Events in the amoebal-plasmodial transition ofPhysarum polycephalum studied by enrichment for committed cells

Summary Amoebae of strain CLof Physarum polycephalum undergo apogamic development to form multinucleate plasmodia. During the amoebalplasmodial transition, large uninucleate cells become irreversibly committed to plasmodium development. In developing cultures, amoebae lose the ability to flagellate before they become committed. Enriched suspensions of committed cells can be obtained by inducing asynchronous differentiating cultures to flagellate and passing the cells through a glass bead column. Committed cells can be cultured to form plasmodia on bacterial lawns or in axenic liquid medium but cannot be cultured on axenic agar medium. Uninucleate committed cells express tubulin isotypes characteristic of amoebae, but after culture in axenic liquid medium, the cells express plasmodial specific tubulin isotypes.Abbrevations SDM Semi-defined medium - DSDM Dilute semidefined medium - LIA Liver infusion agar - SBS Standard bacterial suspension - IEF Isoelectric focussing - SDS Sodium dodecyl sulphate - PAUF Precommitted amoebae unable to flagellate (for the explanation of these cells see text).     

AN IMPROVED METHOD FOR OBTAINING AXENIC CLONES OF PLANKTONIC BLUE-GREEN ALGAE1,2

Axenic clones from 5 isolates of Anabaena flosaquae, 1 isolate of Microcystis acruginosa, and 1 isolate of Aphanizomenon flos-aquae were obtained by a combination of steps that provided a 1000-fold reduction in the bacteria-algae ratio and permitted bacteria-free filaments or cells to be isolated and grown from agar pour plates. The first step consisted of the addition of phenol to a dark-treated culture to selectively reduce the numbers of actively growing bacteria while leaving the resting algal cells viable. The next steps involved washing the treated algal suspension on a Millipore filter pad or membrane followed by plating in washed agar containing buffered mineral medium plus vitamins and soil extract. The final steps consisted of incubating the agar pour plates, coring bacteria-free filaments or cells, culturing the agar cores in a buffered mineral medium, and rigorously testing the resulting cultures for bacteriological contamination. Between 50 and 90% of the cores grew, and of these about 50% were judged axenic. The method, with appropriate adaptations, should be suitable for obtaining axenic clones of other freshwater and marine algae.     

Giardia lamblia: isolation and axenic cultivation.

Giardia lamblia trophozoites have been axenically cultured for more than a year. Initially, organisms were established in a complex liquid medium in the presence of the host's intestinal fungi; subcultures were made of these protozoa-fungus mixtures. G. lamblia trophozoites, free of yeast, were obtained by inoculating a protozoafungus culture in one arm of a U-tube, then later removing, from the other arm of the tube, Giardia trophozoites that had migrated across the base. Medium was changed at 2- or 3-day intervals; numerous subcultures were made. Tests for the possible presence of other organisms in these axenic cultures were negative. Trophozoite cultures remained viable, after freezing in the presence of glycerol, for 14 months. This is the first reported axenic culture of this common human intestinal parasite and pathogen; its study in pure culture is now possible.     

Monoxenic and axenic cultivation of carrier and patient strains of Entamoeba histolytica.

All of five strains of Entamoeba histolytica, isolated from symptomatic cases of amoebiasis, could be adapted to axenic growth on the TP-S-1 medium of Diamond (1968). Four axenic strains were started from amoeba-Crithidia cultures; one could be axenized directly after isolation from a case of cutaneous amoebiasis. Attempts to monoxenize, resp. axenize strains, isolated from Dutch, asymptomatic carriers, were less successful. Only three out of ten strains could be submitted to bacteria-free growth. These three strains, however, originated probably from a recent case of intestinal amoebiasis. The results, suggesting that highly virulent strains can be easier cultivated bacteria-free than those with low or no virulence, are further discussed. The yield of axenic amoebae per tube fluctuates largely depending on many factors such as the strain, the number of transfers (i.e. degree of establishment), the quality of Panmede liver digest and serum in the TP-S-1 medium, and the care of manipulating the cultures. For optimal growth, a more acid medium was required in an amoeba-Crithidia culture than in an axenic culture. Multinucleated, giant amoebae were frequently observed in axenic cultures.     

我国沿海四爿藻的室内培养

在水产养殖中,四爿藻是一种重要的饵料资源,从我国沿海(包括广东、浙江、山东等省)采集到的海洋单细胞绿藻四爿藻,用毛细管法挑取进行单种培养,然后在固体培养基中稀释划线分离得到无菌纯培养物,利用改良的Guillard &; Ryther(Guillard F)培养基、海洋Ⅲ号培养基和改良的海洋Ⅲ号培养基对四爿藻进行了实验室培养。结果发现,改良的海洋Ⅲ号培养基中四爿藻的生长速率最快,最适pH范围为7．0～8．0,加入维生素B12(10μg·L^-1),也可以适当增加四爿藻的生长速率。这种改良的海洋Ⅲ号培养基配制简单,适合于水产养殖中四爿藻的规模生产。     

Somatic embryogenesis and plant regeneration from cell suspension cultures of Cucumis sativus L.

A procedure for the regeneration of whole cucumber plants (Cucumis sativus L. cv. Poinsett 76) by embryogenesis from cell suspension cultures is described. Embryogenic callus was initiated from the primary leaves of 14–17 day old plants. Suspension cultures of embryogenic cells were grown in liquid Murashige and Skoog basal medium containing 5 uM 2,4,5-trichlorophenoxyacetic acid and 4 uM 6-benzylaminopurine. Suspension cultures were composed of a population of cells that were densely cytoplasmic and potentially embryogenic. Differentiation of embryos was enhanced by washing the suspension culture cells with MS basal medium containing 0.5% activated charcoal and twice with MS basal medium followed by liquid shake cultures in MS basal medium. Sixty to 70 percent of the embryos prewashed with activated charcoal germinated into plantlets with normal morphology. Embryos obtained from suspension cultured cells without prewashing with activated charcoal organized into plantlets with abnormal primary leaves. Morphologically normal plantlets were obtained by excising the shoot tips and transferring them to fresh medium.Abbreviation BAP 6-benzylaminopurine - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - MS Murashige and Skoog     

“A simple and rapid method for isolating lichen photobionts“

For decades, lichenologists have developed numerous and varied methods to isolate lichen photobionts. Most procedures are tedious, slow, and require several months after the initial isolation to obtain clones. Furthermore, the purity of the isolated photobionts obtained by more rapid methods is not sufficient to establish phycobiont axenic cultures. We have developed a new method for isolating lichen photobionts from fruticose, foliose and crustose lichens. Basically, it involves homogenization of lichen thalli (from 15 mg to 2 g), a one-step centrifugation through Percoll ®, followed by washing with Tween 20 and sonication. With this simple and rapid method (which takes less than 2 h), photobiont cells are obtained in sufficient quantity and purity to obtain dozens of axenic algal cultures.     

Agrobacterium rhizogenes mediated transformation of grapevine (Vitis vinifera L.)

Genetically transformed grapevine (Vitis vinifera L.) roots were obtained after inocultation of in vitro grown whole plants (cv. Grenache) with Agrobacterium rhizogenes. The strain used contains two plasmids: the wild-type Ri plasmid pRi 15834 and a Ti-derived plasmid which carries a chimaeric neomycin phosphotrans-ferase gene (NPT II) and the nopaline synthase gene. Expression of the NPT II gene can confer kanamycin resistance to transformed plant cells. Slowly growing axenic root cultures derived from single root tips were obtained. Opine analysis indicated the presence of agropine and/or nopaline in established root cultures. For one culture, the presence of T-DNA was confirmed by dot-blot hybridization with pRi 15834 TL-DNA. Callogenesis was induced by subculturing root fragments on medium supplemented with benzylaminopurine and indoleacetic acid.Transformation of in vitro cultured grapevine cells has recently been reported (baribault T.J. et al., Plant Cell Rep (1989) 8: 137–140). In contrast with the results presented here, expession of the NPT II gene Conferred kanamycin resistance to Vitis vinifera calli that was sufficient for selection of trasformed cells.Abbreviations BAP benzylaminopurine - IAA indoleacetic acid - NAA naphtaleneacetic acid - NPT II neomycin phosphostransferase II - EDTA ethylenediaminetetraacetic acid     

Time studies of ecdysone-action on in vitro apolysis of Chilo suppressalis integument

The relationship between induction of in vitro apolysis and the duration of hormone treatment, and the effects of metabolic inhibitors on the ecdysone-induced apolysis were investigated in the cultured integument taken from the rice stem borer larva, Chilo suppressalis. When fragments of integument were subjected to 0.3 μg/ml β-ecdysone for more than 5 hr and then transferred to hormone free medium, they were induced to apolyse one day after treatment. If the fragments of integument were treated with hormone for 1 to 4 hr at first and then treated with hormone for 2 to 5 hr again after a 5 day interval in hormone free medium, almost all the fragments were induced to apolyse one day after treatment. This result suggests that the action of β-ecdysone on the cultured integument is accumulative. If the fragments of integument were cultured in the medium containing actinomycin-D and then transferred to medium containing β-ecdysone, a strong inhibitory effect on the apolysis of the integument was observed. Similarly, an inhibitory effect appeared when fragments of integument were treated first with hormone and then with puromycin. These results show that the m-RNA synthesis necessary for apolysis was completed within 6 hr after hormone treatment. However, the protein synthesis required for apolysis was not. The relationship of the results obtained from these in vitro experiments to the mode of action of ecdysone is discussed.     

Variations in cytotoxicity and isoenzyme patterns of uncloned and cloned cultures of axenic Entamoeba histolytica

Five clones of axenic Entamoeba histolytica (HMI) grown as discrete colonies in semisolid agar medium were adapted in liquid medium and labelled as HMI-C121, HMI-C131, HMI-C143, HMI-C144 and HMI-C145. The clone HMI-C121 was more cytotoxic to the cultured Baby Hamster Kidney (BHK) cells while all other clones were significantly (P less than 0.001) less cytotoxic as compared to the cloned HMI-C121 and uncloned E. histolytica (HMI). The uncloned Indian axenic E. histolytica (KCG:0986:11) as well as E. histolytica (NIH:200) cultures were significantly (P less than 0.001) less cytotoxic to cultured BHK cells. No difference in the electromobility of maleate NADP oxidoreductase (ME) or glucophosphate isomerase (GPI) isoenzyme in the lysates of all the cloned and uncloned cultures of E. histolytica was observed. The clones HMI-C121, HMI-C131, HMI-G143 and HMI-C144 had three bands of hexokinase (HK) while all uncloned cultures and one of clones, HMI-C145 had only two bands. Though cloned and uncloned cultures had a single PGM band, the relative electromobility (rf) of phosphoglucomutase (PGM) for clone HMI-C131, HMI-C143 HMI-C144 was relatively less (rf 0.075) and these were also significantly (P less than 0.001) less cytotoxic to BHK cells as compared to clone HMI-C121. It is felt that axenic E. histolytica culture consists of several populations (clones) and expression of isoenzymes pattern or cytotoxic potentials would depend upon the population which predominantly multiples and outgrows other populations in the culture system.     

Continuous in vitro culture of the carpet shell clam Tapes decussatus protozoan parasite Perkinsus atlanticus

Continuous in vitro cultures of the clam Tapes decussatus parasite Perkinsus atlanticus were established from infected gill fragments, infected haemolymph and parasite hypnospores isolated from infected gill fragments following incubation in Ray's fluid thioglycollate medium (RFTM). No continuous cultures could be initiated from P. atlanticus zoospores. Cultures initiated from hypnospores yielded the highest percentage of continuous cultures (100%, 6/6), followed by cultures initiated from gill fragments (93%, 43/46) and from haemolymph (30%, 3/10). Failures to establish continuous cultures were due to microbial contamination. The source of parasite influenced the success rate, the time taken to establish cultures and the size of cultured cells. In vitro proliferation of parasite cells was mainly by vegetative multiplication. Zoosporulation, yielding motile biflagellated zoospores, was observed at a low frequency (< 1% of dividing cells) in every culture. Morphology of cultured cells examined with light and transmission electron microscopy corresponded to that of P. atlanticus found in clam tissues. Cultured cells enlarged in RFTM and stained blue-black with Lugol's solution, which are characteristics of the Perkinsus species cells. DNA sequences of the internal transcribed spacer (ITS) region of the ribosomal RNA gene complex matched those of P. atlanticus. All cultures were established in a medium designated JL-ODRP-2A that was similar in composition to the culture medium JL-ODRP-1 originally used to propagate Perkinsus marinus in vitro. Proliferation of P. atlanticus in vitro could be supported by the commercial culture medium (1:2 v/v) DME:Ham's F-12 with fetuin.     

Mononuclear cell-modulation of granulosa cell steroidogenesis in rat: influence of ovarian cycle.

The mononuclear cells exert a paracrine influence on the ovarian function, including steroidogenesis. This study examines the ability of the conditioned medium from the cultures of splenic mononuclear cells, obtained during various phases of the ovarian cycle, on progesterone accumulation by the granulosa cells in the culture medium. Female Wistar rats, aged twenty-five days, were made pseudopregnant by an injection of pregnant mare's serum gonadotropin. The splenic mononuclear cells were isolated at follicular phase, early luteal phase, mid luteal phase and late luteal phase and cultured for 48 h. The ammonium sulphate precipitated fraction of the conditioned medium was added to the granulosa cells obtained from immature rats treated with diethylstillboestrol. The granulosa cells were cultured for 48 h, and the progesterone accumulated in the medium was assayed. The conditioned medium from the cultures of the mononuclear cells obtained during follicular phase and late luteal phase inhibited FSH-induced progesterone secretion, whereas conditioned medium obtained from mid luteal phase mononuclear cells enhanced the effect of FSH. The stimulatory effect of db-cAMP on progesterone accumulation in the culture medium is inhibited by conditioned medium obtained from all the phases of the ovarian cycle. This study demonstrates a cyclicity in the behaviour of the splenic mononuclear cells on ovarian steroidogenesis, suggesting a bi-directional paracrine and/or endocrine relationship between ovary and the mononuclear cells.     

Some probiotic properties of chicken lactobacilli

The beneficial effect of lactobacilli has been attributed to their ability to colonize human and animal gastrointestinal tracts. In this work, adhesion assays with three lactobacillus strains and intestinal fragments obtained from chickens were assessed. Lactobacillus animalis and L. fermentum were able to adhere to three kinds of epithelial cells (crop, small and large intestines) with predominance to small intestine. Among the strains considered, L. fermentum subsp. cellobiosus showed the lowest and L. animalis the highest adhesion ability. Scanning electron microphotographs showing L. animalis and L. fermentum adhering to intestinal cells were obtained. The characterization of L. animalis adhesion indicated that lectin-like structure of this strain has glucose/mannose as specific sugars of binding. However, a calcium requirement was not observed. The adhesion of L. fermentum was reduced by addition of sialic acid or mannose (P < 0.01). These carbohydrates can be involved in the interaction between adhesin and epithelial surface. In this case, the dependence on bivalent cations was demonstrated. Lactobacillus fermentum was effective in reducing the attachment of Salmonella pullorum by 77%, while L. animalis was able to inhibit (90%, 88%, and 78%) the adhesion of S. pullorum, S. enteritidis, and S. gallinarum to host-specific epithelial fragments respectively. Our results from this in vitro model suggest that these lactobacilli are able to block the binding sites for Salmonella adhesion.     

Experimental Microbial Populations Thirty-five Years Later: The Influence of Food on the Symbiosis of Paramecium aurelia Syngen 4, 51.7

Changes in the nutrition of Paramecium aurelia affect its ability to serve as host for the bacteroid parasite, kappa, and the presence or absence of kappa affects its ability to grow in axenic culture. Loss of kappa, tested by the presence or absence of killer reaction, occurred in cultures of P. aurelia growing at a reduced division rate on autoclaved Enterobacter aerogenes in suspensions of lettuce and yeast autolysate 14–17 days after they had been rendered bacteria-free by washing. Killer Paramecium sterilized of bacteria by treatment with an antibiotic mixture of penicillin-G and streptomycin in combination with a nonbacterial nonliving culture medium, lost the ability to kill after from 6 to 48 hours in the sterilizing medium. The ciliates from which kappa had been lost during exposure to antibiotics could be transferred immediately and maintained in axenic culture, but those washed free of bacteria could not be maintained axenically until kappa had been lost during cultivation in a medium containing killed bacteria. It is suggested that a knowledge of the nutritional requirements of symbiotic microorganisms is essential for understanding the ecological aspects of eutrophication of aquatic environments.     

In vitro sodium nitroprusside-mediated toxicity towards Leishmania amazonensis promastigotes and axenic amastigotes

Leishmania parasites survive despite exposure to the toxic nitrosative oxidants during phagocytosis by the host cell. In this work, the authors investigated comparatively the resistance of Leishmania amazonensis promastigotes and axenic amastigotes to a relatively strong nitrosating agent that acts as a nitric oxide (NO) donor, sodium nitroprusside (SNP). Results demonstrate that SNP is able to decrease, in vitro, the number of L. amazonensis promastigotes and axenic amastigotes in a dose-dependent maner. Promastigotes, cultured in the presence of 0.25, 0.5, and 1 mmol L(-1) SNP for 24 h showed about 75% growth inhibition, and 97-100% when the cultures were treated with >2 mmol L(-1) SNP. In contrast, when axenic amastigotes were growing in the presence of 0.25-8 mM SNP added to the culture medium, 50% was the maximum of growth inhibition observed. Treated promastigotes presented reduced motility and became round in shape further confirming the leishmanicidal activity of SNP. On the other hand, axenic amastigotes, besides being much more resistant to SNP-mediated cytotoxicity, did not show marked morphological alteration when incubated for 24 h, until 8 mM concentrations of this nitrosating agent were used. The cytotoxicity toward L. amazonensis was attenuated by reduced glutathione (GSH), supporting the view that SNP-mediated toxicity triggered multiple oxidative mechanisms, including oxidation of thiols groups and metal-independent oxidation of biomolecules to free radical intermediates.     

Application of a growth-promoting bacteria for stable mass culture of three marine microalgae

The practical mass culture of marine microalgae, facesoccasionally unexpected problems or collapse. The effect of amarine bacterium, Flavobacterium sp., which was found topromote growth of a marine diatom Chaetoceros gracilisin the axenic culture condition, was examined on the masscultures of three marine microalgae.Three marine microalgae (C. gracilis, Isochrysisgalbana, and Pavlova lutheri) were mass cultured in 3 lflatbottom flasks (2.5 l capacity of culture medium), in anindoor culture room at a commercial pearl oyster hatchery. Themicroalgal cells and the bacterium were inoculated at the sametime, in the culture media. The specific growth rate andmaximal cell density were determined in treated cultures (withadded bacterial strain) and in controls (without addedbacterial strain). The specific growth rate of C.gracilis in treated cultures was significantly higher thanthat of control cultures, and the stationary growth phase inthe treated cultures lasted longer till the end of the cultureperiod. However, the bacterium had no apparent effect on theexponential growth phase of two phytoflagellates, I.galbana and P. lutheri, but kept longer the high celldensity in the stationary growth phases. The added bacterialstrain (Flavobacterium sp.) was the dominant species(more than 45%) among the bacterial flora during the cultureperiod.     

Leishmania infantum: stage-specific activity of pentavalent antimony related with the assay conditions

The determination of the intrinsic sensitivity of Leishmania strains to pentavalent antimonials in clinical trials, before treatment is begun, is essential in order to avoid failures and to allow alternative drugs to be chosen. A comparative study of SbV activity on promastigotes, axenic amastigote-like cells, and intracellular amastigotes of Leishmania infantum, when administered in the form of meglumine antimoniate and free, in hydrochloric solution, was performed. Results indicate that the conditions under which the promastigotes were cultured affect the IC(50) obtained, although results were homogeneous when the products were assayed on axenic-like and intracellular amastigotes. The IC(50) obtained for SbV in the form of meglumine antimoniate or in hydrochloric solution on promastigotes cultured in Schneider's medium depends on the growth rate of the culture and therefore could be regulated by modifying the fetal calf serum concentration in the medium. The pH of the culture medium strongly affected the activity of meglumine antimoniate but not that of the SbV hydrochloric solution on promastigotes cultured in Schneider's medium. This influence of pH was observed to a much lesser extent when promastigotes were cultured on M199 or RPMI media. In homogeneous culture conditions, which included the regulation of the promastigote growth rate through the heat-inactivated fetal calf serum concentration in the medium and the dilution of the meglumine antimoniate with Schneider's medium at pH 6.5, the activity of SbV, free or in the form of meglumine antimoniate, was the same in promastigotes, intracellular amastigotes, and axenic amastigote-like cells.     

Identification of Mycobacterium xenopi by gas chromatography

For the purposes of the following study we cultured 32 strains of Mycobacterium xenopi isolated from clinical specimens and several strains of other slowly growing mycobacteria. The cultures were grown in liquid medium and then analysed--after saponification, methylation, extraction with organic solvent and washing of the organic phase--using a highly sensitive manual gas-liquid chromatographic assay for the determination of secondary alcohol 2-OH-docosanol. The percentage of this compound was compared with that previously measured in strains of Mycobacterium xenopi grown on solid medium. The presence of this specific alcohol was always apparent, even though its quantity was lower than that obtained by growing mycobacteria on solid medium. The absence of interference peaks around the compound was checked by analyzing strains of other slowly growing mycobacteria in the same conditions.     

Axenic Cultivation of Trichomonas tenax, the Oral Flagellate of Man I. Establishment of Cultures

SYNOPSIS. A technique for the axenic cultivation of Trichomonas tenax , the oral flagellate of man, is presented. The medium employed consists of a nutrient broth supplemented with horse serum and a cell-free extract of chick embryo. Three strains established in this medium have been maintained for 16, 23 and 48 months respectively.
All the cultures were initiated with trichomonads grown in association with Trypanosoma cruzi. Attempts to establish axenic cultures with trichomonads obtained from xenic cultures containing a bacterial flora of unknown composition met with failure. This suggests that the successful cutcome of the process of axenization is to a certain extent dependent upon the type of organism(s) with which T. tenax is associated in culture. Furthermore, these findings may serve to explain earlier failures since not only were all of these attempts made with media lacking tissue extract supplements but all were made using bacteria-trichomonad cultures as a source of trichomonads.     

Non-axenic and Axenic Growth of Vorticella microstoma

SYNOPSIS. Vorticella microstoma was grown non-axenically and axenically at pH 6.4. Vorticellas were maintained indefinitely on Bacillus cereus in a medium composed of Proteose-Peptone, Cerophyl, and the filtrate from boiled wheat kernels. Prolific growth occurred in 2-membered cultures. A medium containing hydrolyzed gelatin, aqueous liver extract, yeast nucleic acid hydrolysate, glucose, and penicillin is recommended for axenic growth.
The potential value of vorticellids as research tools is discussed together with metabolic implications of supplementing sterile Proteose-Peptone broth with natural substances in particle form. The ineffectiveness of adding tryptophan, thiamine, glycine, and chelating agents to the axenic medium was considered. Refinements of the axenic medium are on trial.