Biodegradation during contaminant transport in porous media: 4. Impact of microbial lag and bacterial cell growth.

Miscible-displacement experiments were conducted to examine the impact of microbial lag and bacterial cell growth on the transport of salicylate, a model hydrocarbon compound. The impacts of these processes were examined separately, as well as jointly, to determine their relative effects on biodegradation dynamics. For each experiment, a column was packed with porous medium that was first inoculated with bacteria that contained the NAH plasmid encoding genes for the degradation of naphthalene and salicylate, and then subjected to a step input of salicylate solution. The transport behavior of salicylate was non-steady for all cases examined, and was clearly influenced by a delay (lag) in the onset of biodegradation. This microbial lag, which was consistent with the results of batch experiments, is attributed to the induction and synthesis of the enzymes required for biodegradation of salicylate. The effect of microbial lag on salicylate transport was eliminated by exposing the column to two successive pulses of salicylate, thereby allowing the cells to acclimate to the carbon source during the first pulse. Elimination of microbial lag effects allowed the impact of bacterial growth on salicylate transport to be quantified, which was accomplished by determining a cell mass balance. Conversely, the impact of microbial lag was further investigated by performing a similar double-pulse experiment under no-growth conditions. Significant cell elution was observed and quantified for all conditions/systems. The results of these experiments allowed us to differentiate the effects associated with microbial lag and growth, two coupled processes whose impacts on the biodegradation and transport of contaminants can be difficult to distinguish.

Remediation of metal-contaminated soil and sludge using biosurfactant technology.

Development of environmentally benign approaches to remediation of metal-contaminated soils and sewage sludges are needed to replace currently used techniques of either landfilling or metal extraction using caustic or toxic agents. We report results from four application technologies that use a metal-chelating biosurfactant, rhamnolipid, for removal of metals or metal-associated toxicity from metal-contaminated waste. The four applications include: 1) removal of metals from sewage sludge; 2) removal of metals from historically contaminated soils; 3) combined biosurfactant/phytoremediation of metal-contaminated soil; and 4) use of biosurfactant to facilitate biodegradation of the organic component of a metal-organic co-contaminated soil (in this case the biosurfactant reduces metal toxicity). These four technologies are nondestructive options for situations where the final goal is the removal of bioavailable and leachable metal contamination while maintaining a healthy ecosystem. Some of the approaches outlined may require multiple treatments or long treatment times which must be acceptable to site land-use plans and to the stakeholders involved. However, the end-product is a soil, sediment, or sludge available for a broad range of land use applications.

Development of an agar lift-DNA/DNA hybridization technique for use in visualization of the spatial distribution of Eubacteria on soil surfaces.

While microbial growth is well-understood in pure culture systems, less is known about growth in intact soil systems. The objective of this work was to develop a technique to allow visualization of the two-dimensional spatial distribution of bacterial growth on a homogenous soil surface. This technique is a two-step process wherein an agar lift is taken and analyzed using a universal gene probe. An agar lift is comprised of a thin layer of soil that is removed from a soil surface using an agar slab. The agar is incubated to allow for microbial growth, after which, colonies are transferred to a membrane for conventional bacterial colony DNA/DNA hybridization analysis. In this study, a eubacterial specific probe was used to demonstrate that growing bacterial populations on soil surfaces could be visualized. Results show that microbial growth and distribution was nonuniform across the soil surface. Spot supplementation of the soil with benzoate or glucose resulted in a localized microbial growth response. Since only growing colonies are detected, this technique should facilitate a greater understanding of the microbial distribution and its response to substrate addition in more heterogenous soil systems.

Distribution and expression of G-protein in rat cerebral cortical cells. I. Intact tissue.

Localization and developmental expression of guanine nucleotide-binding regulatory protein (G-protein) were investigated in cerebral cortical tissue of rat. Immunohistochemical techniques using a rabbit polyclonal antibody were applied to reveal the pattern of G-protein distribution. Fluorescent staining in cortical sections was localized mainly to neurons; cell bodies were stained along with the proximal region of processes, including apical dendrites. All neurons appeared to be equally stained, and thus the staining activity reflected the laminar pattern of these cells in cortex. Moreover, areas of neuropil, white matter, and layer I were immunonegative, although synaptic labelling could not be directly eliminated. Most glial cells also lacked staining; however, moderately intense labelling was observed in the choroid plexus, ependymal cells and a subpopulation of microglia. Using Western immunoblot analysis, the antigens recognized in rat cerebral cortex were shown to co-electrophorese with the alpha- and beta-subunits of bovine brain G-protein, thus supporting the specificity of the antibody preparation utilized. In contrast to the adult tissue, in fetal and neonatal cortex faint to moderate staining was uniformly exhibited by essentially all cells. As determined by the immunoblot procedure, the overall level of G-protein increased from the fetal stage through the neonatal and adolescent periods to reach a maximum concentration in young adult tissue. Based on cellular distribution, G-protein may function in the overall regulation of neuronal activity within cerebral cortex. Further, these results suggest that there is a selective developmental increase in expression of G-protein in specific cell populations, particularly neurons.

Distribution and expression of G-protein in rat cerebral cortical cells. II. Primary tissue culture.

In continuing an assessment of the cellular distribution and developmental expression of brain guanine nucleotide-binding protein (G-protein) (Go), this component was investigated in primary cultures of dissociated cerebral cortical cells derived from fetal rat. Using a polyclonal antibody recognizing brain Gi/Go, G-protein was detected through immunohistochemical staining and a Western immunoblot procedure. In initial studies, the presence of G-protein in cultures containing mature cells was demonstrated using the immunoblot technique; this G-protein corresponded predominately to the Go form as the alpha- and beta-subunits were revealed, and these co-migrated with similar antigens from the intact tissue and of purified bovine brain G-protein. The pattern of cellular localization or distribution in stained cultures was very similar to that observed in vivo, as differentially high staining was present in neurons, ependymal cells and microglia. In neurons, the immunofluorescent staining was relatively uniform over the cell body and at least the initial segment of processes. When cultures of different ages were assessed, the G-protein exhibited a developmental pattern of increase in expression similar to that observed in whole tissue, as determined by immunoblotting. The cortical cultures reflect the distribution and expression of G-protein in vivo and, thus, specific cell types therein may be useful in investigating the functional role of the predominate type of this protein in brain tissue (Go).

Morphological diversity of ependymal cells in tissue culture.

Ependymal cells were visualized in primary cultures of cerebral cortex from rat using an immunohistochemical staining technique. Five different morphological subtypes of cuboidal ependyma were recognized: 1) round, 2) triangular, 3) columnar, 4) cone- and spindle-shaped, and 5) large pleomorphic cells. These cells varied in size and almost all possessed cilia. Two distinct forms of tanycyte ependyma were detected based on the presence of cilia. These features reflect a significant level of development of the ependymal cells in culture and may correspond to functional diversity within this group.

Localization of G-protein in rat occipital cerebral cortex.

Localization of guanine nucleotide-binding regulatory protein (G-protein) was investigated in occipital cerebral cortical tissue of rat. An immunofluorescence histochemical staining technique was employed using a rabbit polyclonal antibody raised against bovine brain G-protein. Fluorescent staining was primarily localized to neurons, and labelled the cell body and the proximal region of processes. It appeared that all neuronal cells were equally stained, at least in layers II-VI where cell density permitted a direct assessment. The possibility of synaptic labelling could not be eliminated; however, no significant staining of glial elements was detected. The specific nature of staining was supported on Western blots of cortical extract, where the antibody recognized essentially two bands which coelectrophoresed with the alpha- and beta-subunits of purified G-protein. These results suggest that one potential role for the major species of G-protein present in cerebral cortex (Go) may be the overall regulation of neuronal activity.

Method for the identification of brain macrophages/phagocytic cells in vitro.

The use of labelled latex beads, with acute incubation conditions, for the identification of active macrophages in mixed cell cultures based on their phagocytic activity is described. Fluorescent beads provided the best results and selectively labelled active macrophage cells in cultures of blood monocytes. When this technique was applied to primary cultures of rat cerebral cortex, a specific cell type was significantly labelled. This was a small round cell, previously uncharacterized, which in addition to phagocytic activity indicated by the ingestion of beads also possessed macrophage biochemical markers. Thus, the procedure appears useful for phagocytic macrophage identification in vitro, and should be generally applicable to any tissue culture system. Additionally, this procedure can be rendered compatible with cell viability and, therefore, be utilized for long-term monitoring of macrophages.

Further studies on the identification of microglia in mixed brain cell cultures.

The investigation of brain microglia in primary cultures of dissociated cerebral cortical cells has been continued here utilizing a histochemical procedure for thiamine pyrophosphatase, which selectively stains microglia in whole tissue. This procedure also selectively stained a subpopulation of cells in the cortical cultures. The stained cells were small and exhibited quite variable morphology; from these features, a corresponding cell population could be identified in viable cultures. The stained cells, which were distinct from previously identified macrophage forms, were also distinguished from similar appearing cell types--small neurons and oligodendrocytes. Based on their staining properties, morphology, and distinction from other cell types, the identified cells are proposed as equivalents of ramified microglia.

Brain macrophages: questions of origin and interrelationship.

Brain tissue appears to contain several distinct types of macrophages. An effort is made here to present a description of the complete cohort of macrophages and sources of phagocytic activity in this tissue. Initially, the criteria and methods used for the identification of tissue macrophages in general are considered. These include some morphological and ultrastructural features, assessment of phagocytic activity, and histochemistry for intracellular and surface components. Each of these methods or criteria has certain advantages but also associated problems and limitations; all have been applied in various instances to brain tissue. In a final analysis, the most reliable means of identification of tissue macrophages involves a combination of all of these approaches. The identification and characterization of macrophages have been rendered extremely confusing in the brain because of so many different sources of these cells, both intrinsic and blood-derived. The classes of macrophages or phagocytic cells in brain tissue are microglia, supraependymal cells, epiplexus cells, meningeal macrophages, pericytes, and direct blood-derived macrophages. The morphology, location, and functional properties of each of these classes is described. In an overall view, brain tissue is very well protected by intrinsic macrophages, and the locations and distribution of these cells are consistent with other tissues. Finally, in a consideration of origin and interrelationship, the idea is presented that the most likely source for all or most brain macrophages is monocytic blood cells. The latter cells appear to migrate into the tissue from several sites during embryogenesis and may continue to enter, at least from blood vessels, in the adult state.

Presence and development of ependymal cells in primary tissue cultures derived from embryonic rat cerebral cortex.

Using indirect immunohistochemistry, a secondary antibody was detected in a commercial preparation of antiserum against vasoactive intestinal polypeptide. The secondary antibody selectively stained ependymal cells during the first 3 weeks in vitro in cultures of dissociated cerebral cortical tissue from rat. This staining provided a convenient mechanism for investigating the development and properties of these cells in cultures. The overall level of immunofluorescent staining during the initial 3-week time period appeared to directly reflect the proliferation and development of ependymal cells. Fluorescent staining was initially detected in cells which appeared to correspond to matrix cells or progenitor cells from the ependyma. These cells underwent rapid cell division, as evidenced by distinct morphological stages, to yield daughter cells which were the precursors of mature ependymal cells. Three different morphological classes of mature ependymal cells were observed in the cortical cultures. These classes corresponded to the cuboidal, tanycyte and secretory ependymal cell types described in vivo. Direct counting of stained cells showed that these morphological classes were represented in the cultures in roughly the same proportions seen in vivo (cuboidal 75%, tanycyte 19% and secretory 6%). The temporal aspects of ependyma development permitted the staining of developmental stages corresponding to the various morphological classes or types. The morphological sequence of development of the cuboidal cell and tanycyte from the precursor cell or matrix cell--daughter cell was determined. These two cell types displayed marked differences in their developmental sequence. The developmental sequence of the secretory cell could not be resolved; however, what appeared to be multiple morphological subtypes of this cell class were encountered.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of microglia in primary cultures of mixed cerebral cortical cells.

Microglia were investigated in tissue cultures of dissociated cerebral cortex from embryonic rat. Three different forms of microglia were identified in relatively mature cultures on the basis of morphological criteria, esterase histochemistry, and immunohistochemical staining for Fc receptors. Two of these forms corresponded to the amoeboid and ramified microglia; the amoeboid form stained intensely for esterase activity and ramified cells possessed Fc receptors. The third microglial cell form was distinguished by its morphological properties and was characterized by the presence of numerous filopodial processes. This morphological form occasionally possessed esterase staining and was the predominate type of microglia observed in the cultures. The identification of these cell types supports the potential use of this culture system to investigate functional aspects of microglia and their interaction with other brain cells.

Identification of somatostatin-containing neurons in primary cultures of rat cerebral cortex.

The expression of somatostatin-like immunoreactivity was investigated in primary cultures of dissociated cerebral cortical tissue from the rat. A subpopulation of neurons in the cortical cultures exhibited intense staining for somatostatin. These somatostatin-immunoreactive neurons corresponded to 1.25% of the total neuronal population. Stained neurons were typically small with a soma size of 10-20 micron. The majority of somatostatin-containing cells had stellate and bipolar morphology, with the bipolar class predominating.

The status of the study of invertebrate neurons in tissue culture--phylum Arthropoda.

1. Neurobiological studies employing tissue culture are discussed for all invertebrate organisms in phylum Arthropoda and above. Only arthropod species have been investigated in these studies. 2. The members of phylum Arthropoda utilized were Periplaneta, Locusta, Drosophila, and Schistocerca. 3. Some of the initial studies of nerve cells in tissue culture were performed with Periplaneta and Drosophila; these studies were primarily concerned with differentiation, growth and development. 4. Cultured neurons from cockroach and locust have been used in pharmacological investigations of neurotransmitter receptors. 5. Embryos of the grasshopper Schistocerca were cultured in series of developmental studies related to axon guidance and peripheral nerve formation. 6. Recent studies with Drosophila neurons in vitro have applied a genetic approach to the investigation of molecular properties and function of the axonal sodium channel. 7. An overall view of neuronal tissue culture of invertebrates and analysis of the various areas of research is presented.

Ultrastructure of ependymal cells in primary cultures of cerebral cortex.

Ultrastructural features of ependymal cells growing in primary cultures of dissociated cerebral cortical tissue were investigated using electron microscopy. The ependyma exhibited a specific orientation in tissue culture such that the cell surface corresponding to the apical surface in situ was directed toward the culture medium. The membrane on the apical surface of cultured ependyma was characterized by the presence of cilia and microvilli. The lateral margins were marked by a variety of junctions: zonula occludens, zonula adherens, and membrane interdigitations. The cell cytoplasm contained pleomorphic mitochondria, Golgi profiles, clusters of ribosomes, rough endoplasmic reticulum, and particularly profuse scattered microfilaments and microtubules. These features support the identity of the cells as ependymal in the cultures and establish them as a relatively accurate reflection of ependyma in situ.