Staining Mitochondria With Hematoxylin After Formalin-Sublimate Fixation; A Rapid Method

Mitochondria were stained in liver, kidney, pancreas, adrenal and intestinal mucosa of rat and mouse. Tissues 1 mm thick, were fixed in a mixture of saturated aqueous HgCl₂, 90 ml; formalin (37-38% HCHO), 10 ml, at room temperature (25°C) for 1 hr. Deparaffinized sections 3-4μ thick were treated with Lugol's iodine (U.S.P.) followed by Na₂S₂O₃ (5%), rinsed in water and the ribonucleic acid removed by any of the following procedures: 0.2 M McIlavaine's buffer, pH 7.0, 2 hr, or 0.2 M phosphate buffer, pH 7.0, 2 hr at 37°C; 0.1% aqueous ribonuclease, 2 hr at 37°C; 5% aqueous trichloracetic acid overnight at 37°C; or 1% KOH at room temperature for 1 hr. After washing in water, sections were treated with a saturated solution of ferric ammonium alum at 37°C for 8-12 hr and colored by Regaud's ripened hematoxylin for 18 hr. They were then differentiated in 1% ferric ammonium alum solution while under microscopic observation.     

Gelatinization mechanism of potato starch

The non-Newtonian behavior and dynamic viscoelasticity of potato starch (Jaga kids red ’90, 21.0% amylose content) solutions after storage at 25 and 4°C for 24 h were measured with a rheogoniometer. The flow curves, at 25°C, of potato starch showed plastic behavior >1.0% (w/v) after heating at 100°C for 30 min. A gelatinization of potato starch occurred above 1.0% at room temperature. A very large dynamic viscoelasticity was observed when potato starch solution (3.0%) was stored at 4°C for 24 h and stayed at a constant value with increasing temperature. A small dynamic modulus of potato starch was observed upon addition of urea (4.0 M) at low temperature (0°C) even after storage at 25 and 4°C for 24 h. A small dynamic modulus was also observed in 0.05 M NaOH solution. Possible models of gelatinization and retrogradation mechanism of potato starch were proposed.     

Effect of galactomannan addition on the thermal behaviour of κ-carrageenan gels

Gelation/melting cycles of κ-carrageenan/galactomannan (guar, tara and locust bean gums) binary systems have been studied by measuring dynamic rheological parameters. Two experimental conditions were used, (i) the total polysaccharide concentration was kept at 1% and the κ-carrageenan/galactomannan ratio fixed at 4:1 and (ii) the κ-carrageenan concentration was fixed at 0·75% and the galactomannan content varied from 0% to 1·2%. A thermal hysteresis was observed for all mixed systems and was found to depend on the galactomannan used. From a comparison of the gelation temperature (T_g) and melting temperature (T_m) to values obtained with κ-carrageenan alone, it was suggested that galactomannan interferes with gel structure by the formation of a secondary network provided that the M/G ratio is high enough.     

Structure of potato starch pastes in the ageing process by the measurement of their dynamic moduli

The effect of ageing on the rheological properties of potato starch pastes was investigated by measurement of dynamic viscoelasticity. The value of the storage modulus (G′) increased rapidly with time for the first few hours, but attained equilibrium after a long period of ageing. The relation between the concentration and the storage modulus showed a c^1·6 dependence. The Avrami exponent (n) obtained in this study was 0·66 and deviated significantly from the value (n = 1), corresponding to rod-like growth of crystal. The rate of storage modulus increase decreased with increasing concentrations up to 10%, and at higher concentrations little or no increase was observed. In addition, the rate of storage modulus increase increased dramatically when the NaCl concentration was raised to levels of 0·01 or above. An even greater increase in rate occurs on addition of AlCl₃.     

Pretreatment of Cells of Klebsiella pneumoniae with 50% (v/v) Dimethylsulfoxide Yields Purified Deoxyribonucleic Acid of Low Polysaccharide Content

The flow behavior and dynamic viscoelasticity of welan gum solutions were measured with a rheogoniometer. The welan gum showed shear-thinning behavior at a concentration of 0.1%, but plastic behavior above 0.3% at 25°C. The dynamic viscoelasticity increased with increasing concentration, and was scarcely changeable with increasing temperature even at 80°C. Gelation did not occur even in a polysaccharide concentration of 1.0% at low temperature (0°C). An increase of the dynamic modulus was not observed on the addition of CaCl₂ (6.8 mm). The dynamic viscoelasticity of welan gum solution was scarcely changeable in a wide range of pH from 2 to 12. The dynamic modulus was also scarcely changeable on addition of urea (4.0 m). Possible mode of intramolecular associations between the OH-4 of the d-glucosyl residue and the adjacent hemiacetal oxygen atom of the l-rhamnosyl residue, and between the methyl group of the l-rhamnosyl residue and the adjacent hemiacetal oxygen atom of the d-glucosyl residue were proposed.     

Staining of fresh, Undecalcified, thin Bone Sections

Fresh, undecalcified bone sections can be reproducibly and reliably stained by any of the following procedures: (A) Basic fuchsin, 1% in 30% alcohol, 48 hr, 22°C. (B) AgNO₃, 0.033 M, 48 hr, 22°C; washing 48 hr in a large volume of distilled water; exposure to light to develop the color. (C) Metallic sulfides (Co⁺⁺, Pb⁺⁺, Hg⁺⁺, Cu⁺⁺): the nitrate of the metal, 0.033 M, 48 hr, 22°C; then Na₂S, 0.033 M, 48 hr, 22° C. (D) Alizarin Red S, 0.1% solution in distilled water, 48 hr, 22°C; differentiated 48 hr at 22°C in weakly alkaline water, pH about 8. (E) KMnO₄: boiling 8-10 min in a 0.1 N, solution. With the exception of D the surface stain must be ground off the section for microscopic examination of its interior. Stain concentration, time and temperature can be altered to suit specific needs.     

Activity of magnetite-immobilized catalase in hydrogen peroxide decomposition

The present work analyzes the activity in decomposition of H₂O₂ using magnetite-immobilized catalase. The support of catalase is a glutaraldehyde-treated magnetite (Fe₃O₄). The data obtained in the H₂O₂ decomposition are analyzed. The fitting of the initial rate of the H₂O₂ decomposition versus hydrogen peroxide concentration data is discussed using a specific program for enzyme kinetics modeling (Leonora). The free catalase from Aspergillus niger (3.5 or 10 U/mL) does not show substrate inactivation up to 0.4 M H₂O₂. The immobilized catalase at low catalyst concentration shows substrate inhibition. Using 1 mg/mL of supported catalase the predicted maximum activity is higher than in the case of the free catalase at similar catalase concentration, although the optimum temperature is lower (40 °C versus 60 °C).     

Behavioral effects of low dissolved oxygen on the bivalve Macoma balthica

Hypoxia, a dissolved oxygen concentration (DO) below 2 mg l^– 1, is a significant stressor in many estuarine ecosystems. Many sedentary organisms, unable to move to avoid hypoxic areas, have metabolic and behavioral adaptations to hypoxic stress. We tested the effects of hypoxia on the behavior and mortality of the clam Macoma balthica, using four levels of dissolved oxygen in flow-through tanks. We used five replicates of each of four treatments: (1) Hypoxic (DO mean ± SE = 1.1 ± 0.06 mg O₂ l^– 1), (2) Moderately hypoxic (DO 2.6 ± 0.05 mg O₂ l^– 1), (3) Nearly normoxic (DO 3.2 ± 0.04 mg O₂ l^– 1), (4) Normoxic (DO = 4.9 ± 0.13 mg O₂ l^– 1). We lowered the dissolved oxygen with a novel fluidized mud-bed, designed to mimic field conditions more closely than the common practice of solely bubbling nitrogen or other gasses. This method for lowering the DO concentrations for a laboratory setup was effective, producing 1.4 l min^–1 of water with a DO of 0.8 mg O₂ l^– 1 throughout the experiment. The setup greatly reduced the use of compressed nitrogen and could easily be scaled up to produce more low-DO water if necessary. The lethal concentration for 50% of the M. balthica population (LC₅₀) was 1.7 mg O₂ l^– 1 for the 28-day experimental period. M. balthica decreased its burial depth under hypoxic and moderately hypoxic (~2.5 mg O₂ l^– 1) conditions within 72 hours of the onset of hypoxia. By the sixth day of hypoxia the burial depth had been reduced by 26 mm in the hypoxic tanks and 10 mm in the moderately hypoxic tanks. Because reduced burial depth makes the clams more vulnerable to predators, these results indicate that the sub-lethal effects of hypoxia could change the rate of predation on M. balthica in the field.     

Staining Tissue of the Central Nervous System with Luxol Fast Blue and Neutral Red

The tissue is fixed in 10% neutral saline formalin for 1 day to 3 wk depending on the size of the block, dehydrated and embedded in paraffin. The sections are stained at 57° C for 2 hr, then at 22° C for 30 min, in a 0.0125% solution of Luxol fast blue in 95% alcohol acidified by 0.1% acetic acid. They are differentiated in a solution consisting of: Li₂CO₃, 5.0 gm; LiOH-H₂O, 0.01 gm; and distilled water, 1 liter at 0-1° C, followed by 70% alcohol, and then treated with 0.2% NaHSO₃. They are soaked 1 min in an acetic acid-sodium acetate buffer 0.1 N, pH 5.6, then stained with 0.03% buffered aqueous neutral red. Sections are washed in distilled water, 1 sec, then treated with the following solution: CuSO₄·5H₂O, 0.5 gm; CrK(SO₄)₂·12H₂O, 0.5 gm; 10% acetic acid, 3 ml; and distilled water, 250 ml. Dehydration, clearing and covering complete the process. Myelin sheaths are stained bright blue; meninges and the adventitia of blood vessels are blue; red blood cells are green. Nissl material is stained brilliant red; axon hillocks, axis cylinders, ependyma, nuclei and some cytoplasm of neuroglia, media and endothelium of blood vessels are pink.     

A gel network constituted by rigid schizophyllan chains and nonpermanent cross-links

This work reports a gel network formed by rigid schizophyllan (SPG) chains with Borax as a cross-linking agent. The formed cross-links are non-permanent and somewhat dynamic in nature because the cross-linking reaction is governed by a complexation equilibrium. Gelation processes are traced by dynamic viscoelastic measurements to examine the effects of Borax content, SPG concentration, temperature, salt concentration, salt type, and strain. The first-order kinetic model containing three parameters, t(0) (induction time), 1/tau(c) (gelation rate), and (saturated storage modulus), is successfully applied to describe the gelation of the SPG-Borax system. Gelation occurs faster at higher Borax content, higher SPG concentration, higher salt concentration, or lower temperature. Moreover the gelation is cation-type-specific. Storage modulus is a linear function of both Borax content and SPG concentration. The linear relationship between storage modulus and Borax content can be explained by a modified ideal rubber elasticity theory with a front factor alpha to take into account the presence of ineffective cross-links and the effect of SPG chain rigidity. On the other hand, the linear dependence of storage modulus on SPG concentration could be explained on the basis of chain-chain contacting behavior of extended SPG chains. Apparent activation energy and cross-linking enthalpy are calculated to be -74.5 and -32.4 kJ/mol for the present system. Strain sweep measurements manifest that the elasticity behavior of this gel starts to deviate from Gaussian-chain network at a small strain of 10%.     

In vitro excystment of the metacercaria of Plagiorchis species 1 (Trematoda, Plagiorchiidae)

1986. In vitro excystrnent of the metacercaria of Plagiorchis species 1 (Trematoda, Plagiorchiidae). International Journal for Parasitology 16: 641–645. An optimal hatching success of Plagiorchis species 1 metacercariae (100% excystment, active metacercariae, mean hatching speed 2–10 min, lowest variance of the mean speed) was observed after pretreatment in an HCl-pepsin solution at pH 2.0 and 42°C for 60–70 min, and incubation in a hatching medium at 42 °C and pH 7.3–8.0 with a bile salt (Nacholate), NaHCO₃, and a reductant (cysteine with 100% N₂). The minimum conditions for nearly 100% excystment with lower hatching speeds and higher variances were the presence of NaHCO₃, an oxygen concentration reduced to about 3% in the gas phase, pH> 7.3 and a temperature near 30°C if Na-cholate was absent, or in the presence of the bile salt, a phosphate buffer at pH> 5.0 and room temperature only. Obviously some hatching factors acted interchangeably with compensation for missing stimuli by others. The effect of the bile salt was comparable with that of other surfactants. The metacercariae excysted in nonenzymatic media, which implies an active hatching mechanism.     

Immobilization of soybean seed coat peroxidase on polyaniline: Synthesis optimization and catalytic properties

Soybean seed coat peroxidase (SBP) was immobilized on various polyaniline-based polymers (PANI), activated with glutaraldehyde. The most reduced polymer (PANIG₂) showed the highest immobilization capacity (8.2 mg SBP g^-1 PANIG₂). The optimum pH for immobilization was 6.0 and the maximum retention was achieved after a 6-h reaction period. The efficiency of enzyme activity retention was 82%. When stored at 4°C, the immobilized enzyme retained 80% of its activity for 15 weeks as evidenced by tests performed at 2-week intervals. The immobilized SBP showed the same pH-activity profile as that of the free SBP for pyrogallol oxidation but the optimum temperature (55°C) was 10°C below that of the free enzyme. Kinetic analysis show that the K_m was conserved while the specific V_max dropped from 14.6 to 11.4 µmol min^-1 µg^-1, in agreement with the immobilization efficiency. Substrate specificity was practically the same for both enzymes. Immobilized SBP showed a greatly improved tolerance to different organic solvents; while free SBP lost around 90% of its activity at a 50% organic solvent concentration, immobilized SBP underwent only 30% inactivation at a concentration of 70% acetonitrile. Taking into account that immobilized HRP loses more than 40% of its activity at a 20% organic solvent concentration, immobilized SBP performed much better than its widely used counterpart HRP.     

Synergistic Interaction between Kappa-Carrageenan and Locust-bean Gum in Aqueous Media

Although neither kappa-carrageenan nor locust-bean gum gelled alone, a mixed aqueous solution of the above gums gave a gel at the concentration of 0.6% total gums in a range of low temperatures. The solution also gelled even at the concentration of 0.4% total gums in the presence of 0.1% KC1. The maximum dynamic modulus was obtained with a series of the samples composed of kappa-carrageenan and locust-bean gum in the mixing ratios of 1:1 and 3:1 at the concentration of 0.6 and 0.8% total gums at 0°C. The dynamic modulus of a mixed solution of kappa-carrageenan and locust-bean gum was not influenced by pH between pH 7.0 to 11.5, but decreased in the acidic range.

We concluded that intermolecular interactions, at low temperature, between kappa-carrageenan and locust-bean gum may take place on the K⁺-bridge of the former and the backbone of the latter molecule at low concentrations, but at high concentration of the gums, self-association of kappa-carrageenan molecules might also occurred.     

Improving the Staining Qualities of Toluidin Blue

To a 1.0% filtered aqueous solution of toluidin blue add drop by drop 4-5 ml of either a saturated aqueous solution of HgCl₂ or of KI. Collect the resulting dark precipitate on a filter paper and wash it with numerous small quantities of distilled water applied to both inside and outside of the filter paper. Wash until the drippings are distinctly blue (equivalent to about a 0.05% dye solution). Remove the paper and its contents from the funnel and dry either at room temperature or at 37°C. When dry, the treated dye can be brushed off the paper and stored. To prepare a staining solution add a weighed amount (0.12 gm if derived from the HgCl₂ treatment, or 0.3 gm if from KI) to 100 ml of distilled water. This insures a saturated solution in either case and gives a satisfactory stain with most sections in 10-30 min. Thionin and other members of the thiazine dyes also showed improvement in staining qualities after this treatment.     

Solid substrate cultivation of Gibberella fujikuroi on an inert support

Growth of Gibberella fujikuroi on Amberlite, an inert support, and gibberellic acid (GA₃) production was studied in glass columns under different conditions of temperature and water activity (a_w). Maximum biomass concentration and GA₃ production were respectively 40 (mg/g inert support) and 0.73 (mg/g inert support). While high specific growth rates were obtained, low initial nitrogen resulted in low biomass concentrations. Maximum GA₃ (31°C, a_w=0.985) was not produced by the maximum concentration of biomass (25°C, a_w=0.992). Peaks in the rate curves of either outlet gas, CO₂ or O₂, occurred on exhaustion of urea indicating, for future works, just when to feed the culture additional nitrogen.     

Stabilization of quaternary structure and activity of bovine liver catalase through encapsulation in liposomes

Bovine liver catalase was encapsulated in an aqueous phase of the phospholipid vesicle (liposome) to improve the stability of its tetrameric structure and activity. The catalase-containing liposomes (CALs) prepared were 30, 50 and 100 nm in mean diameters (CAL₃₀, CAL₅₀ and CAL₁₀₀, respectively). The CAL₁₀₀ included the types I, II and III based on the amounts of catalase encapsulated. The CAL₃₀, CAL₅₀ and CAL₁₀₀-I contained one catalase molecule per liposome, and the CAL₁₀₀-II and CAL₁₀₀-III on average 5.2 and 17 molecules, respectively. The storage stability of catalase in either CAL system was significantly increased compared to that of free catalase at 4 °C in a buffer of pH 7.4. At 55 °C, free catalase was much more deactivated especially with decreasing its concentration predominantly due to enhanced dissociation of catalase into subunits while it was so done at excessively high enzyme concentration mainly due to enhanced formation of catalase intermolecular aggregates. Among the three types of CAL₁₀₀, the CAL₁₀₀-II showed the highest thermal stability, indicating that an excess amount of catalase in the CAL₁₀₀-III was also disadvantageous to maintain an active form of the catalase even in liposome. In the CAL₁₀₀-III, however, the stability of catalase was significantly improved compared to that of free catalase at the same concentration. The CAL thermal stability was little affected by the liposome size as observed in the CAL₃₀, CAL₅₀ and CAL₁₀₀-I. An intrinsic tryptophan fluorescence of the catalase recovered from the CAL₁₀₀-II thermally treated at 55 °C revealed that a partially denatured catalase molecule was stabilized through its hydrophobic interaction with liposome membrane. This interaction depressed not only dissociation of catalase into subunits but also formation of an inactive intermolecular aggregate between the catalase molecules in a liposome. Furthermore, either type of CAL₁₀₀ showed a higher stability than free catalase in the successive decompositions of 10 mM H₂O₂ at 25 °C mainly because the H₂O₂ concentration was kept low inside liposomes due to the permeation barrier of the lipid membrane to H₂O₂.     

Multi-temperature effects on Hill reaction activity of barley chloroplasts

1. 1. The relationship between temperature and Hill reaction activity has been investigated in chloroplasts isolated from barley (Hordeum vulgare L. cv. Abyssinian).

2. 2. An Arrhenius plot of the photoreduction of 2,6-dichlorophenolindophenol (DCIP) showed no change in slope over the temperature range 2–38 °C. The apparent Arrhenius activation energy (E_a) for the reaction was 48.1 kJ/mol.

3. 3. In the presence of an uncoupler of photophosphorylation, methylamine, the E_a for DCIP photoreduction went through a series of changes as the temperature was increased. Changes were found at 9, 20, 29 and 36 °C. The E_a was highest below 9 °C at 63.7 kJ/mol. Between 9 and 20 °C the E_a decreased to 40.4 kJ/mol and again to 20.2 kJ/mol between 20 and 29 °C. Between 29 and 36 °C there was no further increase in activity with increasing temperature. The temperature-induced changes at 9, 20 and 29 °C were reversible. At temperatures above 36 °C (2 min) a thermal and largely irreversible inactivation of the Hill reaction occurred.

4. 4. Temperature-induced changes in E_a were also found when ferricyanide was substituted for DCIP or gramicidin D for methylamine. The addition of an uncoupler of photophosphorylation was not required to demonstrate temperature-induced changes in DCIP photoreduction following the exposure of the chloroplasts to a low concentration of cations.

5. 5. The photoreduction of the lipophilic acceptor, oxidized 2, 3, 5, 6-tetramethyl-p-phenylenediamine, also showed changes in E_a in the absence of an uncoupler.

6. 6. The temperature-induced changes in Hill activity at 9 and 29 °C coincided with temperature-induced changes in the fluidity of chloroplast thylakoid membranes as detected by measurements of electron spin resonance spectra. It is suggested that the temperature-induced changes in the properties and activity of chloroplast membranes are part of a control mechanism for regulation of chloroplast development and photosynthesis by temperature.

Determination of the maximum and minimum lethal temperatures (LT50) for Loxosceles intermedia Mello-Leitão, 1934 and L. laeta (Nicolet, 1849) (Araneae, Sicariidae)

In this study, the maximum and minimum lethal temperatures (LT₅₀) of L. intermedia and L. laeta were determined in two treatments: gradual heating (25–50°C) and cooling (25°C to −5°C), and 1 h at a constant temperature. In gradual temperatures change, L. intermedia mortality started at 40°C and the LT₅₀ was 42°C; for L. laeta, mortality began at 35°C and the LT₅₀ was 40°C. At low temperatures, mortality was registered only at −5°C for both species. In the constant temperature L. intermedia showed a maximum LT₅₀ at 35°C and L. laeta at 32°C; the minimum LT for both species was −7°C.     

The reactions of zirconium(IV) bromide and zirconium(III) bromide and chloride with ammonia and zirconium(IV) bromide with ammonium cyanide

Unlike ZrCl₄, ZrBr₄ is not ammonolysed in liquid ammonia at temperatures up to −33 °C. The existence of ammoniates ZrBr₄nH₃ (n = 17, 12 and 9) at −36 °C has been established; at room temperature, the hexammine ZrBr₄ · 6NH₃ is the stable species which becomes ZrBr₄ · 2NH₃ at 200 °C. When treated with an excess of NH₄CN in liquid ammonia, complete replacement of bromide ions by cyanide occurs to give an inseparable mixture of Zr(CN)₄ · 2NH₃ and NH₄Br. The chloride and bromide of zirconium(III) also undergo no ammonolysis in liquid ammonia; the ammoniates stable at room temperature are ZrCl₃ · 2.5NH₃ and ZrBr₃ · 6NH₃.     

Shifts in thermoregulatory patterns with pregnancy in the poikilothermic mammal—The naked mole-rat (Heterocephalus glaber)

1. 1. The naked mole-rat (Heterocephalus glaber) is a poikilothermic mammal. During gestation metabolic shifts that differ from both mammalian and reptilian thermoregulatory patterns occurred.

2. 2. Body temperature was directly dependent on ambient temperature. At low ambient temperatures the temperature differential (T_b − T_a) was approximately 3°C, whereas at higher ambient temperatures the temperature differential diminished.

3. 3. In early pregnancy (prior to week 3) oxygen consumption at low ambient temperatures was greater than that of non-reproductive animals. A maximal metabolic rate (3.2 ± 1.0 ml O₂ . g⁻¹ . h⁻¹) occurred at an ambient temperature of 27°C. Thereafter the endothermic pattern of metabolism with increasing ambient temperatures was evident. Oxygen consumption decreased with increasing ambient temperature to minimal rates of 1.2 ± 0.1 ml O₂ . g⁻¹ . h⁻¹ over the ambient temperature range of 31–34°C.

4. 4. Oxygen consumption in late pregnancy (1.8 ± 0.1 ml O₂ . g⁻¹ . h⁻¹) was not correlated with ambient temperature over the entire ambient temperature range measured (24–36°C).

5. 5. Differences in thermoregulation in early and late pregnancy may be attributed to different rates of heat loss as a consequence of (a) changes in surface area and body mass or (b) vascular changes. Furthermore the thermoregulatory changes in late pregnancy may indicate that maximal overall metabolic capacity had been reached, for peak resting metabolism (expressed per animal rather than per gram body mass) in early pregnancy was similar to observed metabolism in late pregnancy.

6. 6. The dissociation of metabolism from both ambient temperature and body temperature in late pregnancy could confer an energetic advantage to this arid dwelling underground inhabitant; for it may enable the breeding female to partition a greater portion of available energy into reproduction.