A rapid, semi-automated SDS-PAGE identification system for oral anaerobic bacteria

SDS-polyacrylamide gel electrophoresis is a useful technique in bacterial differentiation and identification. A rapid, semi-automated SDS-PAGE system (Phast System) was assessed for identification of formate-fumarate-requiring, asaccharolytic, Gram-negative oral anaerobes. The system permitted loading, separation and staining of gels within 2 h. Percentage similarities between strains were determined using correlation coefficients and cluster analysis. The protein profiles were sufficiently reproducible provided that distorted profiles were disregarded. Strains were successfully separated into their species, with the exception of Bacteroides ureolyticus NCTC 10939, which appeared to be distinct from other strains of that species. Twenty-nine unidentified formate-fumarate-requiring, sub-gingival plaque strains were suitably clustered with the standard strains as verified by a series of physiological and biochemical tests.     

A rapid, semi-automated SDS-PAGE identification system for oral anaerobic bacteria

SDS-polyacrylamide gel electrophoresis is a useful technique in bacterial differentiation and identification. A rapid, semi-automated SDS-PAGE system (Phast System) was assessed for identification of formate-fumarate-requiring, asaccharolytic, Gram-negative oral anaerobes. The system permitted loading, separation and staining of gels within 2 h. Percentage similarities between strains were determined using correlation coefficients and cluster analysis. The protein profiles were sufficiently reproducible provided that distorted profiles were disregarded. Strains were successfully separated into their species, with the exception of Bacteroides ureolyticus NCTC 10939, which appeared to be distinct from other strains of that species. Twenty-nine unidentified formate-fumarate-requiring, sub-gingival plaque strains were suitably clustered with the standard strains as verified by a series of physiological and biochemical tests. and accepted 25 July 1989     

A micronucleus method for detection of meiotic micronuclei in male germ cells of mammals

A new rapid micronucleus method is presented for the detection of chromosomal damage induced in spermatocyte stages of mammals. Analysis of micronuclei is done in early spermatids that have been isolated from testis tubules in a special testis isolation medium supplemented with enzymes (collagenase, trypsin and DNAase).     

A rapid method for production of binucleate cells

Populations of BHK-21 fibroblasts that are 90–95% binucleate can be obtained within 2–3 h by combining mitotic synchrony techniques with use of the drug cytochalasin B. Such populations are potentially useful for the study of nuclear-cytoplasmic balance and control, nuclear synchrony, and the production of tetraploid cell lines.     

SwarmPS: rapid, semi-automated single particle selection software

Single particle analysis (SPA) coupled with high-resolution electron cryo-microscopy is emerging as a powerful technique for the structure determination of membrane protein complexes and soluble macromolecular assemblies. Current estimates suggest that approximately 10(4)-10(5) particle projections are required to attain a 3A resolution 3D reconstruction (symmetry dependent). Selecting this number of molecular projections differing in size, shape and symmetry is a rate-limiting step for the automation of 3D image reconstruction. Here, we present Swarm(PS), a feature rich GUI based software package to manage large scale, semi-automated particle picking projects. The software provides cross-correlation and edge-detection algorithms. Algorithm-specific parameters are transparently and automatically determined through user interaction with the image, rather than by trial and error. Other features include multiple image handling (approximately 10(2)), local and global particle selection options, interactive image freezing, automatic particle centering, and full manual override to correct false positives and negatives. Swarm(PS) is user friendly, flexible, extensible, fast, and capable of exporting boxed out projection images, or particle coordinates, compatible with downstream image processing suites.     

A method for rapid freeze fixation of plant cells

Summary We describe here an apparatus that permits rapid freeze fixation of whole cells, which are then prepared by freeze substitution and resin embedment for examination in the EM. The freezing device utilizes a rotary solenoid that rapidly plunges the specimen holder, a formvar-film-covered thin wire loop, into a well of stirred liquid propane at –180C. The rotary solenoid allows for an adjustable, repeatable immersion rate. Substitution takes place at –80 C in acetone with 2% OsO₄ and is followed by en bloc staining in either hafnium tetrachloride or uranyl acetate. We have utilized these techniques on plant cells, for which there has been relatively little published work when compared to other organisms. The results show that, with the versatile specimen holder and rapid, repeatable immersion rates, different cell types, including pollen, stamen hairs, and germinating moss spores, can be rapidly frozen with repeatable success. The improved preservation achieved with rapid freeze fixation over conventional chemical fixation reveals itself particularly in the structure of the plasmamembrane, the cytoskeleton, chromatin, and certain endomembrane systems.     

An objective, rapid and reproducible method for scoring AFLP peak-height data that minimizes genotyping error

Amplified fragment length polymorphism (AFLP) fingerprint data are now commonly collected using DNA sequencers. AFLP genotypes are still often scored by eye from such data - a time-consuming, error-prone and subjective process. We present a semi-automated method of genotyping sequencer-collected AFLPs at predefined fragment locations (loci) within the fingerprint. Our method uses thresholds of AFLP-polymerase chain reaction-product fluorescence intensity (peak height) in order to: (i) exclude AFLP loci that are likely to contribute high rates of error to data sets, and (ii) determine the AFLP phenotype (fragment presence or absence) at the retained loci. Error rate analysis is an integral part of this process and is used to determine optimal thresholds that minimize genotyping error, while maximizing the numbers of retained loci. We show that application of this method to a large AFLP data set allows genotype calls that are rapid, objective and repeatable, facilitating the extraction of reliable genotype data for molecular ecological studies.     

A quantitative procedure for the dissociation of adult mammalian muscle into mononucleated cells

A new technique for quantitative analysis of dissociated adult skeletal muscle is described. Using adult hamster biceps we obtained cell yields of 1–4 × 10⁶ viable cells/g muscle, 4–10% plating efficiency (% cells attached at 20 h in vitro) and spontaneously contracting myotubes at day 7 which exhibited creatine phosphokinase specific activities of 0.4 μmoles/min/ mg protein at 25 °C. This technique can be applied to other adult tissue.     

A rapid fluorometric method for the estimation of DNA in cultured cells

We present here a procedure for the rapid measurement of both DNA and protein from the same aliquot of cell lysate. DNA estimates obtained by this method were compared to replicate determinations using the method of Kissane and Robins (1). The optimal range for the estimation of DNA (1 to 20 μg) is well suited for use with portions of extracts from individual cell cultures; the remainder of the extract remains available for enzyme assays or other parallel determinations.     

A convenient method for distinguishing human and mouse cells in situ

Animal models play critical roles in the field of biomedical research.Mouse models with transplanted human cells or tissues,such as cell line-derived xenografts(CDX)and patient-derived xenografts(PDX),have been widely used[1].Tracking transplanted cells and understanding the spatial relationship between donor and host are extremely important for the study of these models.To distinguish cell sources in mouse xenograft models at the morphological level,antibody recognition methods(immunohistochemistry and immunofluorescence),transgenic reporter methods,and chromosome-specific probe labeling methods are widely used[2–5].However,these methods all need to consider the issues of label specificity,signal intensity,in situ applicability,and complexity of experimental operations,which increase the difficulty in use.In this report,we described a simple method for accurately distinguishing human and mouse nuclei based on 4′,6-diamidino-2-phenylindole(DAPI)staining.This method can accurately identify species not only in cultured cells,but also in mouse xenograft models.Using short-wavelength excitation and high brightness of DAPI stains,this easy-to-use method is expected to be widely used in the field of biomedical research.