Complex Organic Media Research Articles

Slow relaxation of viscoelastic oil modules

The viscoelastic modules of complex organic media evolve slowly. Studying the dependence of a complex shear modulus on strain amplitude reveals logarithmic growth over time of the nonlinearity parameter of a raw oil sample. The linear reduction in the complex shear modulus upon an increase in the amplitude of shear perturbations is confirmed experimentally; this is possible when there is a linear dependence between the viscoelastic parameters of a sample and the strain modulus.

Asymbiotic germination of the White Mountain Orchid (Pseudorchis albida) from immature seed on media enriched with complex organics or phytohormones

The arctic-alpine White Mountain Orchid (Pseudorchis albida) has so-far resisted attempts at in vitro reproduction from seed, but a practical method is required for its conservation. We hypothesised that, having similar seeds to the notoriously intractable orchid Cypripedium calceolus, immature seeds can germinate in vitro. We collected mature and immature seeds from the Italian Alps, and sowed these in sterile conditions on Malmgren’s basal nutrient medium modified with either phytohormones or complex organic media (COM). Different COM treatments included coconut milk, banana powder, malt extract and yeast extract, all at a concentration of 5%. Phytohormones, all at 20 mg L, included 6-benzylaminopurine (BA), gibberellic acid (GA3), kinetin and thidiazuron (TDZ), added to autoclaved growth media via filter sterilization. After six months we found absolutely no germination of mature seeds for any treatment. Only 3.9% of immature seeds germinated in the control treatment and germination in all COM treatments was not significantly greater than this. However, most phytohormones significantly increased germination of immature seed and promoted protocorm development, BA being most effective (50.5% germination) – this method can therefore be used to propagate the species.

Outbreeding and asymbiotic germination in the conservation of the endangered Italian endemic orchid Ophrys benacensis

The Insubric bee orchid, Ophrys benacensis (Reisigl) O.&E. Danesch & F. Ehrend, occurs in fragmented populations only in northern Italy, and suffers from inbreeding depression. We found that pollen from a depressed population at Monte Barro, Lecco, did not fertilise plants of the same population, nor of a larger population at nearby Valmadrera. Fertilisation was successful at both sites when pollen from Valmadrera was used, although the proportion of seeds containing embryos was almost six times greater at Valmadrera. Embryos produced by both populations were equally likely to develop in vitro. Sowing seed on medium enriched with 50 mL L−1 coconut milk more than doubled the germination rate with respect to a non‐enriched control (i.e. from 14.5% to 39.8%; p = 0.024, Student’s t‐test), whereas other complex organic media inhibited germination. We conclude that both pollen and ovules have inherent developmental problems that can be partially overcome by outbreeding with larger populations. Once seed is produced propagation is relatively easy: sufficient plantlets were produced to enlarge the Monte Barro population to 195 times its current size.

Phylogenetic and metabolic diversity of Planctomycetes from anaerobic, sulfide- and sulfur-rich Zodletone Spring, Oklahoma.

We investigated the phylogenetic diversity and metabolic capabilities of members of the phylum Planctomycetes in the anaerobic, sulfide-saturated sediments of a mesophilic spring (Zodletone Spring) in southwestern Oklahoma. Culture-independent analyses of 16S rRNA gene sequences generated using Planctomycetes-biased primer pairs suggested that an extremely diverse community of Planctomycetes is present at the spring. Although sequences that are phylogenetically affiliated with cultured heterotrophic Planctomycetes were identified, the majority of the sequences belonged to several globally distributed, as-yet-uncultured Planctomycetes lineages. Using complex organic media (aqueous extracts of the spring sediments and rumen fluid), we isolated two novel strains that belonged to the Pirellula-Rhodopirellula-Blastopirellula clade within the Planctomycetes. The two strains had identical 16S rRNA gene sequences, and their closest relatives were isolates from Kiel Fjord (Germany), Keauhou Beach (HI), a marine aquarium, and tissues of marine organisms (Aplysina sp. sponges and postlarvae of the giant tiger prawn Penaeus monodon). The closest recognized cultured relative of strain Zi62 was Blastopirellula marina (93.9% sequence similarity). Detailed characterization of strain Zi62 revealed its ability to reduce elemental sulfur to sulfide under anaerobic conditions, as well as its ability to produce acids from sugars; both characteristics may potentially allow strain Zi62 to survive and grow in the anaerobic, sulfide- and sulfur-rich environment at the spring source. Overall, this work indicates that anaerobic metabolic abilities are widely distributed among all major Planctomycetes lineages and suggests carbohydrate fermentation and sulfur reduction as possible mechanisms employed by heterotrophic Planctomycetes for growth and survival under anaerobic conditions.

Knoellia sinensis gen. nov., sp. nov. and Knoellia subterranea sp. nov., two novel actinobacteria isolated from a cave.

Two novel strains of the class Actinobacteria were isolated from a cave in China. Cells of both strains were gram-positive, non-motile, non-spore-forming and not acid-fast and exhibited a rod/coccus growth cycle. Both isolates grew well on complex organic media under aerobic conditions. Their cell wall peptidoglycan contained meso-diaminopimelic acid as diagnostic diamino acid. The acyl type of the glycan chain of peptidoglycan was acetyl. The major respiratory quinone was MK-8(H4). The cellular fatty acid profile was characterized by the predominance of 13-methyltetradecanoic (i-C15:0), 15-methylhexadecanoic (i-C17:0), 14-methylpentadecanoic (i-C16:0) and 14-methylhexadecanoic (ai-C17:0) acids. The major polar lipids were phosphatidylethanolamine, phosphatidylinositol and diphosphatidylglycerol. Mycolic acids were absent. The DNA G+C composition was 68-69 mol%. 16S rDNA-based phylogenetic analysis revealed an intermediate phylogenetic position of the cave isolates between the genera Janibacter and Tetrasphaera, which did not permit their unambiguous affiliation to either genus. Differences in morphological, physiological and chemotaxonomic properties between the two isolates and their closest phylogenetic neighbours support the proposal of a new genus and two novel species, Knoellia sinensis gen. nov., sp. nov. and Knoellia subterranea sp. nov. The type and only strains of the species are respectively HKI 0119T (= DSM 12331T = CIP 106775T) and HKI 0120T (= DSM 12332T = CIP 106776T).

Media for the detection of Bacillus larvae spores in honey

AbstractBacillus larvae, the causative agent of American foulbrood in honey bees completes its life cycle of germination, outgrowth and sporulation in young honey bee larvae by killing them and often bringing about the destruction of the entire hive. While B. larvae germinates and outgrows on complex organic media in vitro, the literature suggests, for reasons that are not at all clear, that a relatively large number of spores of B. larvae are required to yield each visible colony (colony forming units, CFU) on media. Various researchers have reported that from 16 to 3,000 or more spores of B. larvae are required to yield a single colony on an agar plate. HANSEN in Denmark designed a useful method of spreading approximately 80 mg of honey directly on the surface of a PETRI plate containing “J” agar medium to determine if B. larvae spores are present in the honey. In the present study, selected media were tested for the ability to recover B. larvae spores in honeys in the form of visible colonies (CFU) using HANSEN's strek method. A modification of a medium (TMYGP) developed by DINGMAN and STAHLY, (T‐HCL‐YGP agar), recovered considerably more viable B. larvae spores in the form of visible colonies (CFU) than HANSEN's “J” medium. When “J” medium was fortified with 0.1% sodium pyruvate, it was comparable to modified T‐HCL‐YGP medium in its recovery of B. larvae spores. Brain heart infusion agar (BHIA) with the addition of thiamine recovered more spores in the form of viable colonies than did “J” medium but it was not as efficient as T‐HCL‐YGP medium. Serial dilution from 100 to 10,000 times of weighed samples of honey with deionized water led to higher spore counts (CFU per g honey) than that indicated by undiluted honeys plated at 80 mg levels directly onto the surface of media by the HANSEN procedure.

Maintenance ofRhodotorula rubra isolated from liquid samples of gold mine effluents

TheRhodotorula rubra strain isolated from waste waters of a gold mining plant has demonstrated the ability to grow in the presence of cyanide. The maintenance of this strain in complex organic media leads to a loss of this ability. To preserve the cyanide resistance ofR. rubra we tested the following maintenance methods: subculturing in sterile distilled water, freezing at −20, −40, −70°C, liquid nitrogen freezing and the paper replica method. The ability to grow in the presence of cyanide was preserved and a higher viability level was observed for cells maintained frozen at −70°C, in liquid nitrogen and by the paper replica method. Preservation in distilled water resulted in the lowest viability after twelve months of storage.

Oxygen sensitivity of an Escherichia coli mutant.

Genetic evidence indicates that Oxys-6, an oxygen-sensitive mutant of Escherichia coli AB1157, is defective in the region of the hemB locus. Oxys-6 is capable of growth under aerobic conditions only if cultures are initiated at low-inoculum levels. Aerobic liquid cultures are limited to a cell density of 10(7) cells per ml by the accumulation of a metabolically produced, low-molecular-weight, heat-stable material in complex organic media. Both Oxys-6 and AB1157 cells produce the material, but only aerobic cultures of the mutant are inhibited by it. The material is produced by both intact cells and cell extracts in complex media. This reaction also occurs when the amino acid L-lysine is substituted for complex media.

Chromogenesis mirabilis in Streptomyces griseus.

A number of chromogenic Streptomyces, producing diffusible melanoid pigment on complex organic media, fail to form melanin pigment on conventionally used synthetic tyrosine agar. By means of our new melanin formation test, almost all the chromogenic streptomyces can now be detected in chemically defined medium. In contrast to ordinary chromogenic streptomyces, two streptomyces species of the International Streptomyces Project, S. griseus ISP 5236 and S. ornatus ISP 5307, produce melanin pigment only on synthetic tyrosine agar, without showing chromogenicity on complex organic media. From the results obtained with S. griseus ISP 5236 and S. phaeochromogenes ISP 5073, it was revealed that melanin formation by Streptomyces, in general, is inhibited by L-cysteine present in organic nitrogen sources incorporated into natural media. Most chromogenic species of streptomyces produce a higher level of tyrosinase and rapidly utilize L-cysteine in the culture media which result in the manifestation of good chromogenicity on natural media. Peculiarity of chromogenicity of S. griseus and S. ornatus might be due to the lower ability to produce tyrosinase and to utilize L-cysteine in the culture medium.

Chromogenicity of Streptomyces.

A simplified technique to detect polyphenol oxidase and melanin formation by Streptomyces culture filtrates was developed. The procedure involves the direct assay of pigment formation by the culture filtrate with 3-(3,4-dihydroxyphenyl)-L-alanine (L-dopa) as a substrate. Among cultures of the International Streptomyces Project, 34 failed to produce a diffusible dark brown pigment on peptone-yeast extract-iron-agar and synthetic tyrosine-agar and gave a negative reaction to the melanin formation test. Sixteen cultures produced a diffusible dark brown pigment on both peptone-yeast extract-iron-agar and synthetic tyrosine-agar and gave positive reactions to the test with either L-tyrosine or L-dopa as substrate. Twenty-one cultures produced a diffusible dark brown pigment on peptone-yeast extract-iron-agar, but failed to do so on synthetic tyrosine-agar. Most of these cultures gave a positive reaction to the test when L-dopa was used as the substrate. The correlation between chromogenicity on complex organic media and melanin formation was more clearly established with L-dopa as substrate than with synthetic tyrosine-agar in the present test. The melanin formation test by the present technique, instead of chromogenicity on complex organic media, is recommended as a key feature for the classification of Streptomyces.

Chromogenicity of Streptomyces

A simplified technique to detect polyphenol oxidase and melanin formation by Streptomyces culture filtrates was developed. The procedure involves the direct assay of pigment formation by the culture filtrate with 3-(3,4-dihydroxyphenyl)-L-alanine (L-dopa) as a substrate. Among cultures of the International Streptomyces Project, 34 failed to produce a diffusible dark brown pigment on peptone-yeast extract-iron-agar and synthetic tyrosine-agar and gave a negative reaction to the melanin formation test. Sixteen cultures produced a diffusible dark brown pigment on both peptone-yeast extract-iron-agar and synthetic tyrosine-agar and gave positive reactions to the test with either L-tyrosine or L-dopa as substrate. Twenty-one cultures produced a diffusible dark brown pigment on peptone-yeast extract-iron-agar, but failed to do so on synthetic tyrosine-agar. Most of these cultures gave a positive reaction to the test when L-dopa was used as the substrate. The correlation between chromogenicity on complex organic media and melanin formation was more clearly established with L-dopa as substrate than with synthetic tyrosine-agar in the present test. The melanin formation test by the present technique, instead of chromogenicity on complex organic media, is recommended as a key feature for the classification of Streptomyces.

Chromogenesis mirabilis in Streptomyces griseus

A number of chromogenic Streptomyces, producing diffusible melanoid pigment on complex organic media, fail to form melanin pigment on conventionally used synthetic tyrosine agar. By means of our new melanin formation test, almost all the chromogenic streptomyces can now be detected in chemically defined medium. In contrast to ordinary chromogenic streptomyces, two streptomyces species of the International Streptomyces Project, S. griseus ISP 5236 and S. ornatus ISP 5307, produce melanin pigment only on synthetic tyrosine agar, without showing chromogenicity on complex organic media. From the results obtained with S. griseus ISP 5236 and S. phaeochromogenes ISP 5073, it was revealed that melanin formation by Streptomyces, in general, is inhibited by L-cysteine present in organic nitrogen sources incorporated into natural media. Most chromogenic species of streptomyces produce a higher level of tyrosinase and rapidly utilize L-cysteine in the culture media which result in the manifestation of good chromogenicity on natural media. Peculiarity of chromogenicity of S. griseus and S. ornatus might be due to the lower ability to produce tyrosinase and to utilize L-cysteine in the culture medium.

Patterns of in Vitro Ergot Alkaloid Production by Claviceps paspali and Their Association with Different Growth Rates

An isolate of Claviceps paspali, Ta-1, produced ergot alkaloids after total biomass formation was over (Pattern No. 1) when cultured on complex organic media favoring rapid growth rate (e.g., k > 3...

New Procedures of Bryophyte Culture Which Permit Alteration of the Culture Medium during the Life Cycle

New Procedures of Bryophyte Culture Which Permit Alteration of the Culture Medium during the Life Cycle

Morphogenesis and polymorphism of Ankistrodesmus spp.

Summary: Pure cultures of Ankistrodesmus spp. showed continual morphological fluctuation when growing in complex organic media, and to a lesser (though detectable) extent in mineral media. Clonal populations were no less variable. In mineral media, variation of A. braunii was associated with the stationary phase of growth only, but in the complex organic test medium was seen in logarithmic phase as well. The normal processes governing cell morphogenesis are described for A. braunii. Their normal sequence is disrupted by increasing osmotic pressure of the medium in reverse of their normal order in time and of their hypothetical serial coupling to the metabolism concerned with cell growth. The final uncoupling, that of growth from initiation of cell division, is also accomplished by a wide range of C-N heterocyclic ring structures in very low concentration, which may indicate that nucleic acid metabolism is the system concerned at this point in the sequence.

Antisporulation factors in complex organic media. II. Saturated fatty acids as antisporulation factors.

Antisporulation factors in complex organic media. II. Saturated fatty acids as antisporulation factors.

Antisporulation factors in complex organic media. I. Growth and sporulation studies on Bacillus larvae.

Antisporulation factors in complex organic media. I. Growth and sporulation studies on Bacillus larvae.