexes

Aerial oxidation of the iron-molybdenum cofactor (FeMoco) of Azotobacter vinelandii nitrogenase has been shown to yield either the tetrathiomolybdate ion ([MoS4]2-) or the oxotrithiomolybdate ion ([MoOS3]2-), depending on the reaction conditions. Thus, when N-methylformamide (NMF) solutions of FeMoco either were titrated with measured aliquots of air or were diluted with air-saturated NMF, [MoOS3]2- was found to be the predominant product while dilution of NMF solutions of FeMoco with air-saturated methanol produced [MoS4]2- almost exclusively. Similar aerial oxidation of solutions of chemically synthesized Fe-Mo-S clusters showed that significant information about the molybdenum environment in these species could be deduced from the nature of the elicited thiomolybdates. The differences in decomposition products as a function of solvent are postulated to be due to the loss through precipitation of the reducing agent sodium dithionite on addition of methanol but not NMF. These overall decomposition results are discussed in the context of recent X-ray absorption spectroscopic data which suggest the presence of an 'MoS3' core in FeMoco. A possible mechanism whereby [MoS4]2- might be rapidly formed from this core is presented.     

Effects of methanol on the synthesis of aspartame precursor catalysed byPseudomonas aeruginosa elastase

Summary Pseudomonas elastase was found to be efficient in catalysing the reaction betweenN-benzyloxycarbonyl-L-aspartic acid and L-phenylalanine methyl ester producing the aspartame precursor in aqueous and aqueous methanolic solutions. 25% (v/v) methanol was most favourable for the synthesis where about 100% increase in yield was obtained compared to that in aqueous solution.Abbreviations N-cbz-L-Asp N-benzyloxycarbonyl-L-Aspartic acid - L-Phe-OMe L-phenyl alanine methyl ester - HPLC high performance liquid chromatography. All the % of methanol is a volume % in water unless otherwise specified     

Sequential Use of Wright's and Ziehl-Neelsen's Stains for Demonstrating Phagocytosis of Bacterial Spores

Nongerminating spores, germinating spores, and vegetative cells of Clostridium botulinum type A were observed during phagocytosis in the peritoneal fluid of white mice. Since phagocytes are easily ruptured by heat, the method described avoids heating, as this has been employed in conventional spore staining methods. A thin smear of the fluid is air dried on the slide for 2 hr, and stained by Wright's method: stain, 2 min; dilution water, 2 min; and rinsed; then in 0.005% methylene blue for 30 sec, and rinsed. This is followed by Ziehl-Neelsen's stain for 3-4 min and destained with 1: acetone-95% ethanol for 10 sec. The slide is rinsed, and Wright's staining repeated: stain 1 min, dilution 2-3 min; and thereafter washed about 5 ml of Wright's buffer. Blotting and air drying completes the staining. Non-germinating spores stain light red with a red spore wall, germinating spores are deep red throughout, vegetative cells are blue, and leucocytes show a dark purple nucleus and light blue cytoplasm.     

THE EFFECT OF THE H ION CONCENTRATION ON THE AVAILABILITY OF IRON FOR CHLORELLA SP

The data obtained in these experiments indicate clearly that unless the necessary precautions are taken to keep the iron of the culture medium in solution the results obtained by varying the H ion concentration will not represent the true effect of this factor on growth. The availability of iron in nutrient solutions has been the subject of numerous recent investigations and it is now known that iron is precipitated at the lower hydrogen ion concentrations, that the iron of certain iron salts is less likely to be precipitated than that of others, and that certain salts of organic acids tend to keep the iron in solution. In general, ferric citrate seems to be the most favorable source of iron. In addition to chemical precipitation, however, it is also possible for the iron to be removed by adsorption on an amorphous precipitate such as calcium phosphate. As this precipitate is frequently formed when nutrient solutions are made alkaline, this may account for the discordant results reported in the literature as to the availability of certain forms of iron. By omitting calcium from the culture solution iron can be maintained in a form available for growth in alkaline solutions by the addition of sodium citrate. In such solutions the maximum growth of Chlorella occurred at pH 7.5. The alkaline limit for growth has not been established as yet. In investigating the availability of iron at varying concentrations of the hydrogen ion, changes in the pH value of the solution during the course of an experiment should also be taken into account. This is especially important in unbuffered solutions. The differential absorption of the ions of ammonium salts may cause a marked increase in the hydrogen ion concentration, which in turn will cause an increase in the solubility of iron. In strongly buffered solutions as used in these experiments this effect is slight.     

The Status of Wright's Stain and Some Suggestions as to its use

A questionnaire was sent to a number of the larger hospitals and laboratories of the United States in order to determine the degree of satisfaction experienced in employing Wright's stain. From a survey of the answers received, there seems little justification for poor results with this stain. It is probable that the great majority of unsatisfied users work in laboratories where the stain is used so seldom that proper manipulation is not always attained. The writer offers a few simple, but very important precautions to be observed.     

Detergent compatible alkaline lipase produced by marine Bacillus smithii BTMS 11

Bacillus smithii BTMS 11, isolated from marine sediment, produced alkaline and thermostable lipase. The enzyme was purified to homogeneity by ammonium sulfate precipitation and ion exchange chromatography which resulted in 0.51 % final yield and a 4.33 fold of purification. The purified enzyme was found to have a specific activity of 360 IU/mg protein. SDS-PAGE analyses, under non-reducing and reducing conditions, yielded a single band of 45 kDa indicating the single polypeptide nature of the enzyme and zymogram analysis using methylumbelliferyl butyrate as substrate confirmed the lipolytic activity of the protein band. The enzyme was found to have 50 °C and pH 8.0 as optimum conditions for maximal activity. However, the enzyme was active over wide range of temperatures (30–80 °C) and pH (7.0–10.0). Effect of a number of metal salts, solvents, surfactants, and other typical enzyme inhibitors on lipase activity was studied to determine the novel characteristics of the enzyme. More than 90 % of the enzyme activity was observed even after 3 h of incubation in the presence of commercial detergents Surf, Sunlight, Ariel, Henko, Tide and Ujala indicating the detergent compatibility of B. smithii lipase. The enzyme was also found to be efficient in stain removal from cotton cloths. Further it was observed that the enzyme could catalyse ester synthesis between fatty acids of varying carbon chain lengths and methanol with high preference for medium to long chain fatty acids showing 70 % of esterification. Results of the study indicated scope for application of this marine bacterial lipase in various industries.     

Catalytic properties of E. coli tryptophanase in a 50% aqueous solution of methanol and dimethylformamide

Tryptophanase from E. coli retains its ability to form quinonoid intermediate with L-alanine in water--methanol and water--dimethylformamide (1:1 v/v) solutions. Under these conditions the enzyme catalyzes decomposition of S-o-nitrophenyl-L-cysteine (SOPC) to o-nitrophenylthiol, pyruvate and ammonium ion. The enzyme's affinity for this substrate increases on going from water to water-organic solvents whereas the reaction rate decreases. In 50% methanol tryptophanase catalyzes the formation of L-tryptophan from indole and SOPC; in the mixture of 2H2O and C2H3O2H (1:1) the enzymatic isotope exchange of alpha-proton of L-phenylalanine with complete retention of configuration was observed.     

Effects of Alkali Halides and Other Inorganic Salts on Radiation Inactivation of Taka-amylase in Aqueous System

Effects of alkali halides on radiation inactivation of Taka-amylase A were investigated in connection with their synergistic action to the radiation inactivation of microorganisms. Effective concentrations of these halides in protection against irradiation inactivation of this enzyme were well corresponded to that observed in the synergistic action to microorganisms. Removal of dissolved oxygen from halide solutions decreased their protective effect. Irradiated alkali halide solutions of relatively higher concentrations exhibited the inactivating action to this enzyme, and various types of addition effects of halogens and halogenoxyacids were observed with or without irradiation. Sodium nitrite showed effective protection even with higher radiation doses, and the heavy metal cations, Cu⁺⁺ and Fe⁺⁺⁺, also gave a protective action in low concentrations. From these observations, the possible interpretation on the mode of protection of alkali halides was attempted.     

Production and characterization of haloalkaline protease from ascidian-associated <Emphasis Type="Italic">Virgibacillus halodenitrificans</Emphasis> RSK CAS1 using marine wastes

A marine ascidian-associated bacterium, Virgibacillus halodenitrificans RSK CAS1, was optimized for protease production by response surface methodology using marine waste as substrate. The central composite design was employed, and the optimal medium constituents for maximum protease production (1461.11 U/ml) were determined to be shrimp shell powder (15.32 g/l), casein (5.37 g/l), MgSO₄ (3.0 g/l) and NaCl (55.31 g/l). The protease was purified from the culture supernatant to homogeneity in a three-step procedure consisting of ammonium sulfate precipitation, ion exchange chromatography (DEAE-cellulose column) and gel-filtration chromatography (Sephadex G-75 column), resulting in a 8.7-fold-change in purified protein. This protein had a specific activity of 1,086.78 U/mg and a molecular weight of 21 kDa. It exhibited optimal activity at 50 °C, pH 9 and 25 % NaCl. The significant stability of this protein at higher levels of salt, metal ions, organic solvents and commercial detergents and at higher, temperature, as well as its application as a cleaning additive in blood stain removal, suggests its possible use the laundry detergent industry.     

A study of 2-cyanopyridine addition products in the coordination sphere of Ni(II)

The coordination of 2-cyanopyridine molecule to Ni(II) atom promotes a nucleophilic addition of solvent molecules (water, methanol, ethanol) to the nitrile group. The addition of water leads to the formation of solid complexes containing pyridine-2- carboxamide as a chelate ligand. An analogous reaction of 2-cyanopyridine with NiX₂ (X = Cl, Br, I, NCS) in methanolic solutions gives, however, complexes containing two or three molecules of O-methylpyridine-2-carboximidate. No nucleophilic addition of solvent occurred with 3- and 4-cyanopyridine under the same reaction conditions.The complexes under study exhibit an octahedral geometry. The structure and the mode of the ligand coordination have been determined by IR spectra.     

Stability study of Azure B, Eosin Y and commercial Romanowsky Giemsa stock solutions using high performance liquid chromatography

Summary The stability of Azure B and Eosin Y in stock solutions of the individual compounds as well as in mixtures of the two dyes was studied. The purpose of the study of these two essential constituents of the Romanowsky Giemsa stain, commonly used in cytology and histology, was to select a stable mixture as a definitive stock solution. Two specific high performance liquid chromatographic methods were used to monitor qualitative and quantitative changes in solutions. Several parameters influencing the stability of Azure B were examined e.g. the type of counter ion the presence of Eosin Y and the type of solvent used. The second part focused on the stability of Eosin Y in mixtures with different cationic dyes submitted to high temperatures. In conclusion, an Azure B SCN-Eosin Y acid mixture in dimethylsulphoxide (concentrations 0.75% and 0.12%, respectively) was selected as being the most appropriate composition of a stock solution for the Romanowsky Giemsa stain.     

Theoretical and experimental study of the complexation of valinomycin with ammonium cation

The interactions of valinomycin, macrocyclic depsipeptide antibiotic ionophore, with ammonium cation NH4+ have been investigated. Using quantum mechanical density functional theory (DFT) calculations, the most probable structure of the valinomycin-NH4+ complex species was predicted. In this complex, the ammonium cation is bound partly by three strong hydrogen bonds to three ester carbonyl oxygen atoms of valinomycin and partly by somewhat weaker hydrogen bonds to the remaining three ester carbonyl groups of the valinomycin ligand. The strength of the valinomycin-NH4+ complex was evaluated experimentally by capillary affinity electrophoresis. From the dependence of valinomycin effective electrophoretic mobility on the ammonium ion concentration in the background electrolyte, the apparent binding (association, stability) constant (Kb) of the valinomycin-NH4+ complex in methanol was evaluated as log Kb = 1.52 +/- 0.22.     

Evaluation of sample extraction methods for proteomic analysis of coniferous seeds

In this study, three methods of protein extraction from the seeds of the Chinese fir were compared by examining the quality (including the number of protein spots observed) in the two-dimensional gel electrophoresis (2-DE), obtained by isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis of the total released protein. Three protein extraction methods were: TCA-acetone precipitation, SDS extraction/acetone precipitation, and phenol extraction methanol/ammonium acetate precipitation. The results showed that TCA-acetone precipitation was the most effective method for protein extraction; it gave the highest yield of total protein (8.9 mg protein per g seed weight) and the greatest number of proteins spots (1,034 spots) on the 2-DE gel. Further, several proteins were identified by liquid chromatography mass spectrometry (LC MS/MS), which are legumin-like storage protein, similar to AMP binding/acetate-CoA ligase, similar to 40S ribosomal protein S20, actin, ascorbate peroxidase, Similar to cysteine synthase, and unknown protein. These data demonstrates that TCA-acetone precipitation followed by 2-DE and LC MS/MS is a suitable method for proteomic analysis of coniferous species, such as Chinese fir and provides a valuable starting point for similar proteomic analysis of other coniferous tree species.     

X-ray structure of the quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa: basis of substrate specificity

The homodimeric enzyme form of quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa ATCC 17933 crystallizes readily with the space group R3. The X-ray structure was solved at 2.6 A resolution by molecular replacement.Aside from differences in some loops, the folding of the enzyme is very similar to the large subunit of the quinoprotein methanol dehydrogenases from Methylobacterium extorquens or Methylophilus W3A1. Eight W-shaped beta-sheet motifs are arranged circularly in a propeller-like fashion forming a disk-shaped superbarrel. No electron density for a small subunit like that in methanol dehydrogenase could be found. The prosthetic group is located in the centre of the superbarrel and is coordinated to a calcium ion. Most amino acid residues found in close contact with the prosthetic group pyrroloquinoline quinone and the Ca(2+) are conserved between the quinoprotein ethanol dehydrogenase structure and that of the methanol dehydrogenases. The main differences in the active-site region are a bulky tryptophan residue in the active-site cavity of methanol dehydrogenase, which is replaced by a phenylalanine and a leucine side-chain in the ethanol dehydrogenase structure and a leucine residue right above the pyrrolquinoline quinone group in methanol dehydrogenase which is replaced by a tryptophan side-chain. Both amino acid exchanges appear to have an important influence, causing different substrate specificities of these otherwise very similar enzymes. In addition to the Ca(2+) in the active-site cavity found also in methanol dehydrogenase, ethanol dehydrogenase contains a second Ca(2+)-binding site at the N terminus, which contributes to the stability of the native enzyme.     

Ammonium Ion Currents in the Squid Giant Axon

Voltage-clamp studies on intact and internally perfused squid giant axons demonstrate that ammonium can substitute partially for either sodium or potassium. Ammonium carries the early transient current with 0.3 times the permeability of sodium and it carries the delayed current with 0.3 times the potassium permeability. The conductance changes observed in voltage clamp show approximately the same time course in ammonium solutions as in the normal physiological solutions. These ammonium ion permeabilities account for the known effects of ammonium on nerve excitability. Experiments with the drugs tetrodotoxin (TTX) and tetraethyl ammonium chloride (TEA) demonstrate that these molecules block the early and late components of the current selectively, even when both components are carried by the same ion, ammonium.