Formation of Nonculturable Cells of Mycobacterium tuberculosis and Their Resuscitation

Nonculturable cells were found to occur in populations of Mycobacterium tuberculosis cells during the long poststationary phase. These cells were small (0.6–0.8 m) ovoid and coccoid forms with intact cell walls and negligible respiratory activity, which allows them to be regarded as dormant cells. Nonculturable cells were characterized by low viability after plating onto solid medium; a minor part of the population of these cells could be cultivated in liquid medium. Cell-free culture liquid of an exponential-phase Mycobacterium tuberculosisculture or the bacterial growth factor Rpf exerted a resuscitating effect, increasing substantially the growth capacity of the nonculturable cells in liquid medium. During resuscitation of nonculturable cells, a transition from ovoid to rodlike cell shape occurred. At early stages of resuscitation, ovoid cells formed small aggregates. The recovery of culturability was associated with the formation of rod-shaped cells in the culture. The data obtained demonstrate the in vitro formation of dormant cells of Mycobacterium tuberculosis, which do not grow on solid media but can be resuscitated in liquid medium under the effect of substance(s) secreted by actively growing cells.     

Carbonate and Phosphate Precipitation by Chromohalobacter marismortui

The ability of Chromohalobacter marismortui to precipitate carbonate and phosphate minerals has been demonstrated for the first time. Mineral precipitation in both solid and liquid media at different salts concentrations and different magnesium/calcium ratios occurred whereas crystal formation was not observed in the control. The precipitated minerals were studied by X-ray diffraction, scanning electron microscopy and EDX, and were different in liquid and solid media. In liquid media aragonite, struvite, vaterite and monohydrocalcite were precipitated forming crystals and bioliths. Bioliths accreted preferentially close to organic pellicles, whereas struvite preferentially grows in microenvironments free of such pellicles. Magnesian calcite, calcian-magnesian kutnahorite, “proto-dolomite” and huntite were formed in solid media. The Mg content of the magnesian calcite and of Ca-Mg kutnahorite also varied depending on the salt concentration of the culture media. This is the first report on bacterial precipitation of Ca-Mg kutnahorite and huntite in laboratory cultures. The results of this research show the active role played by C. marismortui in mineral precipitation, and allow us to compare them with those obtained previously using other taxonomic groups of moderately halophilic bacteria.     

Anthurium andraeanum Plantlets Produced from Callus Tissues Cultivated in vitro

During the last 3 years a method has been developed to reproduce Anthurium andraeanum Lind. through callus cultures. The procedural sequence is as follows: callus induction on excised leaf fragments, callus subculture on solid and in liquid media, adventitious sprout formation in callus on solid media, and rooting of excised sprouts. Recently 20 adult genotypes were propagated by this method.     

Extracellular enzymatic activity of aquatic and aero-aquatic conidial fungi

Nineteen species of aquatic and areo-aquatic conidial fungi were tested for their ability to produce extracellular enzymes which degrade cellulose, starch, lipids, proteins and tannic acid. The cellulolytic activity was determined by using both solid and liquid media. The activity of other enzymes was examined using solid media. Two-thirds of the species were able to hydrolyze soluble cellulose (CMC) incorporated in solid and liquid media with varying degrees of activity. Extracellular culture filtrates ofAegerita candida, Helicodendron giganteum andH. tubulosum contained a Cl-Cx enzyme complex that could degrade both soluble cellulose (CMC) and crystalline cellulose (filter paper). Lipase activity was demonstrated by 11 species. Fourteen of the species showed activity for amylase and protease, but only 11 of the 16 were capable of degrading tannic acid.     

Fungi on the hair of large mammals in Egypt

Several isolates of Candida albicans were tested for production of chlamydoconidia and metabolic changes when grown on several different solid and liquid media. A liquid medium, consisting solely of sterilized skimmed milk and a solid medium containing processed cheese stimulated more rapid and greater production of chlamydoconidia than the corn meal agar and the other media tested.     

The use of AlbuMAX II(®) as a blood or serum alternative for the culture of Helicobacter pylori

Background: Growth of Helicobacter pyloriin vitro depends on supplementation of the medium with blood or serum. However, these supplements often require frozen storage and can show batch‐to‐batch variation, resulting in differences in bacterial growth. In this study, we introduce the use of a commercially available, lipid‐rich supplement called AlbuMAX II^® (Gibco BRL, Grand Island, NY, USA) for use as a serum/blood replacement for H. pylori culture. Materials and Methods: The growth of H. pylori on solid and liquid media was examined by comparing growth after supplementation with horse blood, fetal calf serum, β‐cyclodextrin or AlbuMAX II^® (Gibco BRL). Human gastric adenocarcinoma (AGS) cellular responses to H. pylori were measured by NF‐κB luciferase assays and IL‐8 ELISA. Results: We show that the growth of H. pylori on both solid and liquid media containing AlbuMAX II^® (Gibco BRL) were comparable to levels obtained on blood agar or liquid media supplemented with serum. Growth was consistently higher in media supplemented with AlbuMAX II^® (Gibco BRL) than media containing β‐cyclodextrin. Furthermore, bacteria grown in AlbuMAX II^® (Gibco BRL) induced proinflammatory responses in AGS cells. Conclusions: AlbuMAX II^® (Gibco BRL) can be used as a serum/blood replacement for the cultivation of H. pylori in solid and liquid media. This medium could be useful for an improved understanding of H. pylori metabolism or for antigen production. Furthermore, AlbuMAX II^® (Gibco BRL) may be suitable for use in remote locations, particularly in areas where frozen storage of serum may be a problem.     

Obtaining autotetraploids in vitro at a high frequency in Citrus sinensis

Tetraploid plants are essential for interploid hybridization to create triploid seedless citrus. Here we report a simple and efficient in vitro method for generating autotetraploids for sweet orange (Citrus sinensis). Cell-division activity in ‘Anliucheng’ sweet orange callus was analyzed using flow cytometry to determine the peak frequency of cell division at which time callus in a liquid media and solid media was treated with 1000 mg l⁻¹ colchicine. The percentage of the DNA-content-varied cells in the callus increased markedly from 11.0% to 44.4% and to 59.0% for liquid and solid media respectively. A total of 20 tetraploid plantlets were recovered via embryogenesis from 47 plantlets regenerated from the treated callus. All the autotetraploids were derived from different embryoids. Autotetraploids will be useful parents for interploid hybridization to generate commercially valuable seedless triploid citrus cultivars.     

Effects of subculturing and physical condition of medium on the nuclear behavior of a plant tissue culture

A callus of the common garden peony, Paeonia suffruticosa, was subcultured on solid and liquid media and analyzed intensively for a period of 153 days in order to test the effects of subculturing and the physical conditions of culture on the mitotic cycle kinetics of a population of cells, particularly in relation to the degree of heteroploidy. The parameters investigated in the kinetic studies included mitotic index values, cell generation time, and the time required for the cell population to double. The mitotic index of Paeonia cells cultured in liquid medium was found to be about two and a half times higher than for those cultured on solid; successive subculturing did not affect the mitotic index on either type of medium. The most significant results of the study came from the chromosome count data, in which diploid and tetraploid cells fluctuated in predominance in successive subcultures, and the apparent earlier manifestation of polyploidy on liquid medium. Mitotic index, cell generation, and population doubling times remained constant throughout the study.     

Isolationin vitro ofStrongwellsea castrans [Fungi: Entomophthorales] a pathogen of adult cabbage root flies,Delia radicum [Dipt.: Anthomyiidae]

The entomogenous fungusStrongwellsea castrans was isolatedin vitro for the first time, by incubating conidia projected from infected cabbage root flies (Delia radicum) in a simple, semi-defined liquid medium comprising dextrose, yeast extract and lactalbumin hydrolysate buffered to pH 7. The fungus grew as long unitunicate hyphae. After transfer to a solid nutrient medium, multinucleate hyphal bodies were formed which developed a thick, laminated wall. Neither conidia nor resting spores developed in liquid or on solid media and the fungus survived successive sub-culturing only in liquid media. Using the API-ZYM system, tests on extracts on hyphae ofS. castrans were positive for 11 enzymes but there were no consistent differences in enzyme profiles betweenS. castrans and fungi of the related genusErynia.      

TWO DISTINCT COLONIAL MORPHOTYPES OF AMPHORA COFFEAEFORMIS (BACILLARIOPHYCEAE) CULTURED ON SOLID MEDIA1

When cultured on different types of solid media, the marine-fouling diatom Amphora coffeaeformis (Ag.) Kütz. consistently formed two distinct colonial morphotypes named tight and fuzzy. Tight colonies were comprised mainly of small, morphologically distorted, nonmotile cells, whereas morphologically normal and highly motile cells formed the fuzzy colonies. Cells from tight colonies were less adherent to glass, grew more slowly in liquid media, and had a slightly decreased viability on plates with copper than cells from fuzzy colonies. Whereas the protein profiles of the two types of cells were nearly identical in polyacrylamide gels stained with Coomassie blue, cells from tight colonies produced a significantly lower amount of a protease-resistant, low M_r polysaccharide or glycoconjugate as detected in silver-stained gels. The frequency of appearance of the fuzzy and tight morphotypes was not influenced by the mode of nutrition or the type of substratum to which the algal cells adhered. However, certain formulations of solid medium and the presence of growth-inhibitory concentrations of copper in agar plates favored the formation of tight colonies. Due to their frequencies and patterns of appearance, it was clear that the two naturally formed morphotypes were not the consequence of spontaneous mutations, genetic rearrangement, or selection of stable natural variants, and we have hypothesized that they were linked to a normal physiological behavior. The tight colonial morphotype was used as a valuable marker to screen for true motility/adhesion mutants within an ultraviolet-mutagenized population of A. coffeaeformis. Seven mutants were isolated that were non-motile on agar plates, poorly adherent to glass, and distinguished from naturally formed cells from tight colonies by their inability to form fuzzy colonies upon subculture on solid media.     

Preparation and use of media for protease-producing bacterial strains based on by-products from Cuttlefish (Sepia officinalis) and wastewaters from marine-products processing factories

Cuttlefish powder (CFP) from Sepia officinalis by-products was prepared and tested as a fermentation substrate for microbial growth and protease production by several species of bacteria: Bacillus licheniformis, Bacillus subtilis, Pseudomonas aeruginosa, Bacillus cereus BG1, and Vibrio parahaemolyticus. All microorganisms studied grew well and produced protease activity when cultivated in medium containing only CFP indicating that the strains can obtain their carbon and nitrogen source requirements directly from whole by-product proteins. Moreover, it was found that the addition to the cuttlefish medium of diluted fishery wastewaters (FWW), generated by marine-products processing factories, enhanced the production of protease. Maximum activity was obtained when cells were grown in cuttlefish media containing 5-times or 10-times diluted FWW. Five-times diluted FWW enhanced protease production by B. cereus BG1 and B. subtilis by 467% and 75% more than control media, respectively. The enhancement could have been due to the high organic content or high salts in FWW.As a result, cuttlefish by-products powder enriched with diluted FWW was found to be a suitable growth media for protease-producing strains. This new process, which converts underutilized wastes (liquid and solid) into more marketable and acceptable forms, coupled with protease production, can be an alternative way to the biological treatment of solid and liquid wastes generated by the cuttlefish processing industry.     

A protocol for rapid micropropagation of endangered Isoplexis

Summary An efficient protocol for large-scale micropropagation of Isoplexis canariensis (L.) Loud., I. chalcantha Svent, and O'Shan., and I. isabelliana (Webb and Berth.) Masf. (Scrophulariaceae) is reported. Multiple shoots were obtained from shoot tips and nodal segments isolated from seedlings when cultured under varying conditions. Factors such as nutrient media, concentration of growth regulators, and type of induction medium (liquid or solid) strongly influenced shoot proliferation and development. Multiple well-developed shoots were obtained after induction on solid media containing Murashige and Skoog full-strength salts and low cytokinin concentrations. By changing the major salt formulation to half strength and employing liquid media the morphogenic parameters evaluated were affected negatively. Many shoots rooted spontaneously in hormone-free media and plants grew successfully in the greenhouse. Flowering was observed 6 mo. after ex vitro cultures were established.     

Optimizing Shoot Formation in Gentiana kurroo Royle for Gentiopicroside Production

The roots and shoots of Gentiana kurroo Royle are rich sources of gentiopicroside (GPD). The plant is used traditionally for curing many metabolic diseases. The exploitation of G. kurroo in its native habitat has placed the plant on the critically endangered list of plants in India. One of the ways of creating an alternative source of G. kurroo is through in vitro propagation. Although a number of in vitro propagation methods for G. kurroo exist, there are no studies that have optimized methods for rapid in vitro shoot production and the production of GPD. The objective of this study was to develop an effective in vitro shoot multiplication system of G. kurroo. Furthermore, the influence of solid and liquid induction media were investigated. Shoots were regenerated from embryogenic callus and transferred to solid and liquid Murashige and Skoog (MS) and Gamborg (B₅) media fortified with various concentrations of BA containing different auxins. It was observed that the liquid medium produced a higher number of shoots than the solid media. MS supplemented with BA (2 mg/L) and IAA (0.5 mg/L) produced?~?5.58 shoots per explant on the solid medium, while?~?16 shoots per explant was obtained in the liquid medium. High-Performance Liquid Chromatography (HPLC) analysis of in vitro shoots grown in the liquid medium produced 9.13 mg/g dry weight (DW) of GPD which is seven-fold higher than that of naturally growing plant shoots. The in vitro protocol for G. kurroo developed in this study may be used for industrial production of GPD.

     

On the interaction in culture between two pathogenic soil-inhabiting fungi

Summary Interaction betweenFusarium vasinfectum andMacrophomina phaseoli was studied on solid and in liquid media at varying cultural conditions, in an attempt to explain the reduction in virulence of each fungus when the two pathogens were mixed together in the soil. Metabolites produced by each fungus in liquid media were suppressive to the growth of the other. Such suppression was influenced by the pH factor. Saltation ofFusarium was favoured by the presence ofMacrophomina metabolites.Dedicated to ProfessorTibor Benedek on the occasion of his 70th birthday.     

Growth Optimization Procedures for the Phytopathogen <Emphasis Type="Italic">Xylella fastidiosa</Emphasis>

For the first time, growth curves are shown for the phytopathogen Xylella fastidiosa on traditional growth media such as PW (periwinkle wilt), BCYE (buffered charcoal yeast extract), and on new ones such as GYE (glutamate yeast extract) and PYE (phosphate yeast extract) that were developed in this work. The optimal growth conditions on solid and liquid media as well as their measurements are presented, by using total protein content and turbidity determinations. The results demonstrated that yeast extract provided sufficient nutrients for X. fastidiosa, since the cells grew well on PYE medium. Received: 29 March 2002 / Accepted: 30 March 2002     

Effects of cultivation techniques and media on yields and morphology of the basidiomycete Armillaria mellea

Summary The yield of cell mass and the morphology of Armillaria mellea, strain ATCC 11114, was studied using a variety of cultivation methods: solid media, standing liquid culture, shake flasks, tower reactors and impeller-stirred reactors. Two different media, malt extract broth and a glucose/asparagine/peptone-medium, and the corresponding agar media, were used. Yields were higher in the malt extract media than in the glucose media. Generally the highest yields were obtained on solid media while agitated cultures gave the lowest yields. Morphological characteristics such as pellet formation, adhesion to surfaces and pigment production were significantly affected by culture conditions.     

The Effect of the Cultivation Method and the Growth Phase on the Lipopolysaccharide Composition of Yersinia pseudotuberculosis

Effects of the cultivation method (suspension cultures in a liquid nutrient broth or colonies on a solid agarized medium) and the growth phase on the lipopolysaccharide (LPS) composition of Yersinia pseudotuberculosis(O : Ib serovar, strain KS 3058) grown in cold (5°C) were studied. The amount of the LPS synthesized by cells depended on the bacteria growth phase for both media. The LPS acylation degree was constant, whereas the length of the O-specific polysaccharide chain varied with the culture age and for both media achieved maximum in the stationary growth phase. The bacteria cultivation on the nutrient agar stimulated more intensive synthesis of LPS, which were extracted more easily, had longer polysaccharide O-chains, and were more toxic than LPS of the bacteria cultivated in the liquid medium. It was proposed that the cultivation of Yersinia pseudotuberculosisin cold as colonies on the agar surface increases the bacterial virulence.     

Cell division across the volume of colonies of flavobacterium sp. 22

Six-day-old colonies ofFlavobacterium sp. 22 were studied by electron microscopy. Direct evidence was obtained of bacterial cell division across the entire colony volume, indicating that the colony growth ofFlavobacterium sp. 22 is not purely peripheral. It is argued that the colony shape is determined not only by peripheral growth but also by physical forces acting upon a droplet of liquid on the surface. For bacterial colonies developing on solid nutrient media, the intercellular matrix plays the role of such a liquid.     

Growth of Cryptococcus neoformans in a thiamine-free medium

The growth of Cryptococcus neoformans in a minimal liquid synthetic medium with or without thiamine (10 g/ml) was investigated. In these media the presence or absence of thiamine had no effect on the development of C. neoformans. To check these results, we performed a series of experiments on a solid form of the minimal synthetic medium. In this study a series of six serial transfers were carried out to starve the cells of nutrients that may have been carried over from their growth on rich media. In each of the transfers on the solid synthetic medium, C. neoformans showed a similar and scarce growth. This finding indicates that C. neoformans could be autotrophic in respect to thiamine.     

High frequency callus formation from maize protoplasts

Summary A solid feeder layer technique was developed to improve callus formation of Black Mexican Sweet maize (Zea mays L.) suspension culture protoplasts. Protoplasts were plated in 0.2 ml liquid media onto a cellulose nitrate filter on top of agarose-solidified media in which Black Mexican Sweet suspension feeder cells were embedded. Callus colony formation frequencies exceeding 10% of the plated protoplasts were obtained for densities of 10³–10⁵ protoplasts/ 0.2 ml, which was 100- to 1,000-fold higher than colony formation frequencies obtained for conventional protoplast plating methods such as liquid culture or embedding in agarose media. Compared with conventional methods, the feeder layer method gave higher colony formation frequencies for three independently maintained Black Mexican Sweet suspension lines. Differences among the three lines indicated that colony formation frequencies might also be influenced by the suspension culture maintenance regime and length of time on different 2,4-dichlorophenoxyacetic acid concentrations. The callus colony formation frequency reported is an essential prerequesite for recovering rare mutants or genetically transformed maize protoplasts.