Recovery of a bacterial sub-population from sewage using immunofluorescent flow cytometry and cell sorting

Abstract The effectiveness of immunofluorescence flow cytometry and cell sorting to detect, quantify and separate indigenous bacterial populations present in low concentrations in sewage outflow was investigated. Preparatory experiments for targeted recovery revealed indigenous, immunoglobulin-G-binding particles present at low levels in sewage outflow samples taken from Coniston Water. Fluorescence-activated cell sorting of this population was employed to enrich for these particles, which were confirmed as bacterial cells. This cell population comprised approximately 23% of the total plate count on MacConkey agar before cell sorting, rising to approximately 95% after sorting. These results corresponded to cell densities of less than 5% of the total plate count on R2A agar. Taxonomic tests suggested the bacterium to be Ochrobactrum anthropi .     

In vitro dynamics of chromatin organization and migration

The organization of eukaryotic chromatin is not static but changes as a function of cell status during processes such as proliferation, differentiation, and migration. DNA quantification has not been used extensively to investigate chromatin dynamics in combination with cellular migration. In this context, an optimized DNA-specific, nonperturbant method has been developed for studying chromatin organization, using the fluorescent vital bisbenzimidazole probe Hoechst 33342: this property has been described by Hamori et al. (1980). Computer-assisted image analysis was used to follow migratory activity and chromatin organization of L929 fibroblasts during in vitro wound healing. Cell movements were analyzed using an optical flow technique, which consists in the calculation of the velocity field of cells and nuclear movements in the frame. This system allows the correlation of cell migration and position in the cell cycle. It makes it possible to study chromatin dynamics using a quantitative analysis of nuclear differentiation reorganization (nuclear texture) and to correlate this with migration characteristics. The present system would be of interest for studying cell-extracellular matrix interactions using differing substrates, and also the migratory response to chemotactic factors. Such a model is a prerequisite for gaining better understanding of drug action.     

Monoclonal antibodies to type D retrovirus (SRV-2)

Monoclonal antibodies (MoAbs) to the major gag core protein p27 and a viral protein p44 of type D retrovirus (SRV-2) were produced and used in the detection of SRV-2 antigens in infected Raji cells and in tissues from macaques with simian acquired immunodeficiency syndrome (SAIDS) and retroperitoneal fibromatosis (RF). Anti-p44 MoAb showed inhibition of syncytium formation by both SRV-1- and SRV-2-infected Raji cells.     

Total root length as estimated from small sub-samples

A method is proposed for estimating the total length of a root system from sub-samples. This method is based on the measurement of the length and diameter of small pieces of roots, and on the measurements of the bulk density of root sub-samples. It is assumed that roots are cylinders with a given bulk density. The length and diameter of small root pieces are measured by image analysis. A weighted quadratic mean (W.Q.M.) root diameter is then calculated and used in estimating the root length. This W.Q.M. diameter is defined as the real mean diameter of an equivalent single root with the same length and volume as the tested root system. The accuracy of prediction is demonstrated for one theoretical root system. The standard deviation of estimation can be calculated using sampling simulations.     

Cytogenetic diversity in primary human tumors

Cytogenetic patterns from primary short-term culture of breast cancer, renal carcinoma, and tumors of the central nervous system are presented to illustrate the range of karyotypic diversity of human solid tumors as well as their biologic differences in culture systems that support their growth. These studies have illustrated several major issues. 1) Results vary with the tissue of origin: primary cultures from breast are almost uniformly diploid, while renal tumors are near-diploid, mosaic, and show clonal aberrations; and CNS tumors are heterogeneous: some diploid, some near-diploid and some highly aneuploid. 2) Results after short-term culture are selective, representing subpopulations from the heterogeneous cells that are detected on direct analysis of fresh tumors by cytogenetics or flow cytometry (FCM). It is not yet clear whether prognosis depends on the dominant population of the primary tumor or alternatively should be influenced by detection of small aneuploid subpopulations. 3) Evidence from all three tumor types supports the interpretation that cytogenetically normal diploid cells constitute part of some tumor populations, and may be better adapted to routine growth in culture than aneuploid subpopulations from the same primary tumors. These cells may also compose a major portion of the viable population of tumors in vivo and, therefore, could represent a useful model for studies of tumorigenesis and therapeutic regimens.     

Induction of reversible changes in cell-surface glycoconjugates and lung colonization potential by 13-cis retinoic acid

Murine squamous carcinoma cells (KLN205) grown in a medium supplemented with the retinoid, 13-cis retinoic acid (RA), had dose-dependent, selective increases in the expression of certain lectin receptors, which correlated with a dramatic decrease in the ability to form pulmonary colonies (P ?.0003) (Couch MJ, Pauli BU, Weinstein RS, Coon JS: JNCI, 78:971 ?977, 1987). These findings suggest a possible relationship between the RA-induced glycoconjugate alterations and the decreased experimental metastatic behavior. We further define the mechanism of RA's action. The finding that RA treatment (5 × 10^?6 M, 5 × 10^?7 M) did not perturb the cell cycle of KLN205 cells provides further proof that the decreased metastatic behavior is not attributable to any inhibition in the rate of growth or to alterations in the cell cycle. Furthermore, since stable subpopulations with variable lectin binding could not be detected, the mechanism of RA's action does not appear to be due to selection of variant tumor-cell subpopulations. Finally, in a scries of experiments designed to determine the reversibility of the RA treatment, the RA-induced decrease in metastatic behavior reverted back to a more metastatic state in the same time frame (3 days) as the reversion of the RA-induced changes in cell-surface glycoconjugate expression. This reversion provides further evidence for a close relationship between the RA-induced modulation of tumor cell-surface glycoconjugate expression and the decreased metastatic behavior; it suggests that transient, reversible modulation of the tumor cell surface may play a role in determining metastatic behavior.     

Subculture of rabbit articular chondrocytes within a collagen gel: growth and analysis of differentiation

Primary cultures of rabbit articular chondrocytes have been subcultured within three-dimensional (3D) collagen gels. Under these conditions, the cells remained viable and divided, but with a lower proliferation rate than that observed in control monolayer cultures. Flow cytometric analysis of progression of the cells into the cell cycle has confirmed and extended these findings. Also the cellular volume was decreased in 3D-culture, being in the same range as thein vivo size of cartilage cells. Specific staining for proteoglycans and type II collagen immunolocalization on sections of gels showed the expression of differentiated phenotypes and revealed the accumulation of these matrix components in the immediate surroundings of the cells. The use of Ultroser G (a serum substitute) improved the conditions for 3D- culture of rabbit articular chondrocytes.     

An inflatable minirhizotron system for root observations with improved soil/tube contact

Commonly used minirhizotrons consisting of a transparent tube inserted into the soil seldom attain good contact between the tube and the soil, which leads to root growth occurring in a gap rather than in the soil. A new system is described involving an inflatable flexible rubber wall, made from a modified motorcycle tube. Pressure ensures a proper tube/soil contact so that the environmental circumstances for root growth along the tube more closely correspond to those in the undisturbed soil. Before the endoscope slide is introduced into the minirhizotron for taking pictures, the inflatable tube is removed, so that there is no-often opaque-wall between the endoscope and the roots. This improves the picture quality and facilitates the analysis of root images.     

Altered growth kinetics precede calcium-induced differentiation in mouse epidermal cells

Summary Primary cultures of newborn mouse epidermal cells proliferate rapidly and with a high growth fraction for several months when grown in medium with low calcium (0.02 to 0.1 mM). Addition of calcium to levels generally used in culture medium (1.2 mM) was followed by rapid changes in the pattern of proliferation. By using a combination of technics (a stathmokinetic method, autoradiography, [³H]thymidine incorporation into DNA, DNA flow cytometry) it was found that cell flux was blocked for 5 to 6 h, followed by a short rise in the mitotic rate at 10 h, and a gradual fall in all growth parameters until about 32 h after the calcium switch. There was no accumulation of cells in any particular cell cycle phase. The results indicate that the calcium switch is followed by a strong reduction in cell flux from G₁ whereas the majority of the cells that had left G₁ at the time of the switch completed one cell division before cessation of all proliferative activity. Both before and after the switch the primary epidermal cultures consisted of one diploid and one tetraploid G₁ DNA stemline that seemed to react in the same way to calcium. This work reported in this paper was undertaken during the tenure of an American Cancer Society-Eleanor Roosevelt-International Cancer Fellowship awarded by the International Union Against Cancer (K. E.). The project was supported by funds partly provided by the International Cancer Research Data Bank Program of the National Cancer Institute, National Institutes of Health, Bethesda, MD, under contract N01-C0-65341 (International Cancer Research Technology Transfer) and partly by the International Union Against Cancer (O.P.F.C.).     

Computer-aided three-dimensional reconstructions of the arrangement of primary spermatocytes in human seminiferous tubules

Summary With the use of a digital image-processing method three-dimensional reconstructions of the arrangement of spermatocytes in human seminiferous tubules were performed. With this method it was possible to investigate the cellular distribution in the tubule in nearly any given perspective and projection. In addition, by means of simple mathematical procedures, such as by transformation of Cartesian coordinates into cylindrical coordinates, it was possible to vary the shape of a reconstruction, i.e., to convert the cylindrical image of a tubular portion into a right-angled r--z-representation.The present work not only confirms the existence of a complex helical plan of organization of the human seminiferous epithelium but also provides further aspects of the phenomenon of physiological germ-cell loss and its integration into the kinetics of spermatogenesis.Dedicated to Prof. E.C. Roosen-Runge, Seattle, on the occasion of his 75th birthday