Respiration in Organic Acid-Forming Molds

Mycelia of Rhizopus oryzae, a lactate forming fungus, were used to study respiration and glucose degradation in this organism.

The respiration of the mycelia obtained during an early stage of shake culture growth, when only a little lactate was produced, was completely inhibited by KCN, while the respiration of the mycelia obtained from older shake cultures, or from surface cultures, regardless of the age, was not inhibited by KCN. These results suggest that energy for growth of Rhizopus oryzae is predominantly obtained by a respiratory system involving cytochromes and that a loss of this system occurs in older shake cultures which then begin to accumulate lactate. This observation was confirmed by the enzymatic studies on cell-free extracts.     

EFFECTS OF ALUMINUM SALTS ON CULTURED NEUROBLASTOMA CELLS

Experimental induction of neurofibrillary tangles was demonstrated several years ago in the central nervous system of rabbits injected with aluminum salts. In the current studies, neuroblastoma cells, capable of morphologic and biochemical differentiation in monolayer culture, have been exposed to medium containing aluminum phosphate; such treatment resulted in an abundant accumulation of 100 Å neurofilaments after 6 days of continous exposure to the aluminum salt. While growth rates and incorporation of radioactive thymidine in treated cells remained similar to controls, total cellular protein, and incorporation of radioactive leucine were significantly increased. Paradoxically, when the protein content of aluminum-treated cultures was maximal, these cultures contained about 20 per cent less ribosomal RNA per cell than in control cultures. In addition, activity of an important neuronal protein, i.e. acetylcholinesterase, was depressed in treated cultures to a level below control values. Both temporal and morphologic similarities between treated neuroblastoma cultures and animals injected with aluminum salts suggest that the observed changes in macromolecular synthesis in cell culture are relevant to in vivo studies.     

AXENIC CULTURE OF THALASSIA TESTUDINUM BANKS EX KÖNIG (HYDROCHARITACEAE)

Cultures of Thalassia testudinum were established and maintained in the absence of other detectable organisms. Axenic cultures were initiated using surface sterilized seeds which were aseptically dissected from surface sterilized fruits. Seedlings were cultured in 75-ml (25-mm × 200-mm) culture tubes containing 30 ml of rooting substrate and 40 ml of chemically defined seawater media. Seedlings and culture media were analyzed for microbial contamination after 42 days of culture utilizing standard marine bacterial/fungal isolating procedures and by light and scanning electron microscopy. Axenic seagrass cultures allow physiological studies such as nutrient assimilation kinetics, rhizosphere and phyllosphere microbial interactions through mono- and poly-axenic seagrass-microbial cultures.     

Quantitative Studies of Resistance Induced By Avirulent Cultures of Erysiphe graminis f. sp. hordei in Barley

Quantification of resistance induced by avirulent cultures of Erysiphe graminis f. sp. hordei against virulent cultures in barley was attempted. Four mildew cultures and 4 barley varieties with known genes of virulence and resistance respectively were used. Pre, post and simultaneous inoculation of leaves was done with avirulent and virulent cultures. Pre-inoculation with avirulent cultures induced resistance in the host such that the pustule number and spore production by later inoculation of virulent cultures was reduced significantly. Once induced, such resistance was active up to 8 days. There was some indication of induced susceptibility if the inducing culture was characterized by medium virulence. Increase of inceulum density of the avirulent (inducer) culture increased the amount of induced resistance Further studies of the phenomenon of induced resistance are needed in relation to possible applications for disease control through inoculations. varietal mixtures and multilines.     

Selection of a super-growing legume root culture that permits controlled switching between root cloning and direct embryogenesis

 Root cultures, displaying vigorous growth and high embryogenic capacity, were established in the legume forage species Lotus corniculatus (bird’s-foot trefoil). Root cloning as well as plant regeneration was achieved on hormone-free medium, in agitated culture in the dark or under stationary conditions in the light, respectively. These qualities of vigorous growth and regeneration faded with time in hormone-free culture, with slow-growing roots turning brown in color. Addition of the synthetic cytokinin-like hormone benzylaminopurine to the culture medium, however, re-established the aging tissue’s capacity for somatic embryogenesis and plant formation. During continuous initiation of new cultures, it was possible to obtain one root culture (selected from 11 960 seeds at a 65% germination rate) which did not show the typical decline of qualities after prolonged proliferation but distinguished itself by displaying even faster growth and more vigorous embryogenic plant production on hormone-free medium. There was no decline since its initiation 9 months earlier. This super-growing root culture produces plants that show no morphological differences as compared to wild-type regenerants or seedlings. Roots, dissected from plantlets derived from super-root embryogenesis, expressed all the super-root qualities again when cultured in vitro. This is the first report on somatic embryogenesis from sustained root cultures without exogenous hormone application. Such a hormone-free, continuous root culture should provide a superior experimental system for genetic or developmental studies that might be sensitive to exogenous hormones, such as somaclonal variation in transgenesis or, since introduced in a legume species, nodulation in vitro. Received: 22 September 1997 / Accepted: 21 October 1997     

In vitro culture of marine trematodes from their snail first intermediate host

The ability to culture parasites outside their host (i.e. in vitro) is essential for several aspects of parasitological research. Here, a culture medium for marine trematode parthenitae was optimized using Philophthalmus sp. rediae from the intermediate snail host, Zeacumantus subcarinatus. The medium was optimized by sequentially testing the suitability of different levels of osmolality, different commercially available media, and different concentrations of supplemented chicken serum, while controlling for genetic variation among cultures. Philophthalmus sp. rediae survived up to 56 days in cultures of the best tested medium, remaining active and continuously shedding cercariae. The broader suitability of the culture medium was tested using five other trematode species from different families (using either the same or other marine snails as first intermediate hosts): Galactosomum sp., Acanthoparyphium sp., Maritrema novaezealandensis, Curtuteria australis, and an undescribed species of the family Opecoelidae. Survivorship of rediae and sporocysts from these species ranged from eight days to 42 days. The culture procedures developed here can therefore be used in the future as a system under which to culture marine trematode parthenitae for experimental studies.     

Long-term cultures of barley synthesize and correctly deposit seed storage proteins

Summary Long-term cultures of four different cultivars of barley (Hordeum vulgare L.) have been established. Both callus and suspension cultures formed embryogenic structures at high frequency even after more than 18 months of culture. These compact proembryogenic cell clusters synthesize seed storage globulins whereas loose cell aggregates in callus culture and suspension cultures of fine dispersed consistency were free of globulins. Globulin synthesis was especially intense in compact structures of callus cultures established from suspension culture-derived protoplasts. Within the cells storage globulins are deposited in the vacuolar compartment as in zygotic embryos. The molecular data provided recommend the system for studies on factors determining seed protein gene expression and intracellular protein transport.Abbreviations MS Murashige and Skoog (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid     

Recent advancement in research on photoautotrophic micropropagation using large culture vessels with forced ventilation

Summary Successful fundamental or basic research, while being stimulated by applied studies, provides the development of new technologies for the benefit of mankind. Photoautotrophic micropropagation or micropropagation using sugar-free medium is no exception from this generalization. The concept of photoautotrophic micropropagation is derived from research that revealed the relatively high photosynthetic abilities of chlorophyllous cultures such as leafy explants and plantlets in vitro. To meet the ever-increasing demand for quality transplants, the scaling-up of photoautotrophic micropropagation systems, for commercialization, has become necessary. This article reviews the recent advancement in the development and utilization of large culture vessels for photoautotrophic micropropagation with special emphasis on the feasibility of the system for the commercial-scale propagation. The review also includes choices for supporting material, ventilation type, planting density, vessel volume, and vessel sterilization procedure, and problems and solutions to achieve uniform growth in a large culture vessel. A case study of the commercial application of a photoautotrophic micropropagation system using large culture vessles, which recently has been established in Kunming, China, is also presented in this article.     

Effects of different vertebrate growth factors on primary cultures of hemocytes from the gastropod mollusc,Haliotis tuberculata

Summary— A useful experimental system from primary cultures of hemocytes from Haliotis tuberculata has been established. Six days after initiation of the culture, the viability of hemocytes remained constant as measured by the MTT assay. In addition, hemocytes showed physiological responses as judged by protein and DNA syntheses in response to treatment with vertebrate growth factors. Porcine insulin and human epidermal growth factor (EGF) stimulated [³H]-leucine and [³H]-thymidine incorporation in hemocytes in a dose-dependent manner. No additive effect of insulin and EGF is observed either for [³H]-leucine or for [³H]-thymidine incorporation. The response of primary cultures of abalone hemocytes to vertebrate growth factors confirms their growth potential in vitro and provides a suitable model for further studies on regulation of the control of cellular processes such as cell growth, differentiation and migration in invertebrate cells.     

Medium for development of bee cell cultures (<Emphasis Type="Italic">Apis mellifera</Emphasis>: Hymenoptera: Apidae)

A media for the production of cell cultures from hymenopteran species such as honey bee, Apis mellifera L. (Hymenoptera: Apidae) was developed. Multiple bee cell cultures were produced when using bee larvae and pupae as starting material and modified Hert–Hunter 70 media. Cell culture systems for bees solves an impasse that has hindered efforts to isolate and screen pathogens which may be influencing or causing colony collapse disorder of bees. Multiple life stages of maturing larvae to early pupae were used to successfully establish cell cultures from the tissues of the head, thorax, and abdomen. Multiple cell types were observed which included free-floating suspensions, fibroblast-like, and epithelia-like monolayers. The final culture medium, WH2, was originally developed for hemipterans, Asian citrus psyllid, Diaphorina citri, and leafhopper, Homalodisca vitripennis cell cultures but has been shown to work for a diverse range of insect species such as bees. Bee cell cultures had various doubling times at 21–23°C ranging from 9–15 d. Deformed wing virus was detected in the primary explanted tissues, which tested negative by rt-PCR for Israeli acute paralysis virus (IAPV), Kashmir bee virus, acute bee paralysis virus, and black queen cell virus. Culture inoculation with IAPV from an isolate from Florida field samples, was detectable in cell cultures after two subcultures. Cell culture from hymenoptera species, such as bees, greatly advances the approaches available to the field of study on colony collapse disorders.     

Cotton ovule culture: A tool for basic biology, biotechnology and cotton improvement

Summary Nearly 30 years ago the conditions for culturing immature cotton ovules were established to serve as a working research tool for investigating the physiology and biochemistry of fiber development. Not only has this tissue culture method been employed to characterize the biochemistry of plant cell expansion and secondary cell wall synthesis, but ovule cultures have contributed to numerous other aspects of plant cell physiology and development as well. In addition to basic studies on fiber development, cotton ovule cultures have been used to examine plant-fungal interactions, to model low temperature stress responses, to elucidate the pathways responsible for pigment formation in naturally pigmented fiber and to probe how cytoskeletal elements regulate cell wall organization. Success in rescuing Gossypium interspecific hybrids was dependent on ovule culture media formulations that could support early embryo development in ovulo. As tissues produced in culture are analyzed by increasingly more sophisticated techniques, there appear to be some differences between ovule growth in planta and ovule growth in vitro. Discerning how ovule culture fiber development is different from fiber development in field-grown plants can contribute valuable information for crop improvement. Cotton ovule cultures are an especially attractive model system for studying the effects of gravity on cell elongation, cellulose biosynthesis and embryo development and are excellent targets for examining transient expression of introduced gene constructs. With only minor modification, the procedure originally described by C. A. Beasley and I. P. Ting for growing cotton ovules in vitro will continue to be useful research tool for the foreseeable future.     

Factors affecting the yield and properties of bacterial cellulose

Acetobacter xylinum E₂₅ has been applied in our studies in order to find optimal culture conditions for effective bacterial cellulose (BC) production. The strain displays significantly higher stability in BC production under stationary culture conditions. In contrast, intensive agitation and aeration appear to drastically reduce cellulose synthesis since such conditions induced formation of spontaneous cellulose nonproducing mutants (Cel−), which dominated in the culture. Mutation frequency strictly depends on the medium composition in agitated cultures. Enrichment of the standard SH and Yamanaka media with 1% ethanol significantly enhanced BC production in stationary cultures. Horizontal fermentors equipped with rotating discs or rollers were successfully applied in order to improve culture conditions. Relatively slow rotation velocity (4 rpm) and large surface area enabling effective cell attachment are optimal parameters for cellulose production. Physical properties of BC samples synthesized either in stationary cultures or in a horizontal fermentor revealed that cellulose from stationary cultures demonstrated a much higher value of Young's modulus, but a much lower value of water-holding capacity. Journal of Industrial Microbiology & Biotechnology (2002) 29, 189–195 doi:10.1038/sj.jim.7000303 Received 01 March 2002/ Accepted in revised form 18 July 2002     

Detection of non-host viable contaminants in<Emphasis Type="Italic"> Pichia pastoris</Emphasis> cultures and fermentation broths

The ability to detect viable contaminants in cultures propagated from the original host-expression system ensures that the integrity and purity of seed banks, fermentation broths, and ultimately the final product are continually controlled and maintained. The method developed to detect such agents must be selective for a broad spectrum of microbes, which may be present at very low levels, while discriminating from the host organisms. Although Pichia pastoris strains are frequently used as cell lines for the expression of heterologous proteins, a method that is specific for monitoring culture purity has yet to be reported for this type of organism. An assay that is capable of recovering contaminating bacteria, fungi, and closely related yeast from cultures of P. pastoris at parts per million detection limits is described here.     

Variability of azadirachtin inAzarirachta indica (neem) and batch kinetics studies of cell suspension culture

Seeds of neem were collected from different parts of India and analyzed for their azadirachtin content by High Performance Liquid Chromatography (HPLC). In order to assess the effects of genotypic and geographical variation on azadirachtin content in cell cultures, callus development was attempted in the seeds containing high and low concentration of azadirachtin. The concentration of azadirachtin in callus cultures was significantly affected by the explant source. Seed kernels with higher azadirachtin content produced higher azadiractin content in callus cultures and lower azadirachtin content was seen in callus cultures produced from seed kernels with low azadiractin content. The protocol for development of elite stock culture ofAzadirachta indica was established with the objective of selecting a high azadirachtin-producing cell line. The highest azadirachtin-producing cell line was selected and the effects of different media and illumination conditions on growth and azadirachtin production were studied in shake flask suspension culture. Detailed batch growth kinetics was also established. These studies provided elite starter culture and associated protocols for cultivation ofA. indica plant cell culture in the bioreactor.     

Long‐term rearing affects pheromone‐mediated flight behaviour of the Indian meal moth,Plodia interpunctella

The pest Plodia interpunctella (Hübner) is reared in many research laboratories. In a culture established in 1996, attraction of males to the female‐produced sex pheromone in flight tunnel assays gradually decreased after ≈15 years of rearing. A new culture was established to enable comparison with the old culture regarding traits associated with mate finding. Female calling activity, pheromone titre and male antennal response to pheromone components did not differ between cultures. In contrast, very few males from the old culture reached the pheromone source in flight tunnel assays compared with 61%–81% of males from the other culture. Our results highlight the importance of maintaining viable insect cultures for research purposes and suggest frequent evaluation of traits involved in chemical communication in such cultures to ensure reliable results in experiments.     

Strengths and limitations of the neurosphere culture system

After the initial reports of free-floating cultures of neural stem cells termed neurospheres (1,2), a wide array of studies using this promising culture system emerged. In theory, this was a nearperfect system for large-scale production of neural cells for use in cell replacement therapies and to assay for and characterize neural stem cells. More than a decade later, after rigorous scrutiny and ample experimental testing of the neurosphere culture system, it has become apparent that the culture system suffers from several disadvantages, and its usefulness is limited for several applications. Nevertheless, the bulk of high-quality research produced over the last decade has also shown that under the right circumstances and for the appropriate purposes, neurospheres hold up to their initial promise. This article discusses the pros and cons of the neurosphere culture system regarding its three major applications: as an assay for neural stem cells, as a model system for neurogenesis and neural development, and for expansion of neural stem cells for transplantation purposes.     

Exfoliative cytologic evaluation of primary cultured lung carcinomas

This paper describes a tissue culture and exfoliative cell culture system that enables one to (1) evaluate the adequacy of primary lung carcinoma cultures for cytogenetic analysis, and (2) predict the likelihood of viable cells and type of differentiation present in the primary lung tumor cultures used for cytogenetics. Primary lung carcinomas were established from explant outgrowths and maintained in serum supplemented or serum free media on plastic or basement membrane associated protein coated dishes in order to obtain cells for karyotypic analysis (Miura et al., 1990). The media from these cultures that would ordinarily have been discarded was aspirated at each media change and used to prepare cytocentrifuge cytology preparations. Papanicolaou stained cells from the preparations were evaluated by cytotechnologists in order to assess (1) the cellularity and presence of cancer cells in the sample, (2) differentiation of the malignant cells, and (3) adequacy for chromosomal studies. We determined that cytology preparations of cell and explant outgrowth cultures from primary lung tumors are a reliable method for screening and evaluating the suitability of primary lung carcinoma cultures for cytogenetic analysis.Supported in part by an NCI grant CA-45745 (JRT). JRT is a Scholar of the Leukemia Society of America.     

Protein: A proposal for a standard parameter to express cyanobacterial biomass in laboratory experiments

This study aims to check if the protein content of a cyanobacterial culture is a reliable biomass parameter for cyanobacteria in laboratory experiments, and therefore can be proposed as a standard biomass parameter in culture work to facilitate comparison of results from different studies. For this purpose, the cyanobacteria Microcystis aeruginosa PCC 7806 and Planktothrix agardhii PT2 were grown in 10-L batch cultures with O2 medium and under iron-, nitrate- or phosphate-limited conditions. A linear correlation was found between protein and biovolume in all cultures during exponential growth. We conclude that protein is a suitable biomass parameter for cyanobacteria in laboratory experiments during balanced growth.     

Plant regeneration from embryogenic cultures of Phragmites australis (Cav.) Trin. Ex Steud.

Embryogenic cultures of the common reed [Phragmites australis (Cav.) Trin. Ex. Steud.] were induced on Murashige and Skoog (1962)-based medium with 2% (w/v) sucrose, B5 vitamins and 4.5 μM 2,4-dichlorphenoxyacetic acid. Four independent culture lines, two initiated from stem nodes and two from roots, were established. These cultures underwent somatic embryogenesis. In one line of stem node origin, the somatic embryos germinated and developed into plants, following transfer of embryogenic cultures to Murashige and Skoog (1962)-based medium lacking growth regulators, with 108 ± 17 plants being recovered per 100 mg fresh weight of culture. In other lines, the somatic embryos developed roots, but not shoots. Shoot regeneration via somatic embryogenesis offers potential as anin vitro system for physiological studies, including assessments of the response of common reed to environmental pollutants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Application of hanging drop technique for stem cell differentiation and cytotoxicity studies

The aim of our study is to explore the possibility of using an ancient method of culture technique- the hanging drop technique for stem cell differentiation and cytotoxicity testing. We demonstrate here a variety of novel applications of this age old technique not only to harness the differentiation potential of stem cells into specific lineages but also for cytotoxicity studies. Here we have prepared hanging drop cultures by placing 20 μl micro-drops of nutrient media and 10% Fetal Calf Serum (FCS) containing cells of interest on the lids of 60 mm dishes. Bottom plates of the dishes were filled with sterile Phosphate Buffer Saline (PBS) to avoid desiccation of samples. Lids were then placed on the bottom plates to achieve hanging drop cultures. We utilized this technique for cultivation of ciliated epithelia to study cytotoxicity and differentiation of bone marrow stromal cells. Most importantly the modified culture technique presented here is simple, economical and cost effective in terms of the time taken and the reagents required and are amenable to goal specific modification such as cytotoxicity testing. It is advantageous over the existing system in terms of retention of viability and functionality for longer duration and for providing three dimensional growth micro-environment making it useful for organotypic cultures and in vivo simulation.