Pasternack's Paraffin Method Modified for Plant Tissue

This method for preparing paraffin sections of plant material is a modification of Pasternack's one-hour method for animal tissues. Fixation in Randolph's CRAF fixative is hastened by heat and increased vapor pressure obtained by the use of screw top vials. Dehydration with Zirkle's butyl alcohol series likewise is hastened in the same manner. The rapid penetration of paraffin by the use of 1/2 paraffin and 1/2 butyl alcohol in heated screw top vials shortens the embedding process. Sections are held on the slide thru staining by albumen fixative and a coating of 0.2% celloidin in absolute alcohol and ether. Good penetration with freedom from shrinkage or distortion is obtained and root tip chromosome counts can be made in approximately 3 hours.     

The Dry Preservation of Fixed Plant Material

A method for the dry-preservation of fixed plant material, root tips and buds, is described. The method seems to be advantageous on long expeditions and when material has to be sent away.

The material is transferred from the fixative to 70% alcohol (3 changes, 1/2 hour in the last). It is dried on blotting-paper. The dried material may be preserved a long time. Material kept dry for 4 1/2 years has proved to be quite satisfactory. Drying has been tried after fixation with CRAF-solutions (Webber and Randolph modifications) and fixatives containing osmic acid (Fleming-Benda and 2BD).

The dry material is swollen by keeping for 2 days in 10% alcohol. It is embedded in paraffin according to the usual method. A satisfactory staining has been obtained after these fixatives using iodine-gentian-violet and Feulgen stainings. In addition to chromosome counts dry material may be used for chromosome morphology studies.

Dried material fixed in aceto-alcohol (1:3) has not turned out to be specially suitable for squash preparations owing to the fragility of the chromosomes. If strong pressure is not applied, satisfactory results may, however, be obtained.     

Synthesis of veratraldehyde from veratryl alcohol by lignin peroxidase with in situ electrogeneration of hydrogen peroxide in an electrochemical reactor

We report the synthesis of veratraldehyde from veratryl alcohol by Phanerochaete chrysosporium lignin peroxidase with in situ electrogeneration of hydrogen peroxide in an electroenzymatic reactor. The effects of operating parameters such as enzyme level, pH, and electrical potential on the efficiency of veratryl alcohol oxidation were investigated. Furthermore, we compared direct addition of hydrogen peroxide with electrogeneration of the material during enzymatic oxidation of veratryl alcohol. The electroenzymatic method using in situ-generated hydrogen peroxide was found to be effective for oxidation of veratryl alcohol by lignin peroxidase. The new method may be easily applied to biodegradation systems.     

A solvent-free method for isotopically or radioactively labeling cyclodextrins and cyclodextrin-containing polymers

A method for installing a distinguishable label onto cyclodextrins or cyclodextrin-containing polymers is reported. Cyclodextrins (CD) and cyclodextrin-containing polymers are exposed to labeled (2H or 14C) ethylene oxide (EO) vapor and the alcohol groups on the CD ring open the EO to give ether-linked labeled methylenes and a terminal alcohol. This method provides for the incorporation of an easily tracked and quantified label without the use of solvents or purification steps. The method can be generalized for use with materials that contain nucleophiles other than alcohols, e.g., amines.     

Versatile bioelectronic interfaces on flexible non-conductive substrates.

Bioelectronic interfaces that establish electrical communication between redox enzymes and electrodes have potential applications as biosensors, biocatalytic reactors, and biological fuel cells. These interfaces are commonly formed on gold films deposited using physical vapor deposition (PVD) or chemical vapor deposition (CVD). PVD and CVD require deposition of a primer layer, such as titanium or chromium, and require the use of expensive equipment and cannot be used on a wide range of substrates. This paper describes a versatile new bench-top method to form bioelectronic interfaces containing a gold film, electron mediator, cofactor, and dehydrogenase enzyme (secondary alcohol dehydrogenase, and sorbitol dehydrogenase) on nonconductive substrates such as polystyrene and glass. The method combines layer-by-layer deposition of polyelectrolytes, electroless metal deposition, and directed molecular self-assembly. Cyclic voltammetry, chronoamperometry, field emission X-ray dispersive spectroscopy, scanning electron microscopy, and atomic force microscopy were used to characterize the bioelectronic interfaces. Interfaces formed on flexible polystyrene slides were shown to retain their activity after bending to a radius of curvature of 18mm, confirming that the approach can be applied on cheap and flexible substrates for applications where traditional wafer-scale electronics is not suitable, such as personal or structural health monitors and rolled microtube biosensors.     

Rapid Dehydration for Celloidin Embedding by Means of Azeotropic Distiliation

By including plant material in a fractional distillation apparatus containing 95% alcohol and benzene it is possible to eliminate water completely in a constant boiling mixture. The dehydrated material can then be embedded in celloidin by orthodox techniques. In spite of the rapidity of the dehydration quite soft tissue as well as woody material can be processed. The method is cheap because it does not require absolute alcohol and is also suited to the humid climate in which it was developed.     

The Use of Picric Acid with the Gram Stain in Plant Cytology

The technic described involves the use of a saturated solution of picric acid in absolute alcohol in the process of dehydration following the gentian-violet-iodine stain as applied to plant cytological material. The method is suitable for both paraffin sections and smears of pollen mother cells fixed in Navashin's or Flemming's solutions. Differentiation in clove oil is very easy since cytoplasm destains immediately, while chromatic material destains very slowly following picric acid. Chromosomes are stained more distinctly than with the usual Gram stain and do not fade.     

Water vapor, aqueous ethyl alcohol, and heat activation of Bacillus megaterium spore germination

Dormant spores of Bacillus megaterium were activated for germination on glucose by heating them in aqueous suspension (but not if heated dry), by treating them with aqueous ethyl alcohol at 30 C, or by exposing them to water vapor at room temperature. The degree of water vapor activation depended upon the relative humidity, the time, and the temperature of exposure. Activation increased the extent and rate of glucose-induced germination and decreased the average microlag. Extended water vapor treatment also activated spores for germination induced by KI and by l-alanine. Spores activated by any of the three treatments were deactivated by treatment at 66 C, either for 18 hr in 100% ethyl alcohol or for 40 hr over P(2)O(5). Deactivated spores were reactivated by heat, by 5 m ethyl alcohol, or by water vapor. It is postulated that heating and ethyl alcohol may change the structure of liquid water, so that it is more like water vapor and can more readily penetrate to and hydrate a critical (enzymatic?) spore site, leading to activation.     

The FlyBar: Administering Alcohol to Flies

Fruit flies (Drosophila melanogaster) are an established model for both alcohol research and circadian biology. Recently, we showed that the circadian clock modulates alcohol sensitivity, but not the formation of tolerance. Here, we describe our protocol in detail. Alcohol is administered to the flies using the FlyBar. In this setup, saturated alcohol vapor is mixed with humidified air in set proportions, and administered to the flies in four tubes simultaneously. Flies are reared under standardized conditions in order to minimize variation between the replicates. Three-day old flies of different genotypes or treatments are used for the experiments, preferably by matching flies of two different time points (e.g., CT 5 and CT 17) making direct comparisons possible. During the experiment, flies are exposed for 1 hr to the pre-determined percentage of alcohol vapor and the number of flies that exhibit the Loss of Righting reflex (LoRR) or sedation are counted every 5 min. The data can be analyzed using three different statistical approaches. The first is to determine the time at which 50% of the flies have lost their righting reflex and use an Analysis of the Variance (ANOVA) to determine whether significant differences exist between time points. The second is to determine the percentage flies that show LoRR after a specified number of minutes, followed by an ANOVA analysis. The last method is to analyze the whole times series using multivariate statistics. The protocol can also be used for non-circadian experiments or comparisons between genotypes.     

Catalytic Efficiency,Structure, and Recycling Behavior of Electrospun Polyvinyl Alcohol-Xylanase Fibers Cross-Linked by Glutaraldehyde

Food Biophysics - A versatile and effective method of producing polyvinyl alcohol (PVA)-xylanase (XY) fibers cross-linked by glutaraldehyde vapor (GA) is reported in the present study. Crosslinking...     

A Modified Technic for Salivary-Gland Chromosomes

This technic was worked out for making permanent mounts of salivary-gland squashes of strains and species of Drosophilidae, which yield material difficult to work with. It includes a prior fixation in a modified Carnoy's fluid, staining in solutions of orcein in 60% or 70% acetic acid, part dehydration in the vapor and liquid of a 95% solution of alcohol, and direct mounting in Euparal by means of the drainage-method.     

Boiling method for the extraction of gut glucagon-like immunoreactive materials.

The boiling method deviced in accordance with the extraction procedure of secretin was applied to the extraction of gut glucagon-like immunoreactive material (GLI) and compared with the acid alcohol method of Kenny with respect to efficiency of the extraction and property of the extracted materials. GLI was extracted from minced porcine small intestine by each method. The total amount of GLI extracted by the boiling method was 14.45+/-2.07 mug/10g small intestine (mean+/-SE), showing a high yield as compared to 4.07+/-0.29 mug/10 g small intestine obtained by the acid alcohol method. The difference was statistically significant (p less than 0.005). The gel chromatogram of the acid alcohol extract was separated into two peaks; peak 1 appeared before the the insulin marker, while peak II was eluted with the glucagon marker. The chromatogram of the boiling extract has a main broad fraction including insulin marker and a minor second peak corresponding to peak II of the acid alcohol extract. Boiling of the acid alcohol extract did not cause any shift of peak I in chromatogram. GLI present in the first half of the main fraction of the boiling extract was different from that in the latter half, but identical to peak I of the acid alcohol extract with respect to the immunoreactivity against glucagon antibody. It is concluded that in the extraction of GLI not only high yield is achieved but one or more new components is picked up by the boiling procedure as compared to the acid alcohol method.     

Optical bio-sniffer for ethanol vapor using an oxygen-sensitive optical fiber

An optical bio-sniffer for ethanol was constructed by immobilizing alcohol oxidase (AOD) onto a tip of a fiber optic oxygen sensor with a tube-ring, using an oxygen sensitive ruthenium organic complex (excitation, 470 nm; fluorescent, 600 nm). A reaction unit for circulating buffer solution was applied to the tip of the device. After the experiment in the liquid phase, the sniffer-device was applied for gas analysis using a gas flow measurement system with a gas generator. The optical device was applied to detect the oxygen consumption induced by AOD enzymatic reaction with alcohol application. The sensor in the liquid phase was used to measure ethanol solution from 0.50 to 9.09 mmol/l. Then, the bio-sniffer was calibrated against ethanol vapor from 0.71 to 51.49 ppm with good gas-selectivity based on the AOD substrate specificity. The bio-sniffer with the reaction unit was also used to monitor the concentration change of gaseous ethanol by rinsing and cleaning the fiber tip and the enzyme membrane with buffer solution.     

Acquisition of a potential marker for insect transformation: isolation of a novel alcohol dehydrogenase gene from <Emphasis Type="Italic">Bactrocera oleae</Emphasis> by functional complementation in yeast

The alcohol dehydrogenase genes make up one of the best studied gene families in Drosophila, both in terms of expression and evolution. Moreover, alcohol dehydrogenase genes constitute potential versatile markers in insect transformation experiments. However, due to their rapid evolution, these genes cannot be cloned from other insect genera by DNA hybridization or PCR-based strategies. We have therefore explored an alternative strategy: cloning by functional complementation of appropriate yeast mutants. Here we report that two alcohol dehydrogenase genes from the medfly Ceratitis capitata can functionally replace the yeast enzymes, even though the medfly and yeast genes have evolved independently, acquiring their enzymatic function convergently. Using this method, we have cloned an alcohol dehydrogenase gene from the olive pest Bactrocera oleae. We conclude that functional complementation in yeast can be used to clone alcohol dehydrogenase genes that are unrelated in sequence to those of yeast, thus providing a powerful tool for isolation of dominant insect transformation marker genes.     

Application of a method incorporating treatment with daunomycin for the selective isolation of slower-growing fungi

Expanding the diversity of fungi can lead to the discovery of secondary metabolites that can be applied in the pharmaceutical field. For this purpose, a new simple method using an anthracycline antibiotic, daunomycin (DM), as a selection reagent in isolating slower-growing fungi was established. DM treatment significantly prevented the growth of ordinary faster-growing fungi, thereby fascilitating the selective isolation of slower-growing fungi. In addition, the possibility of isolating rare fungus genera was increased when the DM method was employed. Furthermore, the number of genera obtained by the DM method (56) was larger than that by the Rose Bengal (RB) method (38), and the genera isolated by these methods showed some overlap (20 genera).     

Acquisition of a potential marker for insect transformation: isolation of a novel alcohol dehydrogenase gene from Bactrocera oleae by functional complementation in yeast

The alcohol dehydrogenase genes make up one of the best studied gene families in Drosophila, both in terms of expression and evolution. Moreover, alcohol dehydrogenase genes constitute potential versatile markers in insect transformation experiments. However, due to their rapid evolution, these genes cannot be cloned from other insect genera by DNA hybridization or PCR-based strategies. We have therefore explored an alternative strategy: cloning by functional complementation of appropriate yeast mutants. Here we report that two alcohol dehydrogenase genes from the medfly Ceratitis capitata can functionally replace the yeast enzymes, even though the medfly and yeast genes have evolved independently, acquiring their enzymatic function convergently. Using this method, we have cloned an alcohol dehydrogenase gene from the olive pest Bactrocera oleae. We conclude that functional complementation in yeast can be used to clone alcohol dehydrogenase genes that are unrelated in sequence to those of yeast, thus providing a powerful tool for isolation of dominant insect transformation marker genes. Received: 29 June 1999 / Accepted: 27 October 1999     

Numerical methods in Quaternary palaeoecology. IV. Separating mixtures of morphologically similar pollen taxa

It has often been found desirable, to strengthen palaeoecological inferences, to be able to separate mixtures of pollen taxa whose morphological characteristics overlap considerably, as with the species of many circumboreal tree genera (birch and pine being much-studied examples). Statistical methods are potentially valuable in this problematic field. Previously developed numerical approaches are discussed. Methods based on the identification of single grains are shown to be less satisfactory than methods involving the fitting of frequency histograms, corresponding to possible combinations of known reference distributions, to the observed frequency histograms from fossil material, as proposed by Eneroth (1951). A solution of this type based on the maximum likelihood principle is described and applied to some of Eneroth's own data. The assumptions and limitations of the method, particularly with regard to size statistics versus other morphological data, are discussed.     

Chemical Selection of Alcohol Dehydrogenase Negative Mutants in Drosophila

We describe a selection procedure which utilizes the vapor from an unsaturated alcohol, 1-pentene-3-ol, for the detection and isolation of mutant flies with little or no alcohol dehydrogenase activity. ADH-negative flies are unaffected by exposure to the unsaturated alcohol, but ADH positives (wild-types) die after short exposure. The technique can be used to select rare ADH-negative individuals from large populations of wild-type flies.     

NAD + binding by yeast alcohol dehydro-genase in the presence of pyrazole and a new method for the determination of the enzyme active site concentration

The pyrazole method of Theorell and Yonetani ( Biochem. Z., 338: 573-553, 1963) has been adapted for the determination of the enzyme active site concentration in yeast alcohol dehydrogenase (EC 1.1.1.1). This method cannot be applied indiscriminately to other alcohol dehydrogenases without modification.     

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry (SESI-MS)

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for sample pretreatment. The method was first described by Fenn and colleagues¹ and has been applied to the detection of illicit drugs² and explosives^3-4, the characterization of skin volatiles⁵, and the analysis of breath^6-7.SESI ionization occurs by proton transfer reactions between the electrospray solution and the volatile analyte, and is therefore suitable for the analysis of hetero-organic molecules, just as in traditional electrospray ionization (ESI). However, unlike standard ESI, the proton transfer process of SESI occurs in the vapor phase rather than in solution (Fig. 1), and therefore SESI is best suited for detecting organic volatiles and aerosols.We are expanding the use of SESI-MS to the detection of bacterial volatiles as a method for bacterial identification and characterization⁸. We have demonstrated that SESI-MS volatile fingerprinting, combined with a statistical analysis method, can be used to differentiate bacterial genera, species, and mixed cultures in a variety of growth media.⁸ Here we provide the steps for obtaining bacterial volatile fingerprints using SESI-MS, including the instrumental parameters that should be optimized to ensure robust bacterial identification and characterization.