A rapid method for obtaining monomeric glutaraldehyde

Summary A new method for the distillation of glutaradlehyde to obtain the monomeric form is presented. The monomer is obtained after only one distillation and it has a purification index (P_i) smaller than 0.20.     

A rapid method for obtaining monomeric glutaraldehyde

A new method for the distillation of glutaraldehyde to obtain the monomeric form is presented. The monomer is obtained after only one distillation and it has a purification index (Pi) smaller than 0.20.     

A simple, rapid method for demonstrating bacterial flagella

We developed a simple, rapid method for demonstrating flagellation of bacteria using the fluorescent protein stain NanoOrange (Molecular Probes, Eugene, Oreg.). The NanoOrange reagent binds to hydrophobic regions of proteins, which results in substantial enhancement of fluorescence. Unbound reagent is essentially nonfluorescent. NanoOrange fluorescently stained bacterial cell bodies, as well as flagella and other appendages, which could be directly observed by epifluorescence microscopy. Detection of flagella was further improved by using a charge-coupled device camera for image capture and processing. The reliability of the method was tested by using 37 pure cultures of marine bacteria. Detection of flagella on the isolates by NanoOrange staining was compared to detection by transmission electron microscopy (TEM). For 36 of 37 cultures, the two methods yielded the same results. In one case, flagella were detected by TEM but not by NanoOrange, although the difference may be attributable to differences between the culture preparations. NanoOrange staining is rapid (10 to 15 min) and does not require fixation or dehydration, so live samples can be stained. Since NanoOrange is a general protein stain and works directly in seawater, it may also prove to be useful for staining other proteinaceous material that is of interest to aquatic microbial ecologists.     

A simple and rapid assay for heme attachment to apocytochrome c

A method for assaying the covalent attachment of heme to apoprotein of cytochrome c was developed. 125I-labeled apoprotein was chemically prepared from 125I-labeled yeast cytochrome c (iso-1-cytochrome c). After incubation of 125I-apocytochrome c with yeast mitochondria, the product was extracted with Triton X-100, digested with trypsin in the presence or absence of a reducing agent, and then precipitated in trichloroacetic acid. The resulting precipitates were collected on nitrocellulose membranes and counted for radioactivity. The radioactivity correlated well with the appearance of a heme-containing peptide in the trypsin digested peptide fragments of cytochrome c. This procedure is simpler and faster than the previously reported methods.     

A simple method for obtaining blood samples from mature frogs

A technique was developed for obtaining periodic blood samples from mature frogs. Up to 150 microliter of blood was collected at weekly intervals. Anesthetization was not required. The data compiled with this technique indicated that activity-induced hyperglycemia was minimal.     

A rapid, simple method for staining bacterial flagella.

A simple modification of Gray's flagellar staining procedure is described. It can be used on air-dried smears or directly on wet mounts of motile bacteria. The stained bacterial flagella can be observed with phase-contrast or bright-field optics. No rigorous cleaning of slides, counterstains, or any washing procedures are required with the staining method, making it very suitable for routine examinations.     

A simple, rapid, aseptic method for filling micropipettes by centrifugation

A centrifugation method for sterilizing, storing, and filling micropipettes is described. Each micropipette is held in a centrifuge tube by a rubber stopper which clamps the butt end of the micropipette. The pipettes are sterilized and aseptically stored. A pipette is filled by injecting solution into the butt end of the micropipette. The micropipette is returned to a suspended position in the centrifuge tube and the liquid is rapidly forced into the micropipette tip by centrifugation. The technique is simpler and more rapid than presently used centrifugation methods.     

A simple and efficient method for obtaining urine samples from rats

A method for collection of urine from rats was developed that is simple, reliable, and efficient. A 5 ml. polystyrene beaker is placed over the urethra and the base of the tail is stimulated with the fingers of one hand. Depending on the quality and quantity of urine needed the perineal area may be shaved and the beaker may be held by hand or attached with tape.     

A rapid, simple method for nuclei isolation from plant protoplasts

A rapid, simple method for nuclei isolation and purification from soybean (Glycine max L. Merr.) protoplasts is described. The isolated nuclei exhibited active amino acid incorporation and RNA synthesis, but DNA synthesis was not detectable. Analysis by CsCl density gradient centrifugation showed that DNA isolated from nuclei had a single band, while DNA isolated from protoplasts consisted of three bands comprised of nuclear DNA, mitochondrial DNA, and chloroplast DNA.     

A simple and efficient method for obtaining transgenic soybean callus tissues

In the present study, a simple and efficient method for obtaining transgenic callus tissues of soybean [Glycine max (L.) Merr.] was developed based on Agrobacterium-mediated transformation. Hypocotyl segments of soybean were used as the starting material. Several factors such as soybean genotype, Agrobacterium concentration, inoculation time, co-cultivation period and addition of antioxidants in co-cultivation medium affecting the transformation efficiency were examined. The explants were cultured on callus induction medium containing 0.5 mg L^?1 6-benzylaminopurine and 2.0 mg L^?1, 2,4-Dichlorophenoxyacetic acid for callus induction. Callus tissues were induced at both the acropetal and basipetal ends. CaMV35S::GUS and CaMV35S::GFP transgenic callus tissues were obtained using the optimized protocol. The average transformation efficiency reached up to 87.7 % based on GUS detection. From inoculation with Agrobacterium to obtaining transgenic soybean callus will take about 3 weeks. In order to validate this method for gene function investigation, GVG::GmSARK transgenic soybean callus tissues were obtained and their senescence-associated phenotypes were assessed. To our knowledge, this is the first report using hypocotyl segments as starting materials to obtain transgenic callus, and this system provides a method for high-throughput screening of functional genes of interest in transformed soybean callus.     

A simple, fast and reliable method for obtaining dopamine histofluorescence from mesencephalic cultures

A modified glyoxylic acid technique for obtaining dopamine histofluorescence from cultured mesencephalic cells is described. This method requires only two solutions: one contains glyoxylic acid, sucrose and monobasic potassium phosphate and is used at room temperature, the other is a Hepes buffered solution used at 37 C. Relatively high concentrations of a monoamine oxidase inhibitor and dopamine are added to the cultures to load dopaminergic neurons; the cell bodies and their processes take up and hold dopamine quickly and evenly. The cultures are dipped in a glyoxylic acid solution, dried in air, heated for 5 min and coverslipped with mineral oil. Since the cultures remain in their culture dishes, the entire procedure takes less than 2 hr. The green histofluorescence characteristic of dopamine is seen when the cultures are viewed by standard fluorescence microscopy. Various cell body types and sizes can be distinguished, as well as the complete extent of their processes and varicosities.     

A simple,high-yield method for obtaining multipotential mesenchymal progenitor cells from trabecular bone

In vitro cultures of primary, human trabecular bone-derived cells represent a useful system for investigation of the biology of osteoblasts. Our recent discovery of the multilineage mesenchymal differentiation potential of trabecular bone-derived cells suggests the potential application of these cells as mesenchymal progenitors for tissue repair and regeneration. Such applications are crucially dependent on efficient cellisolation protocols to yield cells that optimally proliferate and differentiate. In this study, we describe a simple, high-yield procedure, requiring minimal culture expansion, for the isolation of mesenchymal progenitor cells from human trabecular bone. Moreover, these cells retain their ability to differentiate along multiple mesenchymal lineages through successive subculturing. Cell populations isolated and cultured as described here allow the efficient acquisition of a clinically significant number of cells, which may be used as the cell source for tissue-engineering applications.     

A simple rapid procedure for obtaining axenic cultures from monoxenic cultures of myxomycete plasmodia

Axenic culture of myxomycete plasmodia has been attempted from time to time by various authors, but with very little success. From over 500 known species of myxomycetes, fewer than 20 species have been reported in axenic culture to date, including axenic myxamoebal cultures. In these cultures, the plasmodia required either complex media, or a killed bacterial supplement for growth. Furthermore, the time required for attaining the axenic state varied from several months to years. In the present study, a simple, rapid procedure has been developed to render monoxenic plasmodial cultures axenic. This procedure is based on our discovery that plasmodia have certain unusual substrate preferences that are inhibitory to the associated bacteria using Physarella oblonga as a model. The presence or absence of the bacteria could be ascertained through incubation in four different bacteriological media and by the use of a differential staining technique.