Monolayer Cultures for Immunofluorescent Staining from Tumor Primary Explants

Epithelial cells growing around primary explants of carcinomas in plastic ware are well-suited for modern incident light immunofluorescence microscopy. Epithelial outgrowths in growth culture medium are flushed with phosphate-buffered saline (PBS) and absolute ethanol and snap-frozen in liquid nitrogen-isopentane. the walls of the plastic containers bearing the dried monolayer outgrowths are cut out to form microscope slides. Immunofluorescence testa are made on circular areas demarcated on the monolayers by using small metal cylinders to mask against a water-repellent plastic spray. More than 20 immunofluorescence tests can be performed on a culture 40 × 50 mm.     

Monolayer cultures for immunofluorescent staining from tumor primary explants.

Epithelial cells growing around primary explants of carcinomas in plastic ware are well-suited for modern incident light immunofluorescence microscopy. Epithelial outgrowths in growth culture medium are flushed with phosphate-buffered saline (PBS) and absolute ethanol and snap-frozen in liquid nitrogen-isopentane. The walls of the plastic containers bearing the dried monolayer outgrowths are cut out to form microscopic slides. Immunofluorescence tests are made on circular areas demarcated on the monolayers by using small metal cylinders to mask against a water-repellent plastic spray. More than 20 immunofluorescence tests can be performed on a culture 40 X 50 mm.     

Separation of Epoxy-Embedded Cell Cultures from Plastic Flasks for Electron Microscopy

Monolayers of cells grown in ordinary plastic flasks are fixed and embedded “in situ” into Epon. When polymerized at 40 C for 4 days instead of the usual 60 C., the Epon sheet containing the cells is easily detached from the bottom of the plastic container. The Epon sheet is observed by light microscopy as a histological preparation. Ultrathin sectioning of preselected areas can then be carried out in a horizontal plane.     

Hot Melt “Corner Point Method” for Attaching Large Plastic Sections to Glass Slides

We describe a fast method for firm attachment of large plastic sections to glass slides with EVA-copolymers, commonly known as hot melt sticks. Solid hot melt sticks dissolve slowly in xylene to form an adhesive gel within 6 hours. Small drops of hot melt gel are applied to the corners of the sections and surrounding slide surface at ambient or elevated temperatures. The gel sticks to both the plastic and the glass slides. The hot melt “corner point method” prevented detachment of sections in staining procedures. As an additional technique, we suggest the use of hot melt adhesive for attaching plastic specimen blocks to wooden blocks or metallic specimen holders.     

Scanning electron microscopic visualization of a microexudate prepared by the release of cells by urea

The scanning electron microscope (SEM) was used to examine the residue remaining after human fibroblasts, permitted to attach to plastic dishes in the absence of serum, were removed with buffered 1 M urea. The ‘urea carpet’ is entirely different from the “substrate-attached material” (SAM) of Culp [4, 5, 6] in that it contains no “footpad” material. Furthermore, the SEM pictures clearly indicate that urea carpet stimulates the adhesion and spreading of newly added fibroblasts.     

Notes on Technic: “Clearing” Steel Knife Epon Sections in a Polystyrene Film Sandwich

Serial sectioning epoxy embedments by steel knife permits rapid light microscope survey of large tissue volumes, and preselection of areas of interest for electron microscopy. Acetate film (Hollander 1970) and Turtox plastic slides (West 1972) have been suggested as substrates upon which the sections may be “cleared” with an added layer of cured epoxy. In our experience, these substrates are excessively adherent to Epon, and “cleared” sections thinner than 40-50 μm cannot be released from them reliably. The following method is suitable for processing Epon sections 10 or more microns thick.     

Modification of a Scanning Transmission Electron Microscope Specimen Holder for Large Section Viewing

A modification of a scanning transmission electron microscope specimen holder which permits full viewing of large plastic embedded tissue sections is discussed. The method for producing one-centimeter diameter “giant” grids is explained and the procedure for sample preparation is outlined The modification aids the microscopist in his evaluation of tissue structural relationship by providing large areas of tissue for examination and reduces significantly the time required to prepare and examine standard 1-2 mm² electron microscopy tissue sections. Light and electron microscopic evaluations can be made on the same tissue sections.     

Aluminum Foil Molds for Cryostat Blocks

Inexpensive pyramidal templates may be prepared in a range of sizes from segments of plastic or wooden bars (Fig. 1). Pieces of double-layered aluminum cooking foil are shaped by hand around templates of appropriate size and their surfaces are smoothed with fingernails. In this way, “negative” molds of known cross-section and variable depth can be obtained (Fig. 1). Tissue samples are oriented on the bottom of the molds in a few drops of Ames O.C.T. Compound and just covered with the same embedding medium. Narrow strips of light-weight cardboard with identification data are inserted into the blocks close to a lateral surface. The blocks are solidified on the cold stage of a cryostat (a drop of O.C.T. will improve thermal contact and help keep them upright), or with a liquid gas, such as nitrogen, or Freon 22 (in the latter case the molds are immersed as far as the upper level of the O.C.T., but are not submerged, so that the Freon does not bubble into the liquid O.C.T.). After freezing, the foil is removed and the blocks, handled with cold forceps, are mounted on cryostat chucks with O.C.T.     

Adaptation of mouse leukemic cells (L5178Y) to anisotonic media : II. Cell growth

Mouse lymphoma cells (L5178Y) exposed to hypertonic media for 1 h behave as osmometers, but in hypotonic media, after initial swelling, they shrink back to normal volume and maintain it for long periods of time. The lower limit of osmolarity at which this “volume adaptation” will occur lies between 140 and 185 mosM. The “volume adaptation” is associated with a loss of cellular K⁺ probably due to a transient increase in K⁺ permeability and to loss of associated anions and osmotically obliged water. Partial dissipation of the large gradient of K⁺ between cells and medium by pre-exposure to ouabain or to K⁺-free medium results in a diminished capacity to adapt. After the shrinking phase is completed, a new steady state is established with a reduced cellular K⁺ content, normal Na⁺, normal K⁺-permeability, and a reduced activity of the Na⁺ − K⁺ transport system. When adapted cells are returned to normal medium, an initial shrinking is followed by a re-swelling to normal size, associated with a gain in K⁺ content, presumably due to the return to normal activity of the Na⁺ − K⁺ transport system.     

Effects of sandblasting and salt spray on inland plants transplanted to coastal sand dunes

A transplant experiment was conducted on a sandy beach to elucidate whether salt spray and sandblasting are the major factors inhibiting inland plants from becoming established on coastal sand dunes. Potted inland plants of Miscanthus sinensis and Imperata cylindrica var. koenigii were transplanted in two zones on the beach and in one area far inland from the beach. One zone on the beach (sea side) was located on a front dune that was occupied by native sand-dune plants; the other zone (land side) was located behind the sand dunes, where grassland comprised both sand-dune and inland species. To assess the condition of transplants, we measured changes in the canopy leaf area periodically at all sites. The final dry weight at each site was determined at the end of the experiment. Seasonal changes in sandblasting and salt spray intensities were evaluated periodically at the sites by measurement of the opaqueness of exposed transparent plastic sheets and the amount of sodium trapped in exposed filter papers, respectively. All transplants died in the sea-side zone, where both salt spray and sandblasting were most frequent and intense. The final dry weight was greatest at the inland site, which lacked salt spray and sandblasting. Although salt spray was intense in the land-side zone, the canopy leaf area decreased considerably only in seasons during which salt spray was accompanied by intense sandblasting. We concluded that sandblasting accompanied by salt spray is one of the main factors inhibiting the survival and growth of inland plants on coastal sand dunes.     

A cultivar comparison of plant regeneration from suspension cells,callus, and cormel slices ofGladiolus

Callus was initiated from either cormel slices or in vitro-grown plants of sixGladiolus cultivars cultured on Murashige and Skoog’s basal salts medium supplemented with either 10 mg/liter (53.8 µM) 1-naphthaleneacetic acid, 2 mg/liter (9.3 µM) dicamba, or 0.5 mg/liter (2.2 µM) 2,4-dichlorophenoxyacetic acid. More plants were regenerated from callus of the cultivar “Peter Pears” as compared to “Jenny Lee,” “Florida Flame,” or “Golden Year.” No plants were regenerated from callus of “Rosa Supreme” or “Purity White.” Plants were regenerated from 2 and 6-mo.-old suspension cells of “Jenny Lee” and “Peter Pears” but not from “Florida Flame.” Cormel slices cultured on Murashige and Skoog’s basal salts medium supplemented with 1 mg/liter (4.4 µM) 6-benzylaminopurine regenerated plants from all six cultivars indicating a cultivar-independent system of plant regeneration.     

A Rapid Method for Making Permanent Mounts of Nematodes

A rapid method which can be used to mount and clear nematodes and their eggs is presented. Permanent mounts of certain nematodes and parasite eggs have been prepared using a medium consisting of 56 parts of a stock solution of polyvinyl alcohol (“PVA”), 22 parts phenol and 22 parts of lactic acid. The stock solution of PVA is prepared by dissolving 15 grams of PVA in 100 ml. of distilled water. This medium can be used on material killed and fixed in 10% formalin, any concentration of alcohol, alcohol-glycerin or glycerin. Results have been very satisfactory in most instances. An accompanying plate of photographs shows some of the preparations obtained by using this method.     

Cell substratum modulates responses of preosteoblasts to retinoic acid

The aim of this study was to determine the role of ECM components of bone in regulating the differentiation and function of cells of the osteoblast lineage. Rat UMR 201 cells, phenotypically preosteoblast, were plated onto plastic tissue culture dishes or dishes coated with gelled type I collagen or reconstituted basement membrane (matrigel). Acute cell attachment assays showed that cells adhered to substrates in the following order: collagen > matrigel ? plastic. Proliferation rate up to 96 hr were similar on each substrate. However, if cells were treated with 10^?6 M retinoic acid (RA), proliferation rates were reduced compared with control for cells grown on collagen and matrigel but not on plastic. Morphological changes were matrix-specific; in subconfluent cultures, long thin processes were seen with cells grown on collagen and a pattern of interconnecting cell processes formed when cells were plated on matrigel. Striking differences were observed in the constitutive or RA-induced gene expression of cells grown on the different substrates. When cells plated on collagen were treated with RA, induction of mRNA for alkaline phosphatase (ALP) as well as ALP enzyme activity were much less than with cells grown on plastic. In contrast, RA treatment induced osteopontin (OP) mRNA expression more strongly in cells plated on collagen compared with plastic within 24 hr and this was maintained for 72 hr. RA treatment produced a two fold increase of pro-α 1(I) collagen mRNA in cells grown on plastic and matrigel but not in cells grown on collagen. Growth on collagen produced changes in the way UMR 201 cells responded to RA from which they did not fully recover in subsequent 48-hr growth periods on plastic. These results indicate that ECM components regulate the function of and are capable of modulating RA-induced differentiation of preosteoblasts. © 1993 Wiley-Liss, Inc.     

Control of myogenesis in vitro by chick embryo extract

Factors which control the transition from proliferation to myotube formation of cultured avian myogenic cells have been studied. In the conditions used, most cells divide twice after plating and then fuse to form myotubes, about 11 hr after completing final DNA synthesis as indicated by autoradiographic analysis of [³H]TdR incorporation. Giving fresh medium (“feeding”) after 24 hr causes further divisions and delays fusion, while plating cells in “conditioned medium” (in which fusion has already occurred) has the opposite effect. The rate of medium conditioning depends on the number of myogenic cells present, but nonmyogenic cells can also cause conditioning. The entire effect of feeding is reproduced if the amount of embryo extract (EE) initially present in each culture is added instead. This activity of EE involves a macromolecular component. Once cells have completed their presumptive final round of DNA synthesis, adding EE has little effect on their subsequent behavior. This suggests that well before fusion occurs, the ability of these cells to respond to EE by initiating a new round of DNA synthesis is lost, and that they thus become operationally committed to a course leading to fusion. Whether this commitment becomes fixed before, or immediately after, the last mitosis is not clear.     

Poststaining Sections for Electron Microscopy

“Dirt” on electron microscopic sections can generally be avoided by the simple strategem of preventing dry sections from coming in contract with any solution with a dirty surface layer. Wet sections can be pushed through the surface layer of such solutions without ill effect. The first and last solutions to touch a section must be dean, preferably distilled water from a plastic wash bottle.

Mention of a trademark name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA, nor does it imply its approval to the exclurion of other products that may also be suitable.     

Altered expression of biodegradative threonine dehydratase in Escherichia coli mutants.

A number of strains of Escherichia coli K-12 failed to synthesize significant amounts of biodegradative threonine dehydratase (EC 4.2.1.16) when grown anaerobically in tryptone-yeast extract medium, a condition which is optimal for the induction of this enzyme. However, the addition of 10 mM potassium nitrate to the culture medium enabled a few of these strains, notably MB201, to induce the enzyme. An examination of the kinetic parameters, modifier sensitivity, and immunological cross-reactivity revealed that the enzyme produced by MB201 in nitrate-supplemented medium appeared indistinguishable from the dehydratase of a wild-type strain. The reduced expression of threonine dehydratase in MB201 appeared highly specific; the synthesis of two other inducible enzymes, D-serine deaminase and tryptophanase, and two "anaerobic" proteins, namely, fumarate reductase and cytochrome c551, remained unaffected. The mutation (tdcI) responsible for the altered expression of the dehydratase in MB201 was located at min 91 on the E. coli chromosome and appeared to tightly linked to if not identical with pgi, the gene encoding phosphoglucose isomerase, as judged by growth experiments on glucose and fructose, direct assay of phosphoglucose isomerase activity, spontaneous and simultaneous reversion of MB201 (tdcI) to TdcI+ and Pgi+ phenotype, and cosegregation of the two loci during transduction with P1 phage. Because not all strains lacking the dehydratase showed nitrate-dependent enzyme synthesis or had lesions at the pgi locus, it appears that mutations at multiple loci on the E. coli chromosome may influence the expression of the enzyme in vivo.     

Delivery methods for introducing endophytic bacteria into maize

The effectiveness of fivemethods for delivery of ten endophyticbacteria into maize stem and root tissues wasstudied in greenhouse conditions at EmbrapaMilho Sorgo, Sete Lagoas, MG, Brazil. Thedelivery methods included seed inoculation,soil drench, foliar spray, pruned-root dip andseed inoculation + soil drench. The bacterialendophytes were previously isolated from maizeplants, and reinoculated and recovered aftertreatments from maize, cv BR201. Each of thefive methods led to establishment and recoveryof bacterial endophytes in root tissues, butonly four isolates were recovered by the seedtreatment method. All 10 isolates wererecovered by pruned-root dip and seed treatment+ soil drench. No isolates were recovered instem tissues by the seed treatment method, andin the root by foliar spray method. However,all isolates were recovered in stem tissue bypruned-root dip method. The pruned-root dip wasthe most efficient method to deliver bacterialendophytes into maize. The isolate, BR201, wasrecovered by almost all methods in root andstem tissues. The results demonstrate thatendophytic bacteria can be recovered from maizetissues following inoculation by the differentmethods described, but the delivery depends onthe methods used and the endophytic bacterialisolate.     

Optimization of Peroxidase-Catalyzed Oxidation of Luminol in Reversed Micelles of Surfactants in Octane

Luminol oxidation in the Aerosol OT (AOT) reversed micelles in octane catalyzed by horseradish peroxidase (HRP), or its conjugate with Cortisol (HRP-COR), was optimized. The chemiluminescence intensity during luminol oxidation was strongly dependent on the method of preparation of the reaction mixture and the addition of Triton X-45, cyclohexanol and the chemiluminescence “enhancer”, p-iodophenol, into the micellar system. Five procedures for the preparation of the reaction mixture were compared. The maximum chemiluminescence was observed in the micellar system containing all the reaction components excluding a biocatalyst, addition of which into the system started the reaction. Triton X-45, cyclohexanol or p-iodophenol added to the micellar system enhanced significantly the chemiluminescence intensity. The “enhancing” action of p-iodophenol in AOT reversed micelles was 10-fold less than in an aqueous medium.     

Interactions between herbivory and resource availability on grazing tolerance of Leymus chinensis

Herbivory and resource interact to influence plant regrowth following grazing, but few detailed investigations on grazing tolerance at population levels are available. We conducted two pot experiments along a simulated grazing gradient (0%, 25%, 50% and 75% of shoot removal) at three water or nutrient levels to determine the interaction of resource and herbivory on Leymus chinensis, a perennial, dominant species in the eastern Eurasian steppes. Interactions between water availability and clipping intensity on the relative height growth rate (RHGR) and bud number were significant. Significant interactions between nutrient and clipping on RHGR, total biomass and specific leaf area (SLA) were also found. Total biomass and bud number, showing a unimodal curve along the clipping gradient in resource-rich environments, were highest at light clipping level, suggesting that this species has the plastic compensatory responses from under- to overcompensation. Interactions between herbivory and water or nutrient were opposite to each other. The “cooperative” interactions between water and herbivory magnified the difference in grazing tolerance of L. chinensis between high and low water treatments. The “antagonistic” interactions between nutrient and herbivory, on the other hand, were reflected in the lower tolerance to heavy clipping in the high nutrient than low nutrient treatments. Results partly support the limiting resource model (LRM). A modified and simplified graphic model of the LRM was proposed based on our results. The new LRM clearly demonstrated that “cooperative” interactions between varying water levels and clipping intensities aggravate the detrimental impacts of herbivores on plant growth and reproduction, whereas “antagonistic” interactions between nutrient and grazing alleviate the negative effects of herbivores. Biomass compensation and density compensation were identified as main mechanisms of herbivory tolerance in this clonal species.     

Initiation of meiosis in cell cycle initiation mutants of Saccharomyces cerevisiae

Control of the initiation of meiosis in yeast was examined in diploids homozygous for one of four different temperature-sensitive mutations that affect “start” of the mitotic cell cycle. Two of the mutations, cdc28 and tra3, bring about deficiencies in the initiation of meiosis, while cdc25 and cdc35 do not prevent initiation of normal meiosis at both permissive and restrictive temperatures. Moreover, diploids homozygous for the latter two mutations are capable of initiating meiosis in rich growth media upon transfer to the high, non-permissive temperature. This unique feature contrasts with the behavior of other yeast strains which require a starvation sporulation medium for initiation of meiosis. It is suggested that the initiation of meiosis includes functions that are shared with “start” of the mitotic cell cycle, as well as functions related to the choice between the two processes. Meiosis in vegetative media at the restrictive temperature (in cdc25 or cdc35 homozygotes) may be important for the study of chemical and physiological phenomena resulting from the meiotic process and not from adaptation to the sporulation medium.