New milk medium for germ tube and chlamydoconidia production byCandida albicans

A new medium consisting of UHT milk, tween 80 and agar is described for the development of both germ tube and chlamydoconidia byCandida albicans. In total 172 isolates from clinical specimens, includingC. albicans (112),C. guilliermondii (4),C. krusei (3),C. parasilopsis (16).C. tropicalis (28),Torulopsis glabrata (6) andTrichosporon beigellii (3), were examined in this medium by using the standard method. A higher percentage (98.2%) of germ tube production byC. albicans was found in this medium than in undiluted serum (90.2%). In addition, onlyC. albicans was found to be able to produce a high percentage of chlamydoconidia (95.5%) after 48 hours' incubation. In comparison with the conventional medium, corn meal tween 80 agar (21.4%), this new medium gives a significantly higher percentage and abundance of chlamydoconidia production. Being simple, cheap and easy to prepare, the new milk medium is proposed as very practical in the clinical mycology laboratory.     

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.     

Evaluation of Epicoccum nigrum for growth, morphology and production of natural colorants in liquid media and on a solid rice medium

Four nonpathogenic and nontoxigenic Epicoccum nigrum strains were evaluated for their growth, morphology and pigment producing ability in three complex and one defined liquid media. Epicoccum nigrum IBT 41028 produced pigments in all the four media tested with a maximum pigment of 3.68 AU at 410 nm in M1 medium (unoptimized) containing 5 g/l yeast autolysate. The color hue of the crude pigment extracts ranged from 74 to 102 exhibiting dark orange to green-yellow color. Pelleted morphology was shown to have a positive influence on the pigment production by E. nigrum strain IBT 41028 in the liquid media, and the use of Bis-tris buffer was found to diminish or reduce the pellet formation. Since Monascus is a well known pigment producer on rice. Pigment producing ability of E. nigrum IBT 41028 was tested on rice and compared to liquid media with Monascus ruber IBT 7904 as control. Though, both genera preferred rice but E. nigrum produced 4.6 folds higher pigment in the liquid unoptimized fermentation medium compared to M. ruber. Solid phase extraction and subsequently HPLC-DAD analysis of the crude pigment extracts showed qualitative as well as quantitative variation in the pigment composition under solid and liquid cultivations.     

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.

     

An efficient protocol for micropropagation of <Emphasis Type="Italic">Melaleuca alternifolia</Emphasis> Cheel

Melaleuca alternifolia is cultivated for the production of an essential oil useful in the cosmetic and pharmaceutical industries. Despite the economic importance of this species, there is little knowledge about its in vitro propagation. The aim of this study was to establish an efficient protocol for micropropagation of M. alternifolia. With the goal of in vitro multiplication by axillary shoot proliferation, both solid and liquid MS and WPM media were tested with supplementation with BA at 0, 0.55, 1.11, 2.22, 3.33, and 4.44 μM. The best result for shoot multiplication was obtained when either 0.55 μM BA was added into solid MS medium or 1.11 μM BA was added into liquid MS medium, with 5.6 and 11.8 shoots per explant generated, respectively. On solid or liquid WPM medium supplemented with 0.55 μM BA, the proliferation rates were 5.5 and 4.7, respectively. Three auxins (NAA, IAA, and IBA) were tested at 0.53 and 2.64 μM during the rooting stage. Several sucrose concentrations (15, 30, and 45 g L⁻¹) were compared to a sucrose-free medium. Rooting performances on four culture media were then compared: MS, half-strength MS (MS/2), MS + activated charcoal (AC), and MS/2 + AC. The results showed that auxin addition to culture medium is not necessary for in vitro rooting. Rooted microcuttings from different culture media were acclimatized in a greenhouse, and the survival percentage was evaluated. All shoots cultured in an auxin-free MS medium supplemented with sucrose (30 g L⁻¹) produced roots, and all plants survived during acclimatization. Activated charcoal added in rooting medium reduced rooting rates.     

Comparison of different liquid/solid culture systems in the production of somatic embryos from Narcissus L. ovary explants

Alternative procedures for the production of Narcissus L. somatic embryos were investigated. Somatic embryogenesis was initiated on ovary explants isolated from cv. Carlton bulbs, chilled for 12 weeks at 5°C. The explants were cultured on MS media with 3% sucrose and growth regulators: Picloram or 2,4-D (10 or 25 μM) and BA (1 or 5 μM) for 12 weeks in the culture systems: continuous cultivation on solid media, continuous cultivation in liquid media and sequential cultivation using cycles in liquid and solid media. Two types of somatic embryogenesis, indirect and direct, were observed. The developmental pathway depended on the period of exposure to liquid media. Somatic embryos were formed via embryogenic nodular callus on solid media. 2,4-D and BA stimulated the process. The 4-week and 8-week liquid medium treatments resulted in the development of somatic embryos directly from the ovary explant tissue. The highest number of somatic embryos was noted under the influence of 25 μM 2,4-D and 5 μM BA in explants cultivated for 8 weeks in liquid medium and then, for 4 weeks, on solid medium. The effects of inoculum density on biomass increase and the formation of somatic embryos in cultures obtained on a medium with 25 μM 2,4-D and 5 μM BA were also checked. The highest biomass increase was observed after subculturing in liquid medium containing 0.5 μM NAA and 5 μM BA when the density of inoculum was 0.5 g/25 ml of the medium. The highest number of somatic embryos was noted when the density of inoculum was 1.5 g/25 ml.     

Minituber production from immature seed suspension culture ofOrchis papilionacea

Summary Ripe and immature seeds ofOrchis papilionacea (method I and II, respectively) cultured on modified double strength Curtis medium were assayed for minituber production. Ripe seed germination both on solid and in liquid medium was low and the protocorms obtained developed into white calluses. Germination increased from, 9 to 33% when immature seed suspension culture was used. Protocorms obtained in suspension culture under light developed into minitubers, whereas those obtained on solid media developed into callus. A 30 s ultrasonication of immature seeds 1 wk after suspension, culture initiation further enhanced germination and minituber production. Minitubers had to be transferred and embedded in solid regeneration medium for normal growth.     

Pleomorphism in Cellulomonas acidula

Pleomorphism of Cellulomonas acidula in liquid and on solid media is described. Growth in liquid medium is characterized initially by the formation of club-shaped rods and later by cocci. On solid media the organism formed irregular branched cells and large swollen cells.     

Development of the mosquito pathogen Leptolegnia chapmanii (Straminipila: Peronosporomycetes) on an inexpensive culture medium based on sunflower seed

An inexpensive culture medium based on sunflower seed extract (SSE) for production of Leptolegnia chapmanii was developed. Vegetative growth on solid and liquid SSE was compared with two culture media used routinely (peptone, yeast and glucose (PYG) and Emerson YPss). Results indicate that the oomycete is able to grow on SSE medium, producing more zoospores at a faster rate as well as inducing higher mortality rates in Aedes aegypti larvae.     

Shikonin production and secretion by hairy root cultures of Lithospermum erythrorhizon

Summary Hairy root cultures of Lithospermum erythrorhizon were established by transformation of in vitro grown shoots with Agrobacterium rhizogenes 15834. Hairy roots cultured on Murashige and Skoog solid medium did not produce any red pigments. However, the hairy roots cultured in Root Culture solid or liquid media produced a large amount of red pigments, which were released to the medium. The addition of adsorbents to the culture medium stimulated shikonin production by ca. 3-fold. Using this method an air-lift fermenter system was established, equipped with a XAD-2 column, which continuously produced ca. 5 mg/day of shikonin during a period of more than 220 days.     

Initiation, Growth and Nuclear Characteristics of Tissue Cultures of Paeonia suffruticosa

Tissue cultures of the garden paeony, Paeonia suffruticosa have been established using explants of etiolated stems. Callus formation was induced on agar-solidified media containing ammonium ions or amino acids together with the hormones 2,4-dichlorophenoxyacetic acid and kinetin, but not on media lacking the reduced nitrogen component. Attempts to induce callus from explants from green plants were completely unsuccessful and were characterized by the production of intense brown colorations, both of the explant and the medium. Subcultured tissue without the added hormones produced roots, both on solid and liquid media. Growth was tested on a range of liquid media, SH/2, SH, SH × 2 and SH—M, containing 1250, 2500, 5000 and 2500 mg/l potassium nitrate. The SH—M medium also contained 1650 mg/l ammonium nitrate. Growth measured as increased fresh weight was best in the SH/2, SH and SH—M media and was curtailed in the SH × 2 medium. Soluble protein content was highest at the lowest nitrogen concentration. A histochemical comparison of tissue grown on the SH/2, SH—M and SH—M lacking hormones showed that the cells in all the cultures remained diploid while differences were established in total nuclear protein, measured using the ninhydrin-Schiff procedure. Nuclei from SH—M grown cells have a higher protein content than those from the SH/2 medium while cells from the SH—M medium lacking hormones show a further increase in nuclear protein. This raises the question whether this change in nuclear protein is related to the morphogenesis of roots which occurs in this medium.     

Glycosidases of moulds

Selection of a large number of different strains of hyphal fungi of the genusAspergillus, capable of production of extracellular mannosidase and mannanase type enzymes, was carried out. Before cultivating the strains on liquid synthetic medium containing 0.5%Saccharomyces cerevisiae mannan as the carbon source, they were adapted by multiple passage on solid synthetic media containingd-mannose,d-mannose and α-mannan and lastly only α-mannan. The extracellular enzymatic preparations of the mould fungi were tested for their ability to hydrolyse three different substrates—Saccharomyces cerevisiae, Torulopsis ingeniosa andTorulopsis colliculosa mannan. The production of α-mannosidase was found to be specifically dependent on the character of the substrate used for cultivation of the fungus.     

Microplate Assay for Colletotrichum Spore Production

A simple microplate method was devised to assay spore production by Colletotrichum gloeosporioides by growing the fungus on 1 ml of solid media in the wells of tissue culture plates. Growth and sporulation on microplates were compared at days 4 and 8 with growth and sporulation in 100-ml liquid batch cultures that involved 11 common media. Spore production per unit volume of medium was the same for solid and liquid forms of the media. Qualitative assessment of mycelial growth measured on microplates agreed with that of growth measured in liquid cultures. The microplate assay indicated that V8 juice was the best medium and that an organic content of about 6 mg/ml was optimal for high sporulation and low mycelium production. The assay provides a convenient, rapid, and inexpensive means of screening media for the production of fungal conidia in large numbers, to be used, for example, in biological control programs.     

Urease Activity of Enterobacteriaceae: Which Medium to Choose

Detection and intensity of urease activity in enterobacteriaceae greatly varies as a function of the media or techniques used, or both. A comparative investigation on several solid and liquid media led us to the following conclusions. (i) Detection of Proteus spp. can be adequately performed with the highly selective solid medium described by Cook (1948), as well as with the different liquid media described (Stuart standard and rapid media; Elek medium). (ii) Detection of Klebsiella should be based upon urease production on solid media with low buffer capacity (Christensen, 1946). (iii) For the identification of Yersinia, either the solid Christensen urea agar or the rapid Elek technique give optimal results.     

Jasmonate effects on in vitro tuberization and tuber bulking in two potato cultivars (Solanum tuberosum L.) under different media and photoperiod conditions

Summary Jasmonic acid (JA) effects on in vitro tuberization of potato nodal explants cvs. Sangre and Russet Burbank were tested under liquid and solid media conditions and 0,8, and 16h photoperiod. Explants taken from stock plants grown on 2.5μM JA-supplemented medium tuberized first, particularly in darkness. The most pronounced benefits of the JA pretreatment were recorded under 16h photoperiod, which is known to inhibit tuberization. Cultivar Sangre benefited from the JA preconditioning of stock plants more than Russet Burbank. Russet Burbank required the JA supplement in tuberization media to reach the same degree of stimulation. Overall, microtubers produced either from JA preconditioned stock plants or on the JA-containing tuberization media were more uniform and larger than from other treatments. Eight hours photoperiod was by far the best treatment for the production of high-quality uniform microtubers. JA conditioning of stock plants prior to taking explants for tuberization is being proposed as a treatment enhancing the quality of microtubers.     

Growth, sclerotia and aflatoxin production by Aspergillus parasiticus: influence of food preservatives

The influence of six food preservatives on control of aflatoxin production by Aspergillus parasiticus was tested in SMKY and defined media at three concentrations, viz., 0.1, 0.5 and 1.0%. Propionic acid completely inhibited the yield of mycelia and sclerotia, and aflatoxin production in culture medium, mycelia and sclerotia of A. parasiticus at all concentrations, whereas citric acid showed inhibition only at 0.5 and 1.0% concentrations. Sodium metabisulphite did not permit mycelial growth and aflatoxin biosynthesis in SMKY liquid medium but allowed production of sclerotia and aflatoxin on solid media, while the rest of the food preservatives had only marginal inhibitory effects.     

Lippia dulcis shoot cultures as a source of the sweet sesquiterpene hernandulcin

The axenic shoot culture of Lippia dulcis Trev., Verbenaceae, was established on hormone-free Murashige-Skoog solid medium containing 3% sucrose. Shoots were cultured in various liquid or solid media. Woody Plant liquid medium was best for shoot multiplication, but the production of hernandulcin was relatively low. The highest hernandulcin content (2.9% dry wt) was obtained after 28 days of culture on Murashige-Skoog solid medium containing 2% sucrose. The addition of chitosan to the culture media enhanced the growth of shoots as well as the production of hernandulcin, especially with the liquid medium.Abbreviations MS(2%) Murashige-Skoog medium containing 2 % sucrose - MS(3%) Murashige-Skoog medium containing 3 % sucrose - 1/2MS half strength Murashige-Skoog medium containing 2% sucrose - B5 Gamborg B5 medium containing 2% sucrose - WP Woody Plant medium containing 2% sucrose     

In vitro solid-state production of Steinernema innovationi with cost analysis

Low-cost mass production of entomopathogenic nematodes (EPNs) is an important prerequisite towards their successful commercialisation. This study evaluated six low-cost solid substrate media for in vitro mass production of Steinernema innovationi. Cost analysis was undertaken and an estimated retail price was calculated. This was then compared to the costs of commercial EPN products currently on the market. The highest yield of infective juveniles (IJs) was obtained from a medium containing a puree of house fly, Musca domestica, larvae?+?0.15?g canola oil, (781,678?±?221 IJs/5?g medium). This medium also had the lowest number of adults remaining in the medium and dead IJs (<10%) at the time of harvest (Day 28). The estimated retail price (R243.27 per 50 million IJs) for S. innovationi produced with our solid culture system was considerably lower than the market price for other Steinernema species products sold by E～nema, BASF corporation, Koppert, BioBest and Natural Insect Control. The production system developed in this study offers a competitive technology to produce EPN products without having to invest in large-scale liquid fermentation equipment, by using a relatively cheap production medium and simple solid culture growing conditions using Erlenmeyer flasks.     

Attraction of Mexican fruit flies (Diptera: Tephritidae) to bacteria: effects of culturing medium on odour volatiles

Effects of culturing medium on production and emission of volatiles by Pantoea agglomerans (Beijerinck 1888) Gavini et al. 1989 preparations and on attractiveness of the preparations to the Mexican fruit fly, Anastrepha ludens Loew, were investigated. Bacterial cultures in each of four biochemically different types of liquid media emitted different volatiles. Cultures in a medium containing uric acid as its primary nitrogen source emitted more ammonia and 2‐nonanone than the other media. We postulate that the high production of ammonia was because of uricase activity by this uricase (+) strain. Regardless of media type, supernatants emitted more volatiles than preparations containing cells that had been removed from whole cultures and put into distilled water. Attractiveness varied little with biochemical make‐up of the culturing medium although the uric acid and carbohydrate preparations were as a group more attractive than preparations made from the other two media. Supernatants and whole cultures generally were more attractive than cell preparations and non‐inoculated media. Bacteria grown in aqueous uric acid‐based media emitted volatiles similar but not identical to those emitted by bacteria grown on gel (agar or solid) uric acid‐based media in Petri plates.     

Paecilotoxin production in clinical or terrestrial isolates of Paecilomyces lilacinus strains

The production of paecilotoxin from various isolates of Paecilomyces lilacinus was studied using three different media and high performance liquid chromatography (HPLC). Alkaline medium was found to be suitable for the production of the toxins. Among 20 strains tested, 19 including four clinical isolates were found to produce the toxins. Production patterns of paecilotoxins were very similar in each strain and the main toxins were A and B.