A Paraffin Method for Refractory Plant Materials

A modification of Zirkle's n-butyl alcohol method for dehydrating refractory plant material and embedding it in paraffin is described. The material is cut into small pieces and killed and fixed in CRAF III. It is dehydrated in Zirkle's n-butyl alcohol series, slightly modified. Infiltration is accomplished by adding chips of paraffin of low melting point (52°C.) to the bottle containing butyl alcohol and the tissues at a gradually increasing series of temperatures. The butyl-alcohol-paraffin mixture is gradually replaced by pure paraffin (melting point 56-58°C). The material is embedded in Fisher Tissuemat (56-58°C). Before microtoming, the block of embedded tissue is trimmed so that part of the specimen is exposed, and it is soaked in water until it cuts easily.     

Interim Report of the Committee on Histologic Mounting Media: Resinous Media

The Fisher “Permount” naphthalene polymer, the Hartman Leddon “H.S.R.” terpene polymer resin, a Monsanto polystyrene P-1, the Will Corporation “Diaphane” and “Green Diaphane”, and the du Pont “Lucite” methyl methacrylate polymer were examined, and the possibility of use of some other plastics was also explored. The first 5 mentioned were tested for color preservation of a variety of stains in comparison with Canada balsam and Clarite X. From this point of view polystyrene, the Hartman Leddon “H.S.R.” and the Fisher “Permount” resins were the most satisfactory, then the “Diaphanes”. Both “Permount” and “H.S.R.” show some yellowing. The H.S.R. with a melting point of 115°C, the Permount with 150°C. melting point, and the Polystyrene with a thermal denaturation point above 220°C. all excell Canada balsam in heat resistance. Trimethylbenzene, cymene and monoamylbenzene appear to be the best solvents for polystyrene. Mounts made in a solution of 20 g. polystyrene in 100 ml. trimethylbenzene can be packed flat slide to slide in 24 hours after mounting without sticking together.

This report is not intended to deprecate the use of other resinous mounting media which have not as yet been tested or compared with those mentioned herein.     

Formation and crystallization of amylose–monoglyceride complex in a starch matrix

The formation of amylose–lipid complexes in a gelatinized potato starch matrix was investigated using potato starch and glycerol monopalmitin. These complexes exist in two forms, with the amounts of each of the forms being dependent on the temperatures and durations of the pre-treatments.

Differential scanning calorimetry (DSC) was used to analyze transition temperatures and melting enthalpies, and thereby determine the amount of the complexes in the samples. X-ray diffraction analysis was used to investigate their crystallinity.

In measurements with DSC, form I started to melt at 88.5°C, and form II at 112.9°C. When complex form II was preheated at 100 or 110°C, its melting point rose to 116.3 and 119.7°C, respectively, because of an annealing effect. The same phenomenon occurred with complex form I: when preheated at 90°C, its melting point rose to 96.8°C. The crystal formation of form II appeared to be slower when treated at 110°C than at 100°C. Their maximum melting enthalpies were reached after about 24 h and 4 h of preheating, respectively. In X-ray diffraction analyses, form II showed a V-pattern, but form I did not. This indicates that form II is more crystalline than form I. It was possible to transform form I into form II when it was heat treated, because form I was then partially or totally melted.

As a comparison, the charged substance cetyltrimethylammonium bromide created complex form I with amylose in the starch matrix, but not form II.     

Thermal sensations during simultaneous warming and cooling at theh forearm: A human psychophysical study

1. 1.The forearm of 5 female subjects ws thermally stimulated by 2 sets of interposed servo-thermodes that respectively drove skin temperature at ±0.1°C.s⁻¹ for 25 s and then held it constant. Mean skin temperature remained constant. The sequence was repeated at adapting temperatures between 22.5 and 37.5°C.

2. 2.Thermal sensations, continuously reported by the position of a dial, were warmer for heterogeneous thermal stimuli than for homogeneous stimuli when mean skin temperature was greater than 30°C and cooler when less than 27.5°C.

3. 3.This phenomenon is inconsistent with a single additive contribution of “warm” and “cold” information to thermal sensations.

A Celloidin Infiltration-Frozen Section Sequence for Enhanced Preservation of Phosphatases in Bone

The effect of the following embedding procedures on the acid and alkaline phosphatase content of decalcified mouse tibiae has been studied: embedding in 23% gelatine for 18 hr at 37° C, embedding in paraffin wax in vacuo for 1 hr at 58° C, and impregnation with 4% celloidin in diethyl ether and ethanol at 4° C for 2-3 days. Unsupported tissues were also used to demonstrate these enzymes for comparison with the above procedures. Tibiae were first fixed in 10% neutral formalin at 4° C for 15 hr, decalcified in equal volumes of 2% formic acid and 20% sodium citrate at pH 4.9 for not more than 5 days and then washed in distilled water before carrying out the embedding schedules. The celloidin-impregnated tibiae were placed in 70% ethanol to harden the celloidin and then washed in distilled water for 1-2 hr. These tibiae and those embedded in gelatine were cast in a gelatine block which was then hardened in 10% neutral formalin at 4° C for 2 hr. Sections of these and unsupported tibiae were cut at 15 μ on a freezing microtome. Decalcified tibiae embedded and blocked in paraffin wax were sectioned at 15 μ on a base sledge microtome. The enzymes were demonstrated using the coupling azo dye method given by Pearse (Histochemistry, 1st Ed. 1954). The stable diazotates of 4 benzoyl amino 2-5 diethoxyanilene, 3 nitro toluidine and o-dianisidine were used. Of the embedding procedures paraffin wax embedding produced the greatest loss of both enzymes. Gelatine embedding and infiltration with celloidin were equally good for the demonstration of acid phosphatase but for alkaline phosphatase the celloidin method was superior. The gelatine embedded material did not produce consistently good results. Celloidin-impregnated tibiae could be stored without marked deterioration of the enzyme content for longer than gelatine-embedded tibiae.     

Lipase-catalyzed acylation of naringin with palmitic acid in highly concentrated homogeneous solutions

Acylation reactions of naringin with palmitic acid were performed by a lipase after formation of highly concentrated homogeneous solutions. Their initial naringin concentration was 840–950 mM, which is 20–60 times greater than that in organic solvent media. The overall productivity of highly concentrated solutions was more than 15 times greater than those of organic phase media. The addition of DMSO (20–40%, w/w) to substrate mixtures lowered the melting temperature of a naringin–palmitic acid mixture (1:1 molar ratio) to about 40 °C. Reactions at 80 °C apparently followed Michaelis–Menten kinetics despite extremely high substrate concentrations. As the temperature increased from 60 °C to 80 °C, the apparent viscosity of the highly concentrated solution decreased remarkably from 4.31 Pa s to 0.063 Pa s. An activation energy of 7.65 kcal/mol obtained in a range of 60–75 °C suggests a diffusion-control. On the other hand, an activation energy of 17.09 kcal/mol in a range of 75–90 °C indicates a reaction-control. The highest product conversion yield of 33% (mol/mol) was obtained in a 10 h reaction at 80 °C. Addition of activated molecular sieves to the highly concentrated solution increased the product conversion yield by 7% (mol/mol), suggesting that the original equilibrium was disrupted by removing water and then a new equilibrium was reached.     

Laboratory hints from the Literature: Department Devoted to Abstracts of books and papers from other Journals Dealing with stains and Microscopic Technic in General

A modification of Zirkle's n-butyl alcohol method for dehydrating refractory plant material and embedding it in paraffin is described. The material is cut into small pieces and killed and fixed in CRAF III. It is dehydrated in Zirkle's n-butyl alcohol series, slightly modified. Infiltration is accomplished by adding chips of paraffin of low melting point (52°C.) to the bottle containing butyl alcohol and the tissues at a gradually increasing series of temperatures. The butyl-alcohol-paraffin mixture is gradually replaced by pure paraffin (melting point 56-58°C). The material is embedded in Fisher Tissuemat (56-58°C). Before microtoming, the block of embedded tissue is trimmed so that part of the specimen is exposed, and it is soaked in water until it cuts easily.     

High temperature-induced changes in exopolysaccharides, lipopolysaccharides and protein profile of heat-resistant mutants of Rhizobium sp. (Cajanus)

A thermosensitive wild-type strain (PP201) of Rhizobium sp. (Cajanus) and its 14 heat-resistant mutants were characterized biochemically with regard to their cell surface (exopolysaccharides (EPSs) and lipopolysaccharides (LPSs)) properties and protein profile. Differences were observed between the parent strain and the mutants in all these parameters under high temperature conditions. At normal temperature (30 °C), only half of the mutant strains produced higher amounts of EPSs than the parent strain, but at 43 °C, all the mutants produced higher quantities of EPS. The LPS electrophoretic pattern of the parent strain PP201 and the heat-resistant mutants was almost identical at 30 °C. At 43 °C, the parent strain did not produce LPS but the mutants produced both kinds of LPSs. The protein electrophoretic pattern showed that the parent strain PP201 formed very few proteins at high temperature, whereas the mutants formed additional new proteins. A heat shock protein (Hsp) of 63–74 kDa was overproduced in all mutant strains.     

Temperature dependence of the germination of tomato seeds

1. 1.|The germination of tomato “C38” seeds exposed to periodical white incandescent light occurs from 6.0° ± 0.2°C to 37.5° ± 0.2°C, being rate-limited for 10.3° T 25.9°C, and elsewhere limited by the germination capacity.

2. 2.|Rate averages are linearly T-dependent outside their optimum range (25.9° T 29.5°C) and rate variances are typically heterogeneous.

3. 3.|The smooth curvilinear Arrhenius plot indicates that diffusion processes cannot be rate-limiting outside the interval 25.9° T 29.5°C, whereas phase transitions and (or) transconformation of proteins may limit the rate above 34.9°C and, by opposite effects, below 15.3°C.

4. 4.|The thermal communication between the environment and the germinating seed proceeds by a temperature signal which is quenched by random thermal noise at T 11.2°C and at T 34.0°C.

Geographic variation in field body temperature of sceloporus lizards

1. Using data from the literature, I assessed how broad climatic patterns affected field body temperatures (T_b’s) of lizards in the genus Sceloporus.

2. Sceloporus at temperate latitudes had mean T_b’s of 35°C throughout their elevational range. This pattern is associated with “tropical” temperatures that extend into high north latitudes during the summer and the relatively low elevations occupied by the lizards.

3. At tropical latitudes, mean T_b declined from 35°C at low elevations to 31°C at high elevations. This pattern is associated with low seasonal variation in temperature at tropical latitudes and the relatively high elevations occupied by the lizards.     

Microwave Irradiation as a Generator of Heat for Histological Fixation

The kitchen appliance known as the “radar oven” generates heat quickly in materials containing water. Protoplasm exposed to the irradiation can thus be denatured. The amount of heat generated is a function of the time of exposure and the intensity of the irradiation, and the size and specific heat of the tissue or organism being irradiated. But docs such heating have applicabiity to histological technique? One of four carcas temperaturea (approximately 60°, 70°, 77°, and 85 C) was generated in anaesthetized, adult hairless mice of both sexes. “Control” animals were not irradiated. Specimens of liver, kidney, lung, and (from males) testis were taken from the five groups; the tissue spedmens were dehydrated in tetrahydrofuran, embedded in paraffin, sectioned at 9 μm, and stained with hematoxylin and eosin. The preparations were suitable for histological examination. Each organ had an optimum temperature for histological fixation under the conditions of this experiment: liver, ∼70°; kidney, ∼77°; lung, ∼77°; and testis, ∼85 C. Heat fixation by microwave irradiation also shows some applicability to electron microscopical studies and to investigations of the blood vascular arrangements of organs.     

The effect of incubation temperatuer on oxygen consumption and organ growth in domestic-fowl embryos

1. 1.|Oxygen consumption and organ growth were measured in domestic-fowl embryos incubated at different temperatures (36, 38 and 40°C).

2. 2.|Embryonic oxygen consumption was highest at an incubation temperature of 40°C and lowest at 36°C. These differences were ascribed largely to variations in embryo size at different incubation temperatures.

3. 3.|At incubation temperatuers of 40 and 38°C, there was a plateau in oxygen consumption late in incubation, but this was not apparent at 36°C.

4. 4.|At 36°C, some tissues (e.g. eyeballs) were “spared” the repression of growth that characterized the embryo as a whole, while other tissues (e.g. stomach) incurred a much greater growth reduction. Similarly, at 40°C, stomach growth exceeded that of the embryo as a whole, while the eyeballs were largely spared the enhanced growth.

5. 5.|A simple index of tissue age revealed that, in general, there were consensual changes in tissue maturity and growth at different temperatures but that there were some disparities between growth and maturity in individual organs.

Superoxide Production by Stimulated Neutrophils: Temperature Effect

Activation of neutrophils results in a one-electron reduction of oxygen to produce the superoxide anion and other oxygen-derived, microbicidal species. Evidence from many kinetic studies of oxygen-derived radicals generated by stimulated neutrophils in vitro shows that radical production is optimal at 37°C but only lasts several minutes and then rapidly subsides. These findings support the widely held perception that the neutrophil's “oxidative burst” is a transitory event that peaks within minutes of stimulation and ends shortly thereafter. However, while some studies have shown that under controlled conditions stimulated neutrophils can generate superoxide continuously for several hours, others have observed that the superoxide formation by neutrophils stimulated in buffer at 37°C does not persist. To reconcile the conflicting findings and to better understand neutrophil function, we have reinvestigated the effect of temperature on the kinetics of radical generation by PMA-stimulated cells. Electron paramagnetic resonance spectroscopy coupled with spin-trapping and SOD-inhibitable ferricytochrome c reduction were used to monitor superoxide production by neutrophils stimulated at either 25°C or 37°C in RPMI 1640 medium or in Hank's balanced salt solution. When oxygen was supplied continuously, neutrophils stimulated at 25°Cin buffer or in medium generated superoxide for several hours but at 37°C. particularly in HBSS, O₂-formation strikingly and rapidly decreased. This cessation of superoxide generation was reversible by lowering the temperature back to 25°C. These data imply that in vivo neutrophils may be capable of generating oxy-radicals for prolonged periods. In part, our results may also explain the often observed termination of neutrophil-derived radical formation in vitro and help to dispel the perception that neutrophil-derived oxy-radical production is an ephemeral phenomenon.     

Spherulitic crystallization of gelatinized maize starch and its fractions

Binary systems of polymers often display spherulitic morphologies after cooling from the melt, but these phenomena have rarely been reported among food polymers of native-size. Here we report the observation of spherulitic and other morphologies in gelatinized maize starch. The morphology could be manipulated by choosing polymer compositions and kinetic regimes. Spherulites (10 μm diameter) formed from gelatinized high-amylose maize starches and purified amylose at cooling rates of order of magnitude 100 °C/min. They were more numerous and exhibited a higher melting point the greater the ratio of amylose to amylopectin. Rapid cooling rates (150–500 °C/min) resulted in a more even distribution of smaller spherulites. Very rapid (liquid nitrogen quench) or slow (0.1–1 °C/min) cooling rates resulted in mixed morphology, as did addition of 15 or 60% (w/w) sucrose to a 10% (w/w) dispersion of high-amylose starch (HAS). Spherulites were observed in aqueous suspensions of high-amylose maize starch between 5 and 30% (w/w). Lower starch concentrations resulted in a broader size distribution and spherulites of more distinct shape. WAXS patterns of B-type were observed. Negatively birefringent spherulites predominated, but positive spherulites were found. The spherulite melting range overlapped with that for amylose–lipid complex. Evidence indicated that micro-phase separation takes place when a holding period at 95 °C follows gelatinization at 180 °C. Despite the high maximum temperature of treatment (180 °C) there was evidence for a memory effect in samples of 30% HAS. Spherulite morphology closely resembled that of native starch granules in very early stages of development.     

Diethylene Glycol Distearate Embedding and Ultramicrotome Sectioning for Light Microscopy

Diethylene glycol distearate can be used as an embedding medium for light microscopy. Two infiltration changes of about 6 hr each in the melted wax (melting point 47-52 C) are required before the final embedding which is done in 00 gelatin capsules for sectioning in the ultramicrotome by the procedure used in electron microscopy. Serial sections 1-2 μ thick can be cut without difficulty. No cooling devices are necessary for trimming and sectioning at laboratory temperature. Sections rarely become detached from the slides. The staining characteristics of the tissues are the same as when embedded in paraffin. For fluorescence microscopy, essentially the same procedure is followed. Tissues are not distorted and the intracellular structures are well preserved.     

An Aceto-Osmium Method for Demonstrating Nematodes in Citrus Roots

Difficulties are encountered in observing nematodes in citrus feeder roots because of the presence of suberin and other unsaturated compounds. To obviate these difficulties, infected citrus roots, either fresh or preserved, are immersed in a covered jar for 2 hr at 52° C in a solution composed of distilled water, 16 parts; 10% acetic acid, 10 parts; and 2% aqueous osmium tetroxide, 2 parts. The stained, blackened roots are washed in running water for at least 1 hr and then bleached in 10-30% hydrogen peroxide at 32°C for a few seconds until the color of the roots lightens perceptibly. After several washings in water to stop the oxidation reaction, roots are dehydrated in 70, 95, and absolute ethanol held at 52 °C for 30 min at each concentration. After dehydration, roots are cleared in methyl salicylate at 52°C. Examination for nematodes in most cases, can be made after 30 min.     

The effect of partial dehydration on the developmental capacity of mouse embryos stored in the supercooled state

The effect of partial dehydration on the ability of mouse blastocysts to withstand storage at subzero temperatures without freezing was studied. The embryos were equilibrated with a supercooling medium developed at the Centre for Food and Animal Research, containing 3% (Medium A) or 6% (Medium B) methanol and propanediol, and then with the same medium, A or B, containing 0–0.5 mol sucrose. The embryos were placed in 0.25 ml straws, cooled to −5°C or −10°C and stored for up to 3 days. After storage, the embryos were cultured for 48 h in M16 and their ability to develop into expanded blastocysts was used to gauge their survival in supercooled storage.

The maximal beneficial effect of partial dehydration occurred in media supplemented with 0.3–0.5 mol sucrose: the proportions of dehydrated embryos surviving 24 h storage at −5°C and −10°C were 84–85% and 91–100%, respectively, compared with only 58% and 52% of non-dehydrated, supercooled embryos. The corresponding figures for dehydrated embryos after 48 or 72 h storage at −5°C were 86–92% and 38–58% compared with 13% and 4% of non-dehydrated embryos. Similarly, 75–85% and 47–55% of partially dehydrated embryos survived storage for 48 h or 72 h, respectively, at −10°C, compared with 5% and 0% of non-hydrated embryos. Thus, reducing the water content of early mouse blastocysts improved their ability to withstand subzero storage.     

Modification of maize starch by thermal processing in glacial acetic acid

Differential scanning calorimetry (DSC) and X-ray diffraction (XRD) methods were used to determine if corn starch–glacial acetic acid mixtures can be melted and thermally processed at reasonable temperatures. DSC studies showed that the melting temperature of dry starch was reduced from about 280 to 180°C in the presence of >30% acetic acid. Glass transition temperatures varied from 110 to 40°C at 15 and 45% acetic acid, respectively. XRD showed the loss of native starch crystallinity and the formation of V-type complexes. Addition of 10% water decreased the melting temperatures to 140–150°C while addition of a base (sodium acetate) had little effect. Some possible applications of processing starch in glacial acetic acid will be discussed.     

Production, optimization growth conditions and properties of the xylanase from Aspergillus carneus M34

Growth conditions, including incubation times, temperature, agitation rate and initial pH of medium, that affect xylanase production by Aspergillus carneus M34 were studied sequentially use the classical “change-one-factor-at-a-time” method. Our results showed that there was a similar trend between cellular xylanase activity and extracellular xylanase activity. The optimal conditions for xylanase production, different from their cell growth, were on the third day, 30 °C, 100 rpm and pH 4, respectively, in this test. Response surface methodology (RSM) was further introduced to optimize the cultivation conditions and to evaluate the significance of these factors. The optimal cultivation conditions predicted from canonical analysis of this model were achieved by incubation at 35.08 °C with an agitation rate of 111.9 rpm and an initial pH of 5.16. In addition, temperature was the most critical factor for xylanase production by A. carneus M34. Xylanase activity of 22.2 U/mL was verified using the predicted optimal conditions and confirmed the fitness and applicability of the model. The optimal temperature and pH of the crude xylanase activity was observed at 60 °C and acidic pH, respectively. Sustained xylanase activity in the crude extract was also detected over a broad range of pH from 3 to 10. Considering its higher specificity toward agricultural wastes, especially corn cob and coba husk, this strain can be used to develop low-cost media for the mass-production of xylanase.     

Locating Iodine in Tissues Autographically, Especially after Fixation by Freezing and Drying

The ability of radioactive elements to affect photographic emulsions enables the detection of radioactive iodine in the thyroid. By placing unstained histological sections of a thyroid (from an animal treated with radioactive iodine) in contact with the gelatin side of medium lantern slide plates, each accumulation of radioactive iodine in the section affects the photographic plate. After exposures prolonged for several days to several weeks depending on the amount of radioactivity in the tissues, the plate is developed and fixed by routine photographic methods. The histological section is stained and may be compared under the microscope to the reactions on the plate or “autographs”.

In an attempt to detect the location of the inorganic iodine which is displaced during fixation and embedding by ordinary methods because of its solubility, a simplified freezing-drying technic for fixation was devised which, at least with the thyroid, yielded well fixed sections. The quick freezing was obtained with acetone-dry-ice mixtures; and the drying was performed at -25° to -30° C. Preliminary addition of paraffin to the tube in which the drying was performed made possible the inclusion in vacuum by heating the tube when drying was completed. The tissue could then be sectioned at 10ju on the microtome. The slides were placed on photographic plates for detection of radioactive iodine as indicated above. Before staining, the sections were treated with absolute alcohol for denaturation of the proteins.