Effect of culture media and growth phase on the morphology of lactobacilli and on their ability to adhere to epithelial cells

Previous studies have demonstrated that the ability of lactobacilli to attach to and colonize uroepithelial surfaces is an important characteristic that enhances interference against uropathogenic bacteria. This adherence capacity was found to vary amongst lactobacillus strains and with the type of growth medium used to culture the organisms. The present study was undertaken to examine further the effect of culture media and growth phase on lactobacillus adherence to uroepithelial cells in vitro. In addition, a freeze substitution technique was developed to examine the morphology of strainsLactobacillus casei ssrhamnosus RC-17,L. casei GR-1, andL. acidophilus T-13 in relation to growth conditions and adhesion. A growth curve was plotted for strain GR-1, and adherence was found to be lowest for bacteria in early log phase (39 bacteria per uroepithelial cell) and highest in stationary phase (59 bacteria per uroepithelial cell). Strains RC-17 and GR-1 attached in high numbers to uroepithelial cells, whereas T-13 was poorly adherent. The latter formed a long, relatively dense, fibrous capsule after growth in brain heart infusion yeast extract agar, unlike strains GR-1 and RC-17, which formed a short, tightly bound, electron-dense capsule which surrounded the cells in a radial fashion. Growth of RC-17 in batch cultures of human urine, with and without addition of carbohydrates, resulted in formation of an irregular, fibrous extracellular matrix. These experiments illustrate that growth phase and culture conditions affect the extracellular structure of lactobacilli and also affect the adherence capacity of these bacteria. Structural changes mediated by availability of nutrients may partly explain why lactobacilli vary between species and between hosts in their colonization of the urogenital tract.     

The role of regulation of cell speed in the behavior of Physarum polycephalum amoebae

Studies on the behavior of wild-type and mutant Physarum polycephalum amoebae have revealed that regulation of cell speed results in different patterns of cell dispersion in different environments and have shown that P. polycephalum can be used for genetic studies of the mechanisms responsible for this element of cell behavior. Colonies generated by clonal populations of amoebae growing on E. coli display alternate colony morphologies depending on the pH of the culture medium and the presence of live E. coli as a nutrient. In the larger ‘spreading colonies’ cells at the outside of a colony are dispersed over a wide band of bacteria while in the smaller ‘aggregate ring colonies’ most cells moving on bacteria are aggregated in a regularly shaped ring on a narrow band of bacteria at the border of the bacterial lawn created when amoebae completely consume the bacteria available in the colony center. Measurements of cell growth, the rate of colony expansion, and the rate of single cell movement show that cells in contact with bacteria move more slowly in aggregate ring than in spreading colonies. Moreover, since in aggregate ring colonies the rate of movement of cells in contact with bacteria is also reduced relative to that of cells moving on adjacent regions of the agar surface, inhibition of cell speed appears to be at least partially responsible for generating the aggregate ring morphology. Characterization of the behavior of a single locus mutant which generates spreading colonies under conditions where aggregate ring colonies are normally formed has provided additional evidence that a specific mechanism is involved in controlling the distribution of amoebae through regulation of cell speed. Furthermore, the studies of this mutant have shown that aberrant colony morphology can be used as an easily recognized phenotype for identifying and studying mutants with defects which affect the regulation of cell speed.     

Production and purification of an extracellular polyglucan produced byCellulomonas flavigena strain KU

Summary A polysaccharide is synthesized byCellulomonas flavigena strain KU when it is cultured in a synthetic medium which uses ammonium salts as a nitrogen source and contains an excess of a carbon and energy source. Production of the polysaccharide begins in late log phase and reaches a maximimum during stationary phase. In batch cultures it may be produced in yields of up to 9 g dry polysaccharide/liter of culture. It is not secreted into the growth medium but rather remains associated with the cells, resulting, apparently, in their aggregation. When such aggregated cells are extracted with dilute sodium hydroxide solutions the polysaccharide is solubilized. Neutralization of supernatant fluid of such extracts results in sedimentation of the polysaccharide which may then be purified by extensive washing with water. The polysaccharide is insoluble in water, alcohols or acetone, but dissolves in concentrated formic acid, dimethyl-sulfoxide, and dilute sodiumor potassium hydroxide. Thin-layer and gas-liquid chromatographic analysis of hydrolysates indicated that it is a polyglucan. When resuspended in water at concentrations of 2–3% it forms a stable hydrogel.     

Expression of capsule-associated genes of Cryptococcus neoformans

Cryptococcus neoformans produces an extracellular polysaccharide capsule that is related to its virulence. The production of capsular components was reported to be accelerated when cultured on media with lower amount of glucose. In this study, relationship between capsule synthesis and expression of capsule-associated genes (CAP genes) was investigated by quantitative real-time PCR analysis. Normally encapsulated strains and a stable acapsular strain were cultured in 1% polypepton medium with 0.1% or 15% glucose. The results of assessment of the capsule size showed that the capsule of yeast cells cultured in the medium with low amount of glucose was thicker than that with high amount of glucose. The CAP gene expressions of normally encapsulated strains were higher in the medium with 0.1% glucose than in the medium with 15% glucose. Furthermore, CAP10, CAP59 and CAP60 genes were expressed very low in a stable acapsular strain, and CAP64 gene was not expressed. Results of assessment of capsule size and CAP gene expressions by quantitative real-time PCR analysis indicated that CAP gene expressions might be related to the production of capsule, and that glucose concentration in culture media might be related to the expression of CAP genes.     

Transient appearance of lectin receptors onRhizobium trifolii

The appearance on the surface ofRhizobium trifolii 0403 of determinants important to both clover lectin (trifoliin) binding and adherence of the bacteria to clover root epithelial surfaces was studied by quantitative agglutination, immunofluorescence, and direct microscopic techniques. These unique determinants were found for only transient periods of time—as cells left lag phase and as they entered stationary phase of growth in broth. When present, these receptors were associated with a fibrillar polyanionic capsule surrounding the cells when grown on solid medium. These studies support earlier proposals that the architecture of the rhizobial cell surface is not constant in composition, but changes with the phase of growth.     

Inactivation of Candida albicans by ultraviolet radiation

Summary Inactivation of Candida albicans by ultraviolet (uv) light is markedly dependent upon (a) the cell division stage and (b) the nutrition and growth temperatures of cells both before and after irradiation. Cells grown at 37°C after irradiation show lower survivals than those grown at 25°C. At either recovery temperature, cells which had been cultured before irradiation at 37°C are able to sustain less uv damage prior to inactivation than those cultured at 25°C. The radiosensitivities of budding and non-budding cells are the same when survivals are scored at 25°C; at low uv dosages, cells show slightly poorer recoveries on enriched medium than on minimal medium whereas at higher dosages, their recoveries on both kinds of media are equivalent. In contrast, at 37°C, uv treated non-budding cells are much more susceptible to inactivation than budding cells; non-budding cells also express much poorer recovery on enriched medium than on minimal medium at 37°C whereas budding cells survive equally well on either medium. Though non-budding cells grown for irradiation on minimal or enriched media exhibit the same radiosensitivites, budding cells grown for irradiation on enriched medium are more susceptible to inactivation at 37°C than those grown on minimal medium.The particularly poor recovery by irradiated non-budding cells at 37°C is correlated with their unique tendency to undergo a transitory filamentation when initiating growth at that temperature. Evidence is presented that neither the filamentous growth per se nor the temporary inhibition of cell division associated with filamentation causes the poor recovery. Furthermore, while irradiated non-budding cells at 37°C exhibit singular susceptibility to inhibition of recovery by metabolic antagonists which disturb protein synthesis, the course of their filamentous growth is not affected by such agents. It is concluded that recovery from irradiation and the instigation of cytokinesis by non-budding cells of C. albicans result from different metabolic processes which may be related through a common temperature sensitive step. C. albicans does not photoreactivate and observations on recovery by cells prevented from undergoing immediate postirradiation replication do not indicate the existence of a system for dark repair of DNA damage comparable to that occurring in bacteria. Difficulties attending a valid demonstration of DNA dark repair in yeasts are discussed.     

Induction of luciferase synthesis in Beneckea harveyi by other marine bacteria

It has been previously demonstrated that luciferase synthesis in the luminous marine bacteria, Beneckea harveyi and Photobacterium fischeri is induced only when sufficient concentrations of metabolic products (autoinducers) of these bacteria accumulate in growth media. Thus, when cells are cultured in liquid medium there is a lag in luciferase synthesis. A quantitative bioassay for B. harveyi autoinducer was developed and it was shown that many marine bacteria produce a substance that mimics its action, but in different amounts, (20–130% of the activity produced by B. harveyi) depending on the species and strain. This is referred to as alloinduction. None of the bacteria tested produced detectable quantities of inducer for P. fischeri luciferase synthesis. These findings may have significance with respect to the ecology of B. harveyi and P. fischeri.Non-Standard Abbreviation AB medium autoinducer bioassay medium     

CULTURE OF THE TERRESTRIAL CYANOBACTERIUM,NOSTOC FLAGELLIFORME (CYANOPHYCEAE), UNDER AQUATIC CONDITIONS1

Both colonies and free‐living cells of the terrestrial cyanobacterium, Nostoc flagelliforme (Berk. & Curtis) Bornet & Flahault, were cultured under aquatic conditions to develop the techniques for the cultivation and restoration of this endangered resource. The colonial filaments disintegrated with their sheaths ruptured in about 2 days without any desiccating treatments. Periodic desiccation played an important role in preventing the alga from decomposing, with greater delays to sheath rupture with a higher frequency of exposure to air. The bacterial numbers in the culture treated with seven periods of desiccation per day were about 50% less compared with the cultures without the desiccation treatment. When bacteria in the culture were controlled, the colonial filaments did not disintegrate and maintained the integrity of their sheath for about 20 days even without the desiccation treatments, indicating the importance of desiccation for N. flagelliforme to prevent them from being disintegrated by bacteria. On the other hand, when free‐living cells obtained from crushed colonial filaments were cultured in liquid medium, they developed into single filaments with sheaths, within which multiple filaments were formed later on as a colony. Such colonial filaments were developed at 15, 25, and 30° C at either 20 or 60 μmol photons·m^?2·s^?1; colonies did not develop at 180 μmol photons·m^?2·s^?1, though this light level resulted in the most rapid growth of the cells. Conditions of 60 μmol photons·m^?2·s^?1 and 25° C appeared to result in the best colonial development and faster growth of the sheath‐held colonies of N. flagelliforme when cultured indoor under aquatic conditions.     

EFFECTS OF THE LENS CAPSULE ON CELLULAR FLATTENING, CELL GROWTH AND EXPRESSION OF THE DIFFERENTIATED TRAITS OF CHONDROCYTES CULTURED IN VITRO

The morphology, growth and differentiation of chondrocytes cultured on the lens capsule were studied. When incubated in Eagle's MEM with fetal serum, chondrocytes on the surface of the lens capsule became flattened with extended pseudopodia, but most cell remained spherical on the surface of a plastic dish. Thus, the lens capsule promoted cellular flattening of chondrocytes. When grown in Ham's FI2 medium, flattening of cells on the lens capsule was greater and the cells developed the features of fibroblasts without any detectable characteristics of chondrocytes, although their growth rate was not enhanced. This inhibitory effect of the lens capsule on differentiation in this medium was reversed when the cells were separated from the lens capsule and grown on a plastic substrate.     

Biogeochemical Conditions Favoring Magnetite Formation during Anaerobic Iron Reduction

Several anaerobic bacteria isolated from the sediments of Contrary Creek, an iron-rich environment, produced magnetite when cultured in combinations but not when cultured alone in synthetic iron oxyhydroxide medium. When glucose was added as a carbon source, the pH of the medium decreased (to 5.5) and no magnetite was formed. When the same growth medium without glucose was used, the pH increased (to 8.5) and magnetite was formed. In both cases, Fe²⁺ was released into the growth medium. Geochemical equilibrium equations with E_h and pH as master variables were solved for the concentrations of iron and inorganic carbon that were observed in the system. Magnetite was predicted to be the dominant iron oxide formed at high pHs, while free Fe²⁺ or siderite were the dominant forms of iron expected at low pHs. Thus, magnetite formation occurs because of microbial alteration of the local E_h and pH conditions, along with concurrent reduction of ferric iron (direct biological reduction or abiological oxidation-reduction reactions).     

Isolation,origin and properties of enucleate plant microplasts

Summary Auxin-induced highly vacuolated thin-walled callus cells of several plant species, when ruptured, released large numbers of subcellular units most of which were enucleate. These enucleate microplasts are surrounded by an inner membrane of the cell, most probably derived from the tonoplast. When microplasts isolated fromSaintpaulia callus were cultured in a medium supplemented with growth substances they formed a thin wall and underwent budding. Microplasts could be useful for various studies in plant cell biology and for genetic manipulations.     

The Role of Intercellular Contacts in the Initiation of Growth and in the Development of a Transiently Nonculturable State by Cultures of Rhodococcus rhodochrous Grown in Poor Media

It was found that the growth of Rhodococcus rhodochrous cells in a modified Saton’s medium strongly depends on the rate of culture agitation in the flask: agitation at 250 rpm in flasks with baffles stops cell multiplication, whereas slight agitation leads to pronounced culture growth. The growth retardation phenomenon was reversible and did not manifest itself in exponential-phase cultures or when the cells were grown in a rich medium; furthermore, it was not connected with the degree of culture aeration. When agitated at a moderate rate, the bacterial cells formed aggregates in the lag phase, which broke up into single cells in the exponential phase. The inhibitory effect of vigorous agitation was removed by the addition, to the medium, of the supernatant (SN) of a log-phase culture grown in the same medium with moderate agitation. Vigorous agitation is thought to interfere with cell contact, whose establishment is necessary for the development of an R. rhodochrous culture in a poor medium, which occurs in the form of (micro) cryptic growth. When grown in a modified Saton’s medium, R. rhodochrous cells were capable of transition, in the prolonged stationary phase, to a resting and transiently nonculturable state. Such cells could be resuscitated by incubation in a liquid medium with the addition of the supernatant or the Rpf secreted protein. The formation of transiently nonculturable cells was only possible under the conditions of a considerable agitation rate (250–300 rpm), which prevented secondary (cryptic) growth of the culture. This circumstance indicates the importance of intercellular contacts not only for the initiation of growth but also for the transition of the bacteria to a dormant state.__________Translated from Mikrobiologiya, Vol. 74, No. 4, 2005, pp. 489–797.Original Russian Text Copyright © 2005 by Voloshin, Shleeva, Syroeshkin, Kaprelyants.     

Microencapsulation of yeast cells in the calcium alginate membrane

Summary Cells of Saccharomyces cerevisiae (ATCC 24858) were encapsulated in the calcium alginate membrane and cultured. Swelling of the capsule was prevented by adding 0.2 g CaCl₂ to 1 L growth medium. The dry cell concentration based on the inner volume of the capsule reached 309 g/L, which was much higher than could be obtained by cell entrapment. All the cells remained inside the capsule during the cultivation. The flux of CO₂ through the capsule membrane increased approximately twice by adding a nonionic surfactant to the CaCl₂ solution during the step of capsule formation.     

The polysaccharide capsule of the pathogenic fungus Cryptococcus neoformans enlarges by distal growth and is rearranged during budding

The capsule of Cryptococcus neoformans can undergo dramatic enlargement, a phenomenon associated with virulence. A prior study that used Ab to the capsule as a marker for older capsular material concluded that capsule growth involved the intermixing of new and old capsular material with displacement of older capsular polysaccharide towards the surface. Here we have revisited that question using complement (C), which binds to capsular polysaccharide covalently, and cannot redistribute by dissociation and binding at different sites. The experimental approach involved binding of C to cells with small capsules, inducing capsule growth, and following the location of C relative to the cell wall as the capsule enlarged. C remained close to the cell wall during capsule growth, indicating that capsule enlargement occurred by addition of new polysaccharide near the capsule edge. This conclusion was confirmed by an independent method that employed radioactive metabolic labelling of newly synthesized capsule with 3H-mannose followed by gradual capsular stripping with gamma-radiation. Capsule growth proceeded to a certain size, which was a function of cell size, and was not degraded when the cells were transferred to a non-inducing medium. During budding, an opening appeared in the capsule of the mother cell that permitted the nascent bud to separate. Scanning EM suggested that a physical separation formed between the capsules of the mother and daughter cells during budding, which may avoid mixture between both capsules. Our results indicate that C. neoformans capsular enlargement also occurs by apical growth and that budding results in capsular rearrangements.     

Transition of the R factor NR1 and Proteus mirabilis: level of drug resistance of nontransitioned and transitioned cells.

When Proteus mirabilis harboring the R factor NR1 is cultured in Penassay broth containing 100 mug of chloramphenicol (CM) per ml, there is an amplification in the number of copies of the r-determinants per cell. Under these conditions, R factors harboring multiple tandem sequences of r-determinants are formed. Autonomous poly-f-determinants consisting of multiple copies of r-determinants are also formed. This phenomenon has been referred to as the "transition". Transitioned cells have considerably higher levels of resistance to CM and streptomycin (SM), but not to tetracycline (TC), than do nontransitioned cells and grow more rapidly in medium containing either CM or SM. There is essentially no difference in growth rates between transitioned and nontransitioned cells in drug-free medium. The higher level of resistance of transitioned cells to SM has made it possible to investigate the mechanism of the transition. Using replica plating, it has been possible to isolate spontaneously occurring transitioned cells from a nontransitioned population which appear to outgrow the nontransitioned cells during growth in medium containing 100 mug of CM per ml. If transiitoned cells are subsequently cultured in drug-free medium, the cells return gradually to the nontransitioned state, which has been referred to as the "back-transition was monitored by examining the level of resisitance of the cells to SM. In both situations the cell populations were found to be heterogeneous, consisting of a mixture of nontransitioned and transitioned cells. Under the conditions of our experiments, the transition appeared to be due to the more rapid growth of a minor fraction of spontaneously occurring transitioned cells which outgrew the remainder of cells in the population. To obtain the transition, the drug resistance gene must reside on the r-determinants component of the R factor. The transition did not take place when the cells were cultured in medium containing high concentrations of TC. This indicates that the TC resistance genes reside on the resistance transfer factor component of the R factor, which is in agreement with physical studies on R factor deoxyribonucleic acid.     

A growth‐rate composition formula for the growth of E. coli on co‐utilized carbon substrates

When bacteria are cultured in medium with multiple carbon substrates, they frequently consume these substrates simultaneously. Building on recent advances in the understanding of metabolic coordination exhibited by Escherichia coli cells through cAMP‐Crp signaling, we show that this signaling system responds to the total carbon‐uptake flux when substrates are co‐utilized and derive a mathematical formula that accurately predicts the resulting growth rate, based only on the growth rates on individual substrates.     

Successful transplantation of three tissue-engineered cell types using capsule induction technique and fibrin glue as a delivery vehicle

Recent advances in cell biology and tissue engineering have used various delivery vehicles for transplanting varying cell cultures with limited success. These techniques are frequently complicated by tissue necrosis, infection, and resorption. The purpose of this study was to investigate whether urothelium cells, tracheal epithelial cells, and preadipocytes cultured in vitro could be successfully transplanted onto a prefabricated capsule surface by using fibrin glue as a delivery vehicle, with the ultimate goal for use in reconstruction. In the first step of the animal study, tissue specimens (bladder urothelium, tracheal epithelial cells, epididymal fat pad) were harvested for in vitro cell culturing, and a silicone block was implanted subcutaneously or within the anterior rectus sheath to induce capsule formation. After 6 to 10 days, when primary cultures were confluent, the animals were re-anesthetized, the newly formed capsule pouches were incised, and the suspensions of cultured urothelia cells (n = 40), tracheal epithelial cells (n = 32), and preadipocytes (n = 40) were implanted onto the capsule surface in two groups, one using standard culture medium as a delivery vehicle and the second using fibrin glue. Histologic sections were taken, and different histomorphologic studies were performed according to tissue type. Consistently in all animals, a highly vascularized capsule was induced by the silicon material. In all animals in which the authors used fibrin glue as a delivery vehicle, they could demonstrate a successful reimplantation of cultured urothelium cells, tracheal epithelial cells, or preadipocytes. Their animal studies showed that capsule induction in combination with fibrin glue as a delivery vehicle is a successful model for transplantation of different in vivo cultured tissue types.     

Nodulation of soybean byRhizobium japonicum mutants with altered capsule synthesis

Spontaneous mutants with altered capsule synthesis were isolated from a marked strain of the symbiont,Rhizobium japonicum. Differential centrifugation was used to enrich serially for mutants incapable of forming capsules. The desired mutants were detected by altered colony morphology and altered ability to bind host plant lectin. Three mutants failed to form detectable capsules at any growth phase when cultured in vitro or in association with the host (soybean,Glycine max (L.) Merr.) roots. These mutants were all capable of nodulating and attaching to soybean roots, indicating that the presence of a capsule physically surrounding the bacterium is not required for attachment or for infection and nodulation. Nodulation by several of the mutants was linearly proportional to the amount of acidic exopolysaccharide that they released into the culture medium during the exponential growth phase, indicating that such polysaccharide synthesis is important and perhaps required for nodulation. Two of the mutants appeared to synthesize normal lectin-binding capsules when cultured in association with host roots, but not when cultured in vitro. Nodulation by these mutants appeared to depend on how rapidly after inoculation they synthesized capsular polysaccharide.Abbreviations CPS capsular polysaccharide - EPS exopolysaccharide - FITC fluorescein isothiocyanate Contribution No. 719 of the C.F. Kettering Research Laboratory     

Neuronal differentiation of PC12 cells as a result of prevention of cell death by bcl-2

Rat pheochromocytoma PC12 cells die when cultured in serum-free medium. Neurotrophic factors can rescue PC12 cells from cell death, and induce neuronal differentiation. To further investigation the relationship among cell death, survival, and differentiation, the bcl-2 cDNA, which is known to prevent apoptosis in various types of cells, was transfected into PC12 cells. Six monoclonal bcl-2-transfected cell lines were isolated and confirmed to express mRNA and protein product of bcl-2. The wild-type and bcl-2-transfected PC12 cells were kept to adhere to collagen-coated dishes at the inintiation of serum-free experiments to avoid cellular damage due to detachment of the cells by triturtion. Even under the conditions, the control PC12 cells mostly died within 24 h, when cultured in serum-free medium whereas those expressing Bcl-2 survived even for 7 days in serum-free medium. Moreover, outgrowth of long processes in thebcl-2-transfected cells was only observed under the condition to keep the cells attached to the dishes in serum-free medium without any additive neurotrophic or growth factors. Neurofilament medium protein, which is a neuron-specific cytoskeletal component, was also expressed in the differentited cells, suggesting that the long processes in bcl-2-transfected PC12 cells are neurites. However, neuronal differentiation of PC12 cells expressing Bcl-2 was not observed when cultured in serum-containing medium. Accordingly, survival of PC12 cells expressing Bcl-2 under the condition which cells usually die may be accompanied with neuronal differentiation. 1994 John Wiley & Sons, Inc.     

Cytological peculiarities of some extremely halophilic soil archaeobacteria

Ultrastructure and morphogenesis of extremely halophilic neutrophilic (Halobacteriam distributum, Halococcus turkmenicus) and alkaliphilic (Natronobacterium pharaonis, Natronococcus occultus) archaeobacteria were studied. The H. distributum culture was rather polymorphous and produced cells of four types. Due to the irregular cell fission in different planes packages of various numbers of cells surrounded by a common capsule were formed. Resting forms (halocysts) with multilayer covers were present in the population. The N. pharaonis culture consisted of rod-like cells and cyst-like forms. Besides, under conditions of carbon limitation, multicellular aggregated forms were found in the culture. Encapsulated single cells and aggregated forms with a common capsule were observed in H. turkmenicus and N. occultus cultures.