Simplified preparation of mycoplasmas, an acholeplasma, and a spiroplasma for scanning electron microscopy.

A simple, effective procedure was developed for scanning electron microscopic examination of mycoplasmas and similar organisms. Cultivation of several mycoplasmal species, an acholeplasma, and a spiroplasma in broth media in Leighton tubes with cover slips resulted in attachment of the organisms to the cover slips. The attached cells were easily processed for either scanning electron microscopy or light microscopy. By eliminating the need for centrifugation, which was used in previously described techniques, physical stress on the cell is minimized. The effects of different preparative procedures on the morphology of Mycoplasma gallisepticum are described.     

A flow system for the study of shear forces upon cultured endothelial cells

A parallel plate chamber in a flow system has been designed to study the effects of fluid shear stresses on cells. The system was applied to the study of cultured endothelial cells grown on cover slips which were accommodated in recessed wells in the base plate. Dye injection studies in the chamber indicated laminar flow over the cells. Shear rates measured over the cover slips by an electrochemical technique were found to be linear with flow rate. Laser doppler anemometry showed parabolic profiles between the plates. Endothelial cells subjected to flow showed a correlation between the time required for orientation and the magnitude of the shear stress.     

Erratum

We have compared membrane transport of lysine and adenosine by rabbit lung macrophages in suspension and adherent to glass cover slips. The rapid sampling techniques employed permitted measurements to be made over intervals as short as 10 seconds. Suspended cells transported both nutrients at a slower rate, although the Km values for the two transport systems remained unchanged. Colchicine, cytochalasin B and adenosine did not interfere with the enhancement.     

Cell guidance by magnetic nanowires

The phenomenon of contact guidance on thin fibers has been known since the beginning of the 20th century when Harrison studied cells growing on fibers from spider's web. Since then many studies have been performed on structured surfaces and fibers. Here we present a new way to induce guidance of cells or cell processes using magnetic nanowires. We have manufactured magnetic Ni-nanowires (200 nm in diameter and 40 μm long) with a template-based electro-deposition method. Drops of a nanowire/ethanol suspension were placed on glass cover slips. The nanowires were aligned in an external magnetic field and adhered to the cover slips after evaporation of the ethanol. When the wires had adhered, the magnetic field was removed. L929 fibroblasts and dissociated dorsal root ganglia (DRG) neurons from mice were cultured on the nanowire-coated cover slips for 24 h and 72 h respectively. The fibroblasts were affected by the aligned nanowires and displayed contact guidance. Regenerated axons also displayed contact guidance on the wires. There were no overt signs of toxicity caused by Ni-wires. Aligned magnetic nanowires can be useful for lab-on-a-chip devices and medical nerve grafts.     

On tight junction structure: Forssman glycolipid does not flow between MDCK cells in an intact epithelial monolayer

One model of tight junction structure suggests that lipids might flow from cell to cell within shared exoplasmic membrane leaflets. We tested this proposal by co-culturing two clones of MDCK epithelial cells, which differed in their content of Forssman glycolipid, and then staining by immunofluorescence with rabbit anti-Forssman Ig. In co-cultures grown on glass cover slips and on nitrocellulose filters, positive Forssman staining was restricted to sharply demarcated clusters of cells formed by the Forssman-positive clone. Integrity of tight junctions between the two clones was indicated on cover slips by the presence of individual domes (hemicysts) composed of both clones and on filters by the generation of transepithelial potential differences. These results suggest that glycolipids in the exoplasmic leaflet of cells in a tight epithelium do not flow to adjacent cells.     

Permanent Mounting of Unstained and Aceto-Orcein Stained Cells in the Water-Soluble Medium, Abopon

For cellular morphology, mammalian cells were grown on cover slips in Leigh ton tubes, fixed in 1% osmic acid vapor for 2 min, decolorized with 30% H₂O₂ in 5% ammonium oxalate solution (1:7) for 2 min, then washed thoroughly, and finally mounted in a water-soluble medium consisting of a saturated solution of Abopon in 0.2 M phosphate buffer, pH 7.0. For chromosomal analysis of similarly cultured cells, aceto-orcein preparations were made by conventional methods, with the following minor modifications: following pretreatment with colchicine and hypotonic expansion, the cells on the cover slips were fixed in acetic-alcohol (1:3), air dried, incubated at 37° C for 15 min in 2% orcein in 45% acetic acid, rinsed in 45% acetic acid, washed several times in distilled water, and finally mounted in Abopon mounting medium. Both kinds of preparations were allowed to harden for 24 hr before being handled. Such slides will keep for years at room temperature. Studies requiring frequent comparisons of cellular and chromosomal morphology of cultured cells can thus be extended over long periods of time.     

Simplified Radioimmunoassay for Diagnostic Serology

A simplified, indirect radioimmunoassay is described for Escherichia coli, vaccinia virus, and herpesvirus. The antigens were affixed to glass cover slips; thus both the primary and secondary reactions take place on the cover slips, and the unbound antiserum is easily separated from the bound antiserum by rinsing. Rabbit or human immune sera were reacted with the antigens, and the primary immune complex was quantitated by a secondary reaction with (125)I-indicator globulin (anti-rabbit or anti-human). A direct relationship between the antiserum concentration and the (125)I absorption was established. Variations in titers were detectable, and the titers were comparable to complement fixation titers. Homologous and heterologous reactions were distinguishable. The method affords an objective, quantitative, and qualitative evaluation of antibody, and results are reproducible.     

A hot wire device for cutting tissue culture flasks

Summary A small hot wire device for cutting plastic culture ware can be constructed of steel rod, brass screws, nichrome wire and acrylic plastic sheeting and tubing. The nichrome wire is heated using a variable power transformer. Four sequential cuts are made in the culture flask bottom and the bottom separated from the remainder of the flask. Cultures can be stained, air-dried and cover slips affixed with PVP or epoxy resin. This method of cutting culture ware avoids the formation of small bits of polystyrene generated by rotating discs or saws.     

Apparatus for fixing, staining, and rinsing of tissue cultures for fluorescent-antibody testing

A staining tray and tray-housing container have been developed to facilitate fluorescent-antibody staining of tissue cultures on cover slips, which allows fixing, staining, and rinsing with a minimum of handling. Breakage and loss of cells were negligible.     

Morphological changes of giant mitochondria in the unicellular to multicellular phase during parthenogenesis of Ulva partita (Ulvophyceae) revealed by expression of mitochondrial targeting GFP and PEG transformation

A giant mitochondrion that branches and connects as a single mitochondrion in a cell has been observed during specific phases of the cell cycle of unicellular green algae, but has not been observed in multicellular algae. The genus Ulva is a green macroalga in which the haploid and diploid phases are isomorphic and its gametes develop parthenogenetically. The existence or absence of the giant mitochondrion, and its behavior in Ulva partita, were investigated using a parthenogenesis system. To observe the parthenogenesis of gametes and the dynamics of mitochondria by fluorescence microscopy, we developed an experimental system for culturing and observing U. partita on cover slips: gametes were suspended in 6‐well plates filled with artificial seawater, and cover slips were placed on the well bottoms. The gametes settled on the cover slips as spherical cells (1‐cell S phase). These cells grew into larger cells, losing their eyespot (1‐cell L phase), and developed into multicellular thalli. Gene introduction using the polyethylene glycol (PEG) method is available with transformation efficiencies of 9.0–15.1%. Transformation was performed using a plasmid encoding green fluorescent protein (GFP) fused to the mitochondrial targeting sequence, and mitochondria were labeled by GFP fluorescence. This revealed a string‐shaped giant mitochondrion in a cell of the 1‐cell S phase. In the 1‐cell L phase, a reticular mitochondrion was observed. After the initiation of cell division, the reticular mitochondrion was fragmented, and small oval mitochondria were observed in the 5‐cell phase.     

Permanent Mounting of Unstained and Aceto-Orcein Stained Cells in the Water-Soluble Medium,Abopon

For cellular morphology, mammalian cells were grown on cover slips in Leigh ton tubes, fixed in 1% osmic acid vapor for 2 min, decolorized with 30% H₂O₂ in 5% ammonium oxalate solution (1:7) for 2 min, then washed thoroughly, and finally mounted in a water-soluble medium consisting of a saturated solution of Abopon in 0.2 M phosphate buffer, pH 7.0. For chromosomal analysis of similarly cultured cells, aceto-orcein preparations were made by conventional methods, with the following minor modifications: following pretreatment with colchicine and hypotonic expansion, the cells on the cover slips were fixed in acetic-alcohol (1:3), air dried, incubated at 37° C for 15 min in 2% orcein in 45% acetic acid, rinsed in 45% acetic acid, washed several times in distilled water, and finally mounted in Abopon mounting medium. Both kinds of preparations were allowed to harden for 24 hr before being handled. Such slides will keep for years at room temperature. Studies requiring frequent comparisons of cellular and chromosomal morphology of cultured cells can thus be extended over long periods of time.     

Micro Cell Culture Method for Isolation of Chlamydia trachomatis

Presterilized mictotiter plates (96 wells) with BHK-21 cells on 5-mm cover slips were successfully used for cell culture isolation of trachoma from 15 infected conjunctival scrapings.     

Microtechnique for Indirect Immunofluorescence

A microtechnique for staining preparations of cells grown in vitro for indirect immunofluorescence tests is described. Smaller amounts of materials, as well as less handling of cover slips, greatly facilitates the process.     

Freezing by Liquid Carbon Dioxide in Making Slides Permanent

The expansion of liquid CO₂ may be employed in a quick-freeze method for making aqueous slide preparations permanent. An apparatus is described for this purpose which could be duplicated satisfactorily by cutting a 22mm square hole in the top of a standard freezing microtome specimen holder. The edges should be filed smooth to provide a flat surface for the slide to rest on, and clamps added to keep the slide in place while freezing. Once the slide is frozen, the cover slip may be readily removed, leaving practically all of the tissue on the slide. Following simultaneous thawing and dehydration of the slide in 95% alcohol, covering is done with Diaphane or Euparal and a clean, dry cover slip.     

Integral Heating Device for the Rose Culture Chamber

The culture chamber consists of two metal plates held together by four short screws, a thin (film-type) electrical heating unit, a silicone rubber gasket and two cover slips. The order of assembly is bottom plate, heating element, first cover slip, gasket, second cover slip, and top plate. Syringe needles, one containing a thermistor, and others for supply and removal of fluid are inserted into the chamber through the gasket. Temperature is controlled by electrical connections through a Thermistemp temperature controller.     

Growth and Dormancy in Lunularia cruciata (L.) Dum: V. THE CONTROL OF GROWTH BY A NATCRAL, ENDOGENOUS INHIBITOR

The production of an endogenous growth inhibitor by Lunulariahas been demonstrated in tests with diffusates from gemmae andthalli. Increased growth made by bisected gemmae compared withintact controls suggests that self-inhibition also occurs andpoints to the growing tip as the locus of inhibitor production.More inhibitor is produced in short-day (optimum growth) thanin long-day (dormancy inducing) conditions, but even in shortdays the growth of thalli depends on conditions allowing theinhibitor to diffuse away. Thalli prevented from doing thisby resting the apical 2 mm on non-wettable cover slips stoppedgrowth and showed morphological changes of incipient dormancy.Some of the ecological implications of these tests are discussed.     

An Inexpensive Microtome Attachment for Cutting 50-Micron Nonfrozen Sections for Electron Microscopic Histochemistry

The attachment is for an American Optical Co., Model 888 freezing microtome. It consists of right-angle bracket made from % inch metal rod, one end of which M clamped in the freezing stage holder; the other supports a tissue holder whose base lies at a right angle to the usual position. The fixed and blotted tissue specimen is enclosed (without infiltration) in parah, on a piece of filter paper which is attached to the base of the tissue holder, and sectioned across its long axis by 50 μ increments. After sectioning, the filter paper is removed from the base, the paraffin matrix opened, and the sections transferred to the appropriate processing fluid     

Cryostat Sectioning; Modification of the Antiroll Plate

A replaceable antiroll plate and holder have been designed for use in the Ames Lab-Tek cryostat which replace the plastic plate supplied with the instrument and insure a flawless, properly aligned plate for maximum efficiency in thin section cutting. A metal plate holder is attached to the existing screw-driven bracket provided with the instrument by the manufacturer. Glass plates made from one half of a 1.5 × 3 inch microscope slide are coated on the leading edge with spray-on Teflon and provided with tape spacers. These plates slip into the holder and can be adjusted for angular inclination and alignment with the cutting edge by movement within the holder or manipulation of the adjustment screw.     

Preparation of Chromosomes of Tetrahymena Pyriformis for Photomicrography

Conjugating animals of the protozoan, Tetrahymena pryiformis, were affixed to cover slips by means of Nissenbaum's fluid, followed immediately by 1:3 acetic-alcohol for 18-24 hr. After fixation, the material was transferred through a descending alcohol series to water, then hydrolyzed in 1 N HCl, washed in water, followed by immersion in 45% acetic acid and subsequent mounting in aceto-carmine. Photomicrographs were made using a phase-contrast microscope and Microfile film. The schedule resulted in preparations with abundant material, adequate spacing of chromosomes in a single plane, and excellent differentiation of the chromosomes from the cytoplasm.     

Factors Regulating Microbial Biofilm Development in a System with Slowly Flowing Seawater

Microbial biofilm development was followed under growth conditions similar to those of a projected salinity power plant. Microscope glass cover slips were piled in biofilm reactors to imitate the membrane stacks in such a plant. A staining technique closely correlating absorbance values with biofilm dry weight was used for the study. Generally, the biofilms consisted of solitary and filamentous bacteria which were evenly distributed with considerable amounts of various protozoa and entrapped debris of organic origin. Protozoa predation was shown to decrease the amount of biofilm produced. The biofilm development lag phase was longer at lower temperatures. The subsequent growth phase was approximately arithmetic until stationary phase appeared. Adaptation of a hyperbolic saturation function gave curves that agreed well with the logarithm of the amount of biofilm as a function of time. Increased flow velocity, temperature, and nutrient concentration increased the biofilm production rate. An exponential relationship was shown between biofilm production rate and flow velocity within the range of 0 to 15 cm s⁻¹. Intervals in which the biofilms were exposed to fresh water decreased the biofilm production rate more than four times. If the cover slips were inoculated with untreated seawater for 24 h, subsequent UV treatment had an insignificant effect on the biofilm formation.