A method for isolation and purification of nematode larvae.

A new apparatus for isolating, purifying, and collecting larvae of nematodes is described. Results of collecting larvae with Baermann's traditional method and the pasteur pipette cotton plug method are compared to those obtained with the device in this research note. It is simple, easy to use, and a more effective apparatus for the isolation, purification, and collection of nematode larvae.     

A novel method for the isolation of motile bacteria using gradient culture systems.

Isolation of motile bacteria from stream water samples was achieved by using Lutrol F127 (poloxamer 407) as a gelling agent in culture media. This block copolymer has the property of repeatedly liquefying and solidifying at low and high temperatures, respectively. The ability of motile bacteria to move through liquid-state Lutrol F127 towards a higher nutrient concentration was exploited. After establishment of the nutrient gradient and inoculation, the system was cooled to liquefy the medium and kept liquid to allow motile bacteria to move. Raising the temperature allowed solidification and prevented further movement. Colonies could be easily removed. The proportion of motile isolates (determined by microscopic observation) increased from 42% in the indigenous population to 100% after isolation using the gradient system.     

An investigation of small-molecule surfactants to potentially replace pluronic F-68 for reducing bubble-associated cell damage

It is well known that bubble rupture has a detrimental effect on mammalian cells. As a result, Pluronic F-68 (PF-68), a nonionic surfactant, is commonly used to reduce bubble-associated cell damage in sparged bioreactors. While PF-68 is currently effective, there is a concern with respect to its decrease in effectiveness as cell concentrations increase (Ma et al., 2004, Biotechnol Prog 20:1183-1191). In addition, having more than one effective surfactant for cell culture is also highly desirable. Given the empirical nature in which PF-68 was initially discovered as a cell culture additive, a structure-performance study of small molecule surfactants, a distinct group which have been previously investigated for other purposes, was performed in an attempt to find a replacement for PF-68. In this study, a generic platform was established to initially screen both the type and concentration of these surfactants for cytotoxicity. Promising candidates where then evaluated for their ability to rapidly lower the surface tension (dynamic surface tension) of culture media and their ability to prevent cell-bubble attachment in a specially developed bubble creation and collection system. Several promising small- molecule surfactants, and their effective concentration, were identified, which can reduce cell-bubble attachment efficiently without being harmful to cells.     

Stimulation of multiple shoot regeneration from seedling leaves of Hypericum perforatum L. by pluronic F-68

The effects of the non-ionic surfactant, Pluronic F-68, on the growth of shoots regenerated from seedlings (14 days post-germination) of Hypericum perforatum L. were studied. The supplementation of agar-solidified medium with 0.001% (w/v) of Pluronic increased the mean fresh weight of the regenerants after 60 days by 40% and the mean number of plant regenerants recovered per seedling by 34%; a less pronounced increase in the number of regenerants occurred with 0.01% (w/v) of the surfactant. By contrast, the mean fresh weight of the regenerants cultured in the presence of 0.1% (w/v) Pluronic F-68 was 15% lower than untreated controls, although the mean number of regenerants per seedling remained unaltered. The growth of seedling leaf-derived Hypericum callus after 60 days was unaffected by all the concentrations of Pluronic tested. However, there was a tendency for callus cells grown in the presence of Pluronic to be more highly pigmented with anthocyanins. The cultivation of leaf explants with 0.001% or 0.01% (w/v) Pluronic did not affect either the mean fresh weight of the regenerants or the mean number of regenerants per explant. However, decreases in both the mean fresh weight and the mean number of regenerants (both 33.0% lower than the control) occurred following the cultivation with 0.1% (w/v) Pluronic.     

A novel method of plasmid isolation using laundry detergent

Since the discovery of plasmid, various methods have been developed to isolate plasmid DNA. All the methods have one common and important target of isolating plasmid DNA of high quality and quantity in less time. These methods are not completely safe because of use of toxic chemicals compounds. The developed protocol for plasmid extraction is based on the alkaline lysis method of plasmid preparation (extraction atpH 8.0) with slight modifications. Cell lysis reagent sodium dodecyl sulfate is replaced by lipase enzyme present in laundry detergent. A good plasmid preparation can be made, which is well suited for subsequent molecular biology applications. By taking safety measures on count, contaminants like, RNA and protein can be completely avoided with maximized plasmid yield. The resultant plasmid quality and quantity can be well comparable to other prevalent methods.     

A sensitive method for determination of lipase activity from cellulose acetate strips

The agar plate method enables us to locate the enzymatic activity of lipase on the electrophoretic strips and to test it on various substrates. Moreover, it is possible to separate and detect a few lipases which are mixed in a solution and to test their specificity toward various substrates. Finally, this method allows us to locate and rapidly follow lipases with low enzymatic activity.     

A novel method for the isolation of mycobacterial DNA

Abstract DNA was isolated from mycobacteria by a simplified procedure. Cells were suspended in 6 M guanidinium chloride, the suspension was cooled to −70 °C, then incubated at 65 °C for 10 min, cooled in ice, deproteinized by chloroform and DNA was recovered from the supernatant. The procedure was used to obtain DNA from several mycobacteria (1 × 10⁹) or more cells) including Mycobacterium neoaurum M. fortuitum M. phlei and M. smegmatis . Each of the species was shown to have two ribosomal RNA operons per genome, and preliminary evidence was obtained which suggests that one of these operons is homologous with one of the operons of M. smegmatis .     

A novel method for rapid isolation of plasmid DNA

A new disposable chromatographic column, pZ523, has been developed for separating plasmid DNA from bacterial chromosomal DNA. Use of pZ523 spun columns eliminates the need for ethidium bromide-cesium chloride density gradients which require long centrifugation times. pZ523 purified plasmids have been shown to be of purity suitable for restriction analysis, ligation, transfection of mammalian cells and transformation of bacteria. Unlike the traditional ultracentrifugation method, pZ523 offers an extremely rapid alternative method for purifying large amounts of plasmid DNA (2.5 mg to 4.5 mg) from cleared bacterial lysates in only 25 minutes.     

A novel method for the isolation and study of a magnetotactic bacterium

The magnetococcus, a magnetotactic bacterium, has been grown in a complex simulated natural environment. Sufficiently pure samples of cells were obtained magnetically making axenic cultures unnecessary for many purposes. The magnetococcus is a Gram-negative coccus, 1.6 m in diameter and readily distinguished by highly refractile inclusions and its magnetotactic behavior. This organism is actively motile by means of two bundles of flagella. Electron dense ferromagnetic inclusions were localized between the flagellar bundles. Collections of magnetococci were morphologically homogeneous and negligibly contaminated by extraneous bacteria. DNA extracted from pooled collections of cells was homogeneous by analytical CsCl centrifugation. The guanine-cytosine content was 61.7%. Total iron by percent cellular dry weight was 3.8%. Comparisons with a previously described magnetotactic marine coccus were made.Non-Standard Abbreviations Tris Tris (hydroxymethyl) aminomethane buffer - EDTA Dipotassium ethylenediamine tetraacetic acid - GC Guanosine cytosine     

A novel method for repetitive peptide synthesis in solution without isolation of intermediates.

A novel method was developed for the large-scale manufacture of peptides in solution, called DioRaSSP-Diosynth Rapid Solution Synthesis of Peptides. This method combines the advantages of the homogeneous character of classical solution-phase synthesis with the universal character and the amenability to automation inherent to the solid-phase approach. The process consists of repetitive cycles of coupling and deprotection in a permanent organic phase and is further characterized by the fact that intermediates are not isolated. Couplings are mediated by water-soluble carbodiimide. Several types of function may be applied for temporary amino protection depending on the sequence of the actual peptide, including Z, Fmoc, Msc and Nsc. Formate is the preferred hydrogen donor during hydrogenolysis of the Z function, while 1,8-diazabicyclo[5.4.0]undec-7-ene is used to deprotect Fmoc, Msc and Nsc. Morpholine is added during the deprotection of Msc and Nsc to scavenge the arising alkenes. Processes according to this highly efficient synthesis method are easy to scale up and yield products of reproducible high purity, which is guaranteed by a new quenching method for residual activated compounds, applying an anion-forming amine such as a beta-alanine ester. This ester should display a lability similar to that of the temporary amino-protecting function, allowing simultaneous deprotection of the growing peptide and the quenched compound. The DioRaSSP approach assures the completely quantitative removal of deprotected quenched compounds before the coupling step of the next cycle of the synthesis by basic aqueous (that is active) extraction, while the growing peptide remains anchored in the organic phase due to the presence of hydrophobic protecting functions.     

A liquid-based method for the assessment of bacterial pathogenicity using the nematode Caenorhabditis elegans

Caenorhabditis elegans has previously been used as an alternative to mammalian models of infection with bacterial pathogens. We have developed a liquid-based assay to measure the effect of bacteria on the feeding ability of C. elegans. Using this assay we have shown that Pseudomonas aeruginosa strain PA14, Burkholderia pseudomallei and Yersinia pestis were able to inhibit feeding of C. elegans strain N2. An increase in sensitivity of the assay was achieved by using C. elegans mutant phm-2, in place of the wild-type strain. Using this assay,P. aeruginosa PA01 inhibited the feeding of C. elegans mutant phm-2. Such liquid-based feeding assays are ideally suited to the high-throughput screening of mutants of bacterial pathogens.     

A simple spectrophotometric method for measurement of nematode populations.

A convenient automated method for measuring inorganic phosphate based on the malachite green reaction with a phosphomolybdate complex has been developed. Less than 100 pmol of inorganic phosphate can be readily quantitated by the method which utilizes standard AutoAnalyzer equipment. Inorganic phosphate is measured in sample volumes of less than 0.1 ml and without interference by a number of phosphorylated metabolic intermediates or nucleotides. This methodology is especially useful in the analysis of hydrolytic processes involving phosphorylated substrates.     

A simple method for the isolation of allelic series using male-linked translocations

Summary Using Adh null alleles a genetic sexing technique is being developed in Ceratitis capitata. In order to facilitate the isolation of a whole series of null alleles at this locus a technique utilizing male-linked translocations is described. It provides a simple efficient and general method for the isolation of any allelic series in species where little genetic information is available.     

A new method for the isolation of undegraded nuclear and cytoplasmic RNA from liver of Xenopus larvae

In order to obtain undegraded RNA from subcellular components a technique for tissue fractionation at ?20°C was developed by using 50% glycerol in the homogenization medium. RNA extracted from nuclear and cytoplasmic fractions, isolated by this method, revealed no signs of degradation after electrophoresis on exponential gels.