Selective isolation and distribution of the genus Planomonospora in soils

For the screening of bioactive compounds and study of global distribution, a selective isolation method for Planomonospora strains by centrifugation from soil is examined. Planomonospora strains produced characteristic sporangia on the humic acid-trace salts gellan gum medium (pH 9.0) so that this genus was readily recognized on the isolation plate. High yields of motile spores were obtained by using a flooding solution containing 0.1% skim milk in 5 mM N-cyclohexyl-2-amino-ethanesulfonic acid (pH 9.0) followed by incubating the preparation at 32 degreesC for 90 min, centrifuging it at 1000 x g for 10 min, and further incubation at 32 degreesC for 60 min after centrifugation. By combining the techniques described above, we isolated 246 Planomonospora strains from 137 of the 1200 soil samples examined. Ninety-four percent of these strains were recovered from neutral to slightly alkaline soils (pH 7.0 to 9.0). Strains of P. venezuelensis group were obtained from 13 soil samples (1.1%), which were collected in Bolivia, Cyprus, Egypt, Greece, India, Japan, New Caledonia, and Turkey. Strains of this group appear widely distributed in the soil of tropical to temperate regions. To our knowledge, this is the first record that strains of this group have been isolated from a location other than Venezuela.     

Effect of gelling agent on colony formation in solid cultivation of microbial community in lake sediment

Since Robert Koch and colleagues found agar to be an effective gelling agent over a century ago, the pure culture method using agar plates has long been a standard of microbiology. Agar is undoubtedly easy to handle and useful for culture of microorganisms, but recent discovery of the ubiquity of microorganisms that cannot be cultured on agar raises a question: is agar really the best agent? In this study, we investigated the effect of two gelling agents, agar and gellan gum, on colony formation of a diverse array of microorganisms (total 108 strains) newly isolated from freshwater sediments and a representative microorganism as a slow grower on agar medium, Gemmatimonas aurantiaca, to clarify (i) whether they can grow on both agar and gellan gum plates, and (ii) the difference in time required for colony formation between the two gelling agents. Interestingly, 22 of 108 isolates showed no ability to form any visible colonies on the agar medium but did so on the gellan gum medium, and showed low 16S rRNA gene sequence similarities to their closest species. The remaining 86 isolates grew on both agar and gellan gum, but 52 of them grew much faster on gellan gum than on agar. Moreover, gellan gum also significantly stimulated the colony formation of the representative slow‐growing microorganism G. aurantiaca. Our results demonstrate that the gelling agent is a crucial factor for the growth of bacteria on plate media, and that alternatives to agar will be very important for increasing the culturability of yet‐to‐be cultured microorganisms.     

Growth characteristics of the slow-growing actinobacterium Frankia sp. strain CcI3 on solid media

Frankia sp. strains are slow-growing Actinobacteria that form N₂-fixing root nodules on certain angiosperm trees and shrubs. These organisms are generally cultured in liquid media as routine growth on solid media is impractical because of lengthy incubation periods. Liquid media is problematic for filamentous organisms because unicellular spores are difficult to isolate, and mutants are difficult to separate. The goal of this study was to optimize Frankia CcI3 growth on solid media. Starting with an appropriate basal medium containing pyruvate as a carbon source, various peptones were individually tested to determine if the time needed for colonies to appear could be reduced. Bacto™ Proteose Peptone #3 at 1.6 mg ml⁻¹ final concentration promoted the best growth with colonies visible after 3 days, compared with the 7- to 10-day incubation required for control unamended media. Gellan gum gave a more rapid growth response as compared with agar. Increased calcium concentrations were required to promote solidification of gellan gum. Sporulation occurred within 2 weeks of plating CcI3 with colonies changing color from cream to reddish-brown. Scanning and transmission electron micrographs revealed the architecture of colonial development. Colonies developed in a lens-shaped manner mainly by penetration into the gel. Older colonies had numerous empty hyphae with sporangia developing near or at the surface and with crystalline material, presumably pigment, interspersed among the hyphae. Occasional vesicles were seen mixed within the colonies. Colonial development of Frankia CcI3 is reminiscent of that described for other members of the filamentous Suborder Frankineae.     

Rapid purification of commercial gellan gum to highly soluble and gellable monovalent cation salts

Commercial gellan gum contains divalent cation contaminants (mainly Ca²⁺ and Mg²⁺) in levels sufficient to neutralize over one-third of its car☐yl groups. Consequently, in order to dissolve gellan gum in water, the mixtures must be heated to more than 90°C. This has prevented applications of gellan gum to such uses as the immobilization of viable enzymes and cells in beads. A rapid two-step method is described here to purify commercial gellan gum to the monovalent cation salts in an overall yield of 85%, through the intermediate free acid form. The gellan monovalent cation salts were highly soluble at temperatures as low as 5°C, and readily gelled upon exposure to solutions of divalent cations. Laboratory-scale preparations of 100 g of gellan monovalent cation salts were readily achieved in a day.     

Selective Isolation and Distribution of Sporichthya Strains in Soil

A simplified enrichment method in which centrifugation is used for selective isolation of Sporichthya strains from soil is described. Gellan gum plus 2 mM CaCl₂ stimulated growth of Sporichthya polymorpha KCC A0089 so that this organism was readily recognized on an isolation plate. High yields of motile spores were obtained by using a flooding solution containing 0.1% skim milk in 10 mM MOPS (morpholinepropanesulfonic acid) (pH 8.0) and then incubating the preparation at 27°C for 60 min and centrifuging it at 1,000 × g for 10 min. Dry heat treatment at 80°C for 60 min increased the ratio of Sporichthya colonies to nonfilamentous bacteria on a gellan gum plate. Since S. polymorpha was sensitive to 14 antibiotics, including nalidixic acid, addition of these antibiotics was not suitable for isolating Sporichthya strains. Our isolates were identified as Sporichthya strains on the basis of their morphological and chemotaxonomic characteristics. By combining the techniques described above, we isolated a number of Sporichthya strains from 21 soil samples, which were collected in Belgium, France, India, Japan, Papua New Guinea, Spain, Taiwan, the United Kingdom, and the United States. Sporichthya strains are widely distributed in the world. To our knowledge, this is the first time that Sporichthya strains have been isolated from locations other than the United States or Japan.     

Use of a single procedure for selective enrichment, isolation, and identification of plasmid-bearing virulent Yersinia enterocolitica of various serotypes from pork samples.

Many selective enrichment methods for the isolation of Yersinia enterocolitica from foods have been described. However, no single isolation procedure has been described for the recovery and identification of various plasmid-bearing serotypes. A single improved procedure for selective enrichment, isolation, identification, and maintenance of plasmid-bearing virulent serotypes of Y. enterocolitica from pork samples was developed. Enrichment at 12 degrees C in Trypticase soy broth containing yeast extract, bile salts, and Irgasan was found to be an efficient medium for the recovery of plasmid-bearing virulent strains of Y. enterocolitica representing O:3; O:8; O:TACOMA; O:5, O:27; and O:13 serotypes. MacConkey agar proved to be a reliable medium for the isolation of presumptive colonies, which were subsequently confirmed as plasmid-bearing virulent strains by Congo red binding and low calcium response. Further confirmation by multiplex PCR employed primers directed at the chromosomal ail and plasmid-borne virF genes, which are present only in pathogenic strains. The method was applied to pig slaughterhouse samples and was effective in isolating plasmid-bearing virulent strains of Y. enterocolitica from naturally contaminated porcine tongues. Strains isolated from ground pork and tongue expressed plasmid-associated phenotypes and mouse pathogenicity.     

Improved gellan gum production by a newly-isolated Sphingomonas azotifigens GL-1 in a cheese whey and molasses based medium

Gellan gum is a water-soluble exopolysaccharide, it has applications in the food, pharmaceutical and chemical industries. In this study, a gellan gum producing strain was isolated from rice root, and this strain was identified be the species of Sphingomonas azotifigens. The Plackett-Burman design was applied to investigate the main factors affecting gellan gum production by S. azotifigens GL-1 in a molasses and cheese whey based medium; the medium compositions were optimized by response surface methodology. The optimum cheese whey based medium consisted of cheese whey 68.34 g/L, Na₂HPO₄ 14.58 g/L and KH₂PO₄ 7.66 g/L, and the maximum gellan gum production that using this medium was 33.75 ± 1.55 g/L. 14.75 ± 0.65 g/L gellan gum was obtained with an optimized molasses medium, which consisted of molasses 50 g/L, Na₂HPO₄ 9.71 g/L and KH₂PO₄ 5.92 g/L. The molecular weight of gellan gum obtained from two medias were 1.06 × 10⁶ and 0.89 × 10⁶ Da, respectively. The cheese whey-derived gellan gum showed a higher rhamnose, lower glucuronic acid and higher glycerate content compared to the molasses-derived gellan gum. S. azotifigens GL-1 has a high gellan gum production capacity in a cheap medium suggesting it has great potential as an industrial gellan gum producer.     

Soft gel medium solidified with gellan gum for preliminary screening for root-associating,free-living nitrogen-fixing bacteria inhabiting the rhizoplane of plants

For preliminary screening for and characterization of free-living nitrogen-fixing bacteria from rhizoplane microflora, we used Winogradsky's mineral mixture-based nitrogen-free medium solidified with 0.3% gellan gum. The soft gel medium enabled some reference and wild free-living nitrogen-fixing bacteria to grow in characteristic colonies, including their reaction to oxygen and their motility change. Gellan gum is thus likely to be a better gel matrix than agarose for the investigation of root-associating, free-living nitrogen-fixing bacteria to identify their characteristic behaviors.     

A differential medium for the isolation and rapid identification of a plant soft rot pathogen, Erwinia chrysanthemi

A medium was developed for the isolation and differentiation of Erwinia chrysanthemi from other Erwinia spp. based on the production of blue-pigmented indigoidine. The medium, named NGM, consists of nutrient agar supplemented with 1% glycerol, that induces pigment production, and 2 mM MnCl2*4H2O, that further enhances color development. More than fifty E. chrysanthemi strains from six different plant hosts were tested. All tested strains of E. chrysanthemi grew well on the NGM medium, developing dark brownish to blue colonies easily distinguishable from other Erwinia spp. The results indicate that pigment production on the NGM medium is a very stable property and can be used as a phenotypic property to differentiate E. chrysanthemi from other Erwinia spp. In addition, a specific oligonucleotide primer set was designed for the detection of indC, which is involved in indigoidine biosynthesis. All E. chrysanthemi strains tested contained indC as determined by PCR amplification. No amplification was observed with other Erwinia spp. Thus, pigment production of E. chrysanthemi on the NGM medium is consistent with the existence of indC. The NGM medium was used to isolate and identify the causal agent of soft rot lesions of diseased Phalaenopsis orchids from three orchid cultivation areas in Taiwan. The causal agents of Phalaenopsis soft rot were all identified as E. chrysanthemi. The results indicate that the NGM medium is efficient in isolation and identification of E. chrysanthemi from plants with soft rot symptoms and can also be used for epidemiological studies.     

Effects of Potassium Chloride and Sodium Chloride on the Thermal Properties of Gellan Gum Gels

The exothermic and endothermic peaks in cooling and heating curves of differential scanning calorimetry (DSC) for gellan gum gels without and with potassium chloride and sodium chloride were analyzed. The gelling and melting temperatures shifted to higher temperatures with increasing gellan and salt concentration in the concentration range of gellan from 0.3 to 2.0% (w/w). The exothermic and endothermic enthalpy increased with increasing gellan and salt concentrations. Cooling DSC curves showed one exothermic peak for samples with salts and at low gellan concentration. Heating DSC curves showed many peaks for all samples except 0.3% (w/w) gellan gum gels. The sol-gel transition of samples was examined numerically by using a zipper model approach. The introduction of cations increases the number of junction zones or zippers and decreases the rotational freedom of parallel links. This makes the structure of junction zones more heat resistant, and increases the elastic modulus of the gel.     

Secondary Sporangium Formation in Phytophthora Palmivora (Butl.) Butl

Very few sporangia of Phytophthora palmivora germinated directlyand produced secondary sporangia in distilled water and in solutionsof amino acids and carbohydrates at 30 °C. Although 1.0per cent (w / v) peptone and yeast-extract stimulated a highpercentage of germination by formation of germ tubes, less than1.0 per cent of the germinated sporangia produced secondarysporangia. Secondary sporangium formation was induced by transferringgerminated primary sporangia from a nutrient medium of sufficientlyhigh concentration to either distilled water or dilute solutionsof organic and inorganic compounds immediately after emergenceof the germ tubes. The percentage of germinated sporangia formingsecondary sporangia was influenced by both the nature and concentrationof the medium into which they were transferred. The secondarysporangia were significantly smaller than the primary sporangia. Phytophthora palmivora, germination, sporangium, Theobroma cacao L., cocoa     

Reliability of O157:H7 ID agar (O157 H7 ID-F) for the detection and isolation of verocytotoxigenic strains of Escherichia coli belonging to serogroup O157

AIMS: The reliability of the O157:H7 ID agar (O157 H7 ID-F) to detect verocytotoxigenic strains of Escherichia coli (VTEC) of serogroup O157 was investigated. METHODS AND RESULTS: This medium, designed to detect strains belonging to the clone of VTEC O157:H7/H-, contains carbohydrates and two chromogenic substrates to detect beta-d-galactosidase and beta-d-glucuronidase and sodium desoxycholate to increase selectivity for Gram-negative rods. A total of 347 strains of E. coli including a variety of serotypes, verocytotoxigenicity of human and animal sources were tested. The green VTEC O157 colonies were easy to detect among the other dark purple to black E. coli colonies. Of 63 O157:H7/H- strains, 59 (93.7%) gave the characteristic green colour. Three of the failed four strains of O157:H- were not verocytotoxigenic, missing only one VTEC O157. Three non-O157 strains gave the characteristic green colour on the medium and were VTEC OR:H- (2) and Ont:H- (1), possibly being degraded variants of the O157 enterohaemorrhagic E. coli clone. CONCLUSIONS: The O157:H7 ID agar (O157 H7 ID-F) was largely successful in isolating VTEC belonging to the O157:H7/H- clone. SIGNIFICANCE AND IMPACT OF THE STUDY: A medium, suitable for isolating strains of VTEC O157 was successfully evaluated and should be useful for the isolation of these pathogens.     

Humic acid-vitamin agar,a new medium for the selective isolation of soil actinomycetes

A new medium, designated HV agar, containing soil humic acid as the sole source of carbon and nitrogen was developed.The HV agar was superior to other currently used media, including colloidal chitin agar, glycerol-arginine agar and starch-casein-nitrate agar, for the isolation and enumeration of soil actinomycetes: It allowed the growth of the largest numbers of actinomycete colonies belonging to each genus of Streptomyces, Micromonospora, Microbispora, Streptosporangium, Nocardia, Dactylosporangium, Microtetraspora and Thermomonospora on the plate, while restricting the development of true bacteria. The HV agar supported adequate growth and good sporulation for these actinomycetes.Even when spore suspensions were used as the inoculum, the HV agar produced remarkably larger numbers of actinomycetes, especially strains of the genera Micromonospora, Microbispora, Streptosporangium, Dactylosporangium and Saccharomonospora, than did glycerol-arginine agar. It was found that the spores of these actinomycetes were activated upon germination by treatment at 20°C for 30 min with a O.2% solution of humic acid prior to incubation.     

Selective medium for the isolation and enumeration of Mycobacterium avium-intracellulare and M. scrofulaceum

A medium for the selective isolation and enumeration of Mycobacterium avium-intracellulare and M. scrofulaceum (MAIS) was developed, based upon the ability of these mycobacteria to utilize Tween 80 as sole carbon source and grow optimally at pH 5.5 on a simple mineral salts medium. Representative MAIS strains had higher efficiencies of plating on the Tween 80 medium compared with Middlebrook 7H10. It was shown that nonmycobacterial organisms in natural waters had lower efficiencies of plating on the Tween 80 medium and smaller colonies, thus allowing direct isolation and enumeration of the slowly growing mycobacteria without overgrowth.     

Modification of sorbitol MacConkey medium containing cefixime and tellurite for isolation of Escherichia coli O157:H7 from radish sprouts

A modified version of sorbitol MacConkey medium containing cefixime and tellurite (CT-SMAC medium) was produced by adding salicin and 4-methylumbelliferyl-beta-D-galactopyranoside to CT-SMAC medium; this medium was designated CT-SSMAC medium and was used to isolate Escherichia coli O157:H7 from radish sprouts. Of 101 non-E. coli bacteria isolated from radish sprouts that produced colorless colonies similar to colonies of E. coli O157:H7 grown on CT-SMAC medium, 92 (91%) formed colonies that were red to pink or were beta-galactosidase negative and colorless on CT-SSMAC medium. On the other hand, colonies of E. coli O157:H7 strains were colorless and beta-galactosidase positive on CT-SSMAC medium. Our results suggest that CT-SSMAC medium is more selective than CT-SMAC medium for isolating E. coli O157:H7.     

Improvement of the detection of Listeria monocytogenes by the application of ALOA, a diagnostic, chromogenic isolation medium

A new selective agar medium, ALOA, for the selective and differential isolation of Listeria monocytogenes has been evaluated. All stressed cultures of L. monocytogenes serovars tested grew on the medium as bluish colonies surrounded by a distinctive opaque halo and gave a productivity ratio of at least 0.95. Non-pathogenic Listeria sp. produced bluish colonies without a halo as was also the case for some enterococci and bacilli. Special attention must be paid to some Bacillus cereus strains and L. ivanovii since their colony appearance can be misleading. Only some unidentified listeria-like bacteria gave false-positive results. ALOA detected 4. 3% more positives from naturally contaminated dairy and meat samples compared with the ISO procedure when used with GenprobeTM or VidasTM for confirmation of presumptive colonies; 13.9% false negatives were found compared with 38.9% using PALCAM/Oxford. ALOA was also clearly superior to Oxford and PALCAM when samples containing both L. monocytogenes and L. innocua were examined. The introduction of ALOA in standard isolation procedures as an additional medium would enhance the detection ratio and reduce the time and cost of analysis for L. monocytogenes.     

Increased gelling agent concentration promotes somatic embryo maturation in hybrid larch (Larix × eurolepsis): a 2-DE proteomic analysis

An integrated physiological and proteomic approach was used to investigate the effects of high gellan gum concentration in the medium during maturation of somatic embryos (SE) of hybrid larch, by comparing embryos incubated in media with a high gellan gum concentration (8 g l(-1) ) and the standard concentration (4 g l(-1) ) after 1, 3, 6 and 8 weeks of maturation. Because of the reduced availability of water in the 8 g l(-1) medium, the cultured embryos had a lower osmotic water potential (Ψπ) and water contents, but higher dry weights (DWs), at 8 weeks compared with embryos cultured on the standard medium. The high gellan gum concentration induced a desiccation that is characteristic in zygotic embryo maturation. Total soluble proteins were extracted from SE with trichloroacetic acid (TCA)-acetone after 1 and 8 weeks of maturation on media with 4 and 8 g l(-1) of gellan gum, and separated by two-dimensional gel electrophoresis (2-DE) at pH 4-7. More than 1100 proteins were reproducibly detected on each gel. At 1 and 8 weeks respectively, the abundances of 62 and 49 spots detected in analyses of embryos matured at the two gellan gum concentrations, significantly differed. Among 62 significantly differing spots at 1 week of maturation, the corresponding proteins of 56 were reliably identified by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), and were found to be mainly involved in 'carbohydrate metabolism', 'genetic information processing' or 'environmental information processing' according to kegg taxonomy. Both physiological parameters and the proteins identified suggested that the embryos were stressed when they were cultured on 4 g l(-1) of gellan gum.     

Differential medium for selection of bacterial L-asparaginase producer strains

A specific, fast, and easy method for revelation of active plate producers of L-asparaginase using differential medium on the basis of LB or M9 with 1.5% agar was developed. Each 100 ml of LB or M9 medium additionally contained 6-7 ml ofglycerol, 4 g of L-asparagine, 0.2 g of CaCO3, and diagnostic components: 3 ml of 0.2 M CuSO4 x 5H2O and 2.5 ml of 0.1 M K3Fe(CN)6, pH 7.6-7.8. The results were counted 12-20 or 24-48 h after strain growth at 37 degrees C in corresponding mediums. Red color of colonies and colored zone around them showed the ability of the strain under study to destroy asparaginic complexes. The recommended method allows revealing bacterial strains producing L-asparaginase with specific activity of not less than 0.1-3.0 MU/mg of protein.     

Isolation and characterization of novel iron-oxidizing bacteria that grow at circumneutral pH.

A gel-stabilized gradient method that employed opposing gradients of Fe2+ and O2 was used to isolate and characterize two new Fe-oxidizing bacteria from a neutral pH, Fe(2+)-containing groundwater in Michigan. Two separate enrichment cultures were obtained, and in each the cells grew in a distinct, rust-colored band in the gel at the oxic-anoxic interface. The cells were tightly associated with the ferric hydroxides. Repeated serial dilutions of both enrichments resulted in the isolation of two axenic strains, ES-1 and ES-2. The cultures were judged pure based on (i) growth from single colonies in tubes at dilutions of 10(-7) (ES-2) (ES-2) and 10(-8) (ES-1); (ii) uniform cell morphologies, i.e., ES-1 was a motile long thin, bent, or S-shaped rod and ES-2 was a shorter curved rod; and (iii) no growth on a heterotrophic medium. Strain ES-1 grew to a density of 10(8) cells/ml on FeS with a doubling time of 8 h. Strain ES-2 grew to a density of 5 x 10(7) cells/ml with a doubling time of 12.5 h. Both strains also grew on FeCO3. Neither strain grew without Fe2+, nor did they grow with glucose, pyruvate, acetate, Mn, or H2S as an electron donor. Studies with an oxygen microelectrode revealed that both strains grew at the oxic-anoxic interface of the gradients and tracked the O2 minima when subjected to higher O2 concentrations, suggesting they are microaerobes. Phylogenetically the two strains formed a novel lineage within the gamma Proteobacteria. They were very closely related to each other and were equally closely related to PVB OTU 1, a phylotype obtained from an iron-rich hydrothermal vent system at the Loihi Seamount in the Pacific Ocean, and SPB OTU 1, a phylotype obtained from permafrost soil in Siberia. Their closest cultivated relative was Stenotrophomonas maltophilia. In total, this evidence suggests ES-1 and ES-2 are members of a previously untapped group of putatively lithotrophic, unicellular iron-oxidizing bacteria.     

Isolation of new actinomycete strains for the screening of new bioactive compounds

In order to facilitate the discovery of novel bioactive compounds from microorganisms, various techniques for isolation of new actinomycete strains have been attempted. Studies of the vertical distribution of actinomycetes in soil, isolation of actinomycetes from desert soils or fallen leaves, selective isolation of Kitasatospora strains using novobiocin or Actinoplanes strains using the chemotactic method, and the use of gellan gum as a solidifying agent were carried out. We discovered 9 novel bioactive compounds from actinomycete strains isolated under unusual conditions, and proposed two new genera, five new species and one new subspecies.