Use of continuous flow microfluorimetry for DNA determinations in Penicillium

BRIDGE, P. D. & HUDSON, L., 1989. Use of continuous flow microfluorimetry for DNA determinations in Penicillium. Continuous flow microfluorimetry has been used to characterize spore suspensions of fasciculate strains of Penicillium. Fluorescent Feulgen staining combined with appropriate filters allows the measurement of conidial DNA content in samples of 10 000 conidia in 30 seconds. The results obtained for apparently closely related strains and single conidium isolates of the same strains showed significant differences which appear to correspond with phenotypic differences. Some implications of, and possible explanations for, this phenomenon are discussed.     

A simple and rapid technique for fluorescence staining of fungal nuclei

A technique for staining fungal nuclei using fluorescence stain Hoechst Dye 33258 in McIlvaine standard buffer of pH 7.26–7.44 is reported. It is a broad-spectrum fungal nuclear staining tool found to be effective onAgaricus bisporus, Alternaria helianthi, Fusarium oxysporum f. sp.lini, Penicillium binellum, Pythium ultimum, Rhizoctonia solani, andSaccharomyces cerevisiae. Conidial nuclei ofAlternaria helianthi, Fusarium oxysporum f. sp.lini, andPenicillium binellum also stained well.     

A technique for the rapid extraction of microalgal DNA from single live and preserved cells

Molecular methods are increasingly being used in the study of harmful microalgae; however, DNA extraction techniques have imposed limitations on the species and questions studied, with research primarily restricted to cultured specimens. Here we describe a simple method that merges two existing techniques for DNA extraction from live and preserved single dinoflagellate cells. DNA was successfully isolated from live single cells of Gambierdiscus toxicus Adachi et Fukuyo, 1979 and cells preserved using formalin/methanol fixation. This method supplements existing techniques and expands the scope of genetics studies conducted on dinoflagellates to include routine molecular analysis of single cells isolated from field samples.     

A rapid microscale technique for isolation of recombinant plasmid DNA suitable for restriction enzyme analysis

A simple and rapid microscale technique is described for the isolation of plasmid DNA which involves cell lysis with phenol, centrifugation, phenol extraction, ethanol precipitation, and RNase digestion. The plasmid DNA is of suitable purity and quantity for multiple restriction endonuclease digestions and bacterial transformations. This “miniprep” procedure is applicable for a variety of types of plasmids ranging in size from 2900 to 18,400 base pairs (bp) and for a number of Escherichia coli strains. The plasmids are rapidly cleaved by all restriction enzymes (total of 14) tested to date. Recombinant clones have been screened for insertions as small as 10 bp and as large as 5000 bp. The procedure takes ~3 h and has been routinely used to simultaneously analyze 24 candidate clones. This procedure is reliable and useful for rapid screening of recombinant DNA candidates where analysis by restriction endonuclease digestion is necessary.     

Variation in nuclear DNA content in an ex-type isolate of Penicillium measured by continuous flow microfluorimetry

Abstract The effect of purified enterotoxin produced by Clostridium difficile on Chinese hamster ovary (CHO) cells was examined. In a certain concentration range (0.9–3.6 μ g/ml), purified toxin caused CHO elongation, namely a cytotonic effect, which is similar to a typical morphological change in CHO cells induced by cholera enterotoxin. At a higher concentration, purified enterotoxin had a cytotoxic effect on CHO cells which was neutralized by anti- C. difficile enterotoxin, but not by anti- C. difficile cytotoxin. Thus, enterotoxin had both cytotonic and cytotoxic effects on CHO cells. About 60 and 180 min were required for binding of enterotoxin to CHO cells, and its internalization, respectively, both times being much longer than those for C. difficile cytotoxin.     

Vital DNA staining and cell sorting by flow microfluorometry

A procedure has been investigated for sorting viable cells according to their DNA content. Cells are stained with the U.V. activated fluorochromes 4'6-diamidino-2-phenylindole (DAPI), Hoechst 33258 or Hoechst 33342, and sorted with a Fluorescence Activated Cell Sorter. Hoechst 33342 is a suitable vital stain for a variety of cell types. Hoechst 33258 and DAPI, however, are quantitative vital stains for CHO cells only. Cloning efficiency is unaffected by the sorting procedure, and these stains are not mutagenic at concentrations suitable for vital staining. Potential applications of this procedure to cell biology are discussed.     

A rapid method for staining large chylomicrons

In this report, we present a rapid method for producing high-quality micrographs suitable for determining the size distributions of particles in concentrated samples of postprandial chylomicrons and chylomicron remnants. The procedure consists of mixing particles with osmium tetroxide in water to stabilize the lipids of the particles. These fixed and positively stained particles are then negatively stained with phosphotungstate in the presence of dilute sucrose. This dual staining procedure prevents the fusion and clustering of chylomicrons during processing for electron microscopy and is effective with particles of different lipid compositions. In addition, this procedure is simple and rapid, adding only one mixing step and 5 min to the preparation time required for conventional negative stains.     

A new rapid immunohistochemical staining technique using the EnVision antibody complex.

Rapid immunohistochemical investigation, in addition to staining with hematoxylin and eosin, would be useful during intraoperative frozen section diagnosis in some cases. This study was undertaken to investigate whether the recently described EnVision system, a highly sensitive two-step immunohistochemical technique, could be modified for rapid immunostaining of frozen sections. Forty-five primary antibodies were tested on frozen sections from various different tissues. After fixation in acetone for 1 min and air-drying, the sections were incubated for 3 min each with the primary antibody, the EnVision complex (a large number of secondary antibodies and horseradish peroxidase coupled to a dextran backbone), and the chromogen (3,3'diaminobenzidine or 3-amino-9-ethylcarbazole). All reactions were carried out at 37C. Specific staining was seen with 38 antibodies (including HMB-45 and antibodies against keratin, vimentin, leukocyte common antigen, smooth muscle actin, synaptophysin, CD34, CD3, CD20, and prostate-specific antigen). A modification of the EnVision method allows the detection of a broad spectrum of antigens in frozen sections in less than 13 min. This method could be a useful new tool in frozen section diagnosis and research. (J Histochem Cytochem 49:623-630, 2001)     

A non-invasive technique for rapid extraction of DNA from fish scales

DNA markers are being increasingly used in studies related to population genetics and conservation biology of endangered species. DNA isolation for such studies requires a source of biological material that is easy to collect, non-bulky and reliable. Further, the sampling strategies based on non-invasive procedures are desirable, especially for the endangered fish species. In view of above, a rapid DNA extraction method from fish scales has been developed with the use of a modified lysis buffer that require about 2 hr duration. This methodology is non-invasive, less expensive and reproducible with high efficiency of DNA recovery. The DNA extracted by this technique, have been found suitable for performing restriction enzyme digestion and PCR amplification. Therefore, the present DNA extraction procedure can be used as an alternative technique in population genetic studies pertaining to endangered fish species. The technique was also found equally effective for DNA isolation from fresh, dried and ethanol preserved scales.     

A rapid staining technique for the detection of the initiation of germination of bacterial spores

AIMS: We propose to apply the Wirtz-Conklin staining technique to evaluate spore germination. METHODS AND RESULTS: Spores at different stages of germination were stained with modified spore stain (Wirtz-Conklin) and evaluated for staining properties. Bacillus spores suspended in deionized water, which does not support germination, stained greenish-blue. Spores suspended in germination enhancers that did not form bacilli stained pink, indicating the initiation of germination. Spores suspended in culture media, which promotes bacterial outgrowth, formed bacilli and were also stained pink. CONCLUSIONS: Modified spore stain (Wirtz-Conklin) was found to be useful to detect the initiation of spore germination as early as 30 min following incubation in a germination environment. SIGNIFICANCE AND IMPACT OF THE STUDY: This simple staining procedure is useful in detecting the initiation of germination of bacterial spores.     

A protocol for Papanicolaou staining of cytologic specimens following flow analysis

A protocol has been developed for restaining cytologic specimens that have been analyzed on a multidimensional slit-scan flow system. The technique involves Papanicolaou staining of cells on a membrane filter that has been previously stained with acridine orange and fixed with glutaraldehyde buffer. The specimen and staining solutions were sequentially added to a 5-micrometers pore size, 47-mm diameter Gelman "Metricel" filter while it remained in a glass filtration apparatus. The practice of retaining the filter in the filtration apparatus throughout the staining procedure minimizes cell loss and eliminates specimen cross contamination when compared with conventional filter dip staining. The availability of this postflow specimen Papanicolaou staining protocol permits accurate determination of the performance characteristics of a multidimensional slit-scan flow system and should be useful whenever staining of a limited number of cells with minimal cell loss is desired.     

A novel system for rapid screening of effective siRNA target sites by one step transfection with a single vector

siRNA technique has been widely used to study the gene functions and to develop disease therapeutics. One of the challenges of RNAi application is to obtain the most effective target specific siRNA sequences. Currently the process to select and validate optimal siRNA sites for a given gene, which is usually based on screening by using Western blot, Northern blot or Q-PCR, remains empirical and time consuming. Although few fluorescence-based siRNA sequence selection systems have proven useful, the rapid and efficient screening of siRNA target sites is still challenging. In the paper, we developed a quick and efficient method to screen siRNA target sites with a novel single vector system, which contains the following cassettes: (1) an eGFP reporter gene expression cassette followed by a multiple cloning site and SV40 pA for insertion of a target sequence; (2) siRNA expression cassette containing a dual PoI III promoter driving in opposite directions; and (3) an internal loading control, mCherry reporter gene. Based on this one-step transfection with single vector system, we could rapidly screen effective, target specific siRNA fragments in an unbiased manner by judging the fluorescence intensity ratio of eGFP to mCherry. The generation of this novel vector system will promote the application of siRNA in basic research and disease therapy.     

A rapid analytical technique for flow cytometric analysis of cell viability using calcofluor white M2R

Analysis of dead versus live cells is shown to be possible using Calcoflour White M2R (CFW), a fluorescent brightener. Comparison of CFW with both propidium iodide (PI) and fluorescein diacetate (FDA) was performed on a FACS 440 dual laser flow cytometer on several populations of cultured rat and mouse cell lines, peripheral leukocytes, splenocytes, diatoms, and plant protoplasts. As a measure of cell viability, staining results with CFW were strongly associated with PI (correlation coefficient of 0.9886) and FDA (inverse correlation coefficient of 0.9647). With plant and algal cells, controls are necessary as CFW does stain live cells to some extent. CFW (excitation: UV, emission max: 435 nm) can be used in conjunction with two-color immunofluorescence analysis using fluorochromes excited at 488 nm with no interference.     

A rapid computer analysis for multicomponent DNA reassociation kinetics

An iterative program, Gaushaus, has been adapted to analyze complex DNA reassociation kinetics. With valid experimental data, rapid convergence to a solution is always achieved by this program even when the starting parameters are grossly in error. The output of the program also includes useful statistical information.     

An improved method for single step purification of metagenomic DNA

An improved method for purification of intact metagenomic DNA from soil has been developed using Q-Sepharose, which purified the DNA from phenolic and humic acid contaminants in a single step. The entire procedure for purification took only 45 min. A total of 81% of DNA was recovered after purification and there was 84% reduction in humic acid contents. The purified DNA was readily digested with restriction enzymes and can be further used for molecular applications.