广西北部湾茅尾海红树林生境放线菌分离培养基的比较

【背景】红树林生境拥有丰富的微生物资源,分离获得更多的纯培养放线菌为新型抗生素的发现提供菌种资源。【目的】使用新型放线菌分离培养基,发掘广西北部湾茅尾海红树林生境放线菌资源,评估新型放线菌分离培养基分离效果。【方法】设计出新型放线菌分离培养基——椰子汁-海藻糖培养基,对广西北部湾茅尾海红树林根际土壤和红树林根皮放线菌进行分离。基于分子生物学鉴定方法,对获得的纯培养放线菌进行多样性分析。【结果】从红树林根际土壤样品中分离获得65株放线菌,分布在5个目10个科15个属,使用YIM171培养基分离到7个属,MA培养基分离到5个属,椰子汁-海藻糖培养基分离到11个属,其中包括4株潜在新种;从红树林根皮样品中分离获得19株放线菌,分布在4个目7个科10个属,使用YIM171和椰子汁-海藻糖培养基分别分离到7个属,包括4株潜在新种;使用椰子汁-海藻糖培养基在红树林生境两类样品中获得3株潜在新种。【结论】广西北部湾茅尾海红树林根际土壤和根皮中放线菌多样性丰富,使用椰子汁-海藻糖培养基分离放线菌效果较佳,可用作放线菌的分离培养基。     

Selective medium with nalidixic acid for isolating antibiotic-producing Actinomyces

The majority of actinomycetes belonging to various genera proved to be resistant to nalidixic acid concentrations having an inhibitory effect on bacteria with trailing growth i.e. B. subtilis and B. mycoides. The bacteria prevented isolation of actinomycetes as pure cultures. The use of a selective medium with nalidixic acid for isolation of soil actinomycetes resulted in 20 per cent increase in the number of the actinomycetes isolated as pure cultures. Preliminary treatment of the soil samples with calcium carbonate under moist conditions followed by the inoculation to the medium with nalidixic acid made it possible to increase isolation of actinomycetes at most 100-fold. With this complex method 495 actinomycete cultures were isolated, their antibiotic properties were studied and their taxonomic position at the genus level was determined. The complex method including the preliminary treatment of soil samples with calcium carbonate followed by inoculation to the selective medium with nalidixic acid is efficient and may be recommended for screening organisms producing new antibiotics.     

Gastric cancer cell lines AGS before and after CD40 signal activating

The aim of this study was to investigate the molecular mechanisms underlying the antitumour effects of CD40L through analysing the change of genes expression profile in AGS using Affymetrix Gene Chip. Human gastric carcinoma AGS cells were first incubated with 2 μg/ml sCD40L or equal volume of medium (control) in F12 medium. RNA was isolated from AGS and were reverse transcribed, labeled with digoxigenin-11-dUTP, and then hybridized with Clontech Atlas mouse cDNA expression arrays for comparison. Performing clustering analysis, we found that 7 detected genes were down-regulated and 38 were upregulated as the sCD40L acted on AGS. To further verify the results of gene chip screening, Gene Database was searched, finding that the most significantly up-regulated genes were Gadd45a, c-Jun and Bcl-2, and the most significantly down-regulated genes were Cyclin D1, CDC6, TNFR10B, c-IAP2 and ORC5L. Based upon these findings, the signalling pathways that possibly mediate CD40-induced apoptosis are proposed.     

Application of actinomycetes to soil to ameliorate water repellency

AIMS: The aim of this study was to develop a novel isolation technique using a mixture of Bacillus and Streptomyces phages to selectively isolate wax-utilizing non-streptomycete actinomycetes effective in ameliorating water repellency in a problem soil. METHODS AND RESULTS: Phages added to a soil suspension reduced the dominance of Bacillus and Streptomyces isolates and significantly increased the number of non-streptomycete actinomycetes on isolation plates. Promising isolates, grown on a medium containing beeswax as sole carbon source, were selected for application to water repellent soil. Their addition significantly reduced water repellency. CONCLUSIONS: Phage application significantly increased the isolation of non-streptomycete actinomycetes. Wax-utilizing isolates were found to significantly reduce water repellency in a problem soil. SIGNIFICANCE AND IMPACT OF THE STUDY: The phage technique can be used for the routine isolation of non-streptomycete actinomycetes. Beeswax medium can be used to selectively isolate wax-utilizing micro-organisms with the potential to ameliorate water repellency in soil.     

沉默胃泌素基因抑制胃癌细胞的增殖、迁移和侵袭

胃癌细胞分泌的胃泌素与胃癌的发生、发展密切相关.为了探讨胃泌素对胃癌细胞增殖、迁移和侵袭的影响,本文构建靶向胃泌素基因的siRNA表达载体, 转染胃癌细胞AGS, 成功获得沉默胃泌素基因的稳转胃癌细胞株AGS/Gas-siRNA. 用MTT实验、软琼脂集落形成实验、细胞伤愈实验、Transwell实验及ELISA检测沉默胃泌素基因后细胞的增殖、迁移、侵袭及转移相关蛋白基质金属蛋白酶-2（MMP-2）和血管内皮生长因子（VEGF）的含量. 结果显示: 与空载体转染的对照细胞比较, 沉默胃泌素基因的细胞, 其增殖率和克隆形成率显著降低,迁移和侵袭到Transwell下室的细胞数分别降低了31.6 %和34 %. 培养上清液中MMP-2和VEGF含量也低于对照细胞. 结果提示,沉默胃泌素基因的胃癌细胞,通过降低MMP 2和VEGF分泌,抑制了细胞的增殖、迁移和侵袭, 这可能是胃泌素促进胃癌侵袭转移的机制之一.     

A new preferential medium for enumeration and isolation of desert actinomycetes

In order to facilitate the discovery of novel actinomycetes from the Egyptian deserts, which can be useful as new sources for bioactive metabolites, different media for enumeration and isolation of desert actinomycetes have been tested. For this purpose, 30 soil samples from different six sites representing the Western and Eastern deserts of Egypt were collected. The two deserts are considered hyper-arid and the soil characteristics were determined. The media used were glucose–yeast extract agar, soil extract agar and a new minimal medium (MM) containing glucose, yeast extract and mineral salts. The effects of the soil characteristics on the total viable actinomycete counts on the three media were evaluated. The results showed that the highest actinomycete count in samples from five out of six sites was obtained on MM. Also MM was more selective for actinomycetes and significantly decreased the number of fungal colonies and to a lower extent the number of bacterial colonies. Moreover, it supported the development of different and diverse groups of actinomycetes. From the results obtained in this study, MM is a new useful medium for enumeration and selective isolation of actinomycetes from the desert soils.     

A new method for the selective isolation of actinomycetes from soil

Summary A coal-vitamin medium was developed to isolate actinomycetes from soil, which was superior to other currently used media. It increased the number of actinomycetes and inhibited the growth of other soil bacteria. The pretreatment of soil suspension with peptone (6%) and lauryl sulfate (0.05%) at 50°C for 10 min, also greatly increased the number of actinomycetes from soil prior to incubation with new medium.     

微波处理对土壤放线菌分离效果的影响

采用稀释平皿分离测数及琼脂块法研究了微波预处理对土壤放线菌分离效果的影响.结果表明: 1)微波处理能显著增加可培养放线菌数量.随着微波处理时间的增加,高有机质土壤放线菌总数呈先增后减的趋势.处理3～15 min,GA及HA培养基上放线菌总数分别较对照提高8.3%～92.6%及24.4%～108.5%;处理18～24 min,放线菌总数分别较对照降低了62.1%～78.8%及41.4%～79.8%.微波处理对低有机质土壤放线菌数量无明显影响.2)微波处理对可培养放线菌种类有明显影响.随着微波处理时间的增加,高有机质土壤中新出现的放线菌种类呈先增后减趋势. 处理3～24 min,GA与HA培养基上新出现放线菌种类占放线菌总种类的比例为62.5%～85.7%与66.7%～83.3%,新出现了原小单孢菌属、链轮丝菌属等稀有放线菌属;链霉菌类群也有明显变化.低有机质土壤具有类似趋势.3)微波处理对可培养放线菌中拮抗性放线菌株数占供试放线菌总株数的比例也有明显影响.微波处理6、9和15 min,拮抗性放线菌株数所占比例分别较对照提高66.7%、66.7%和83.3%,其中新出现的拮抗性放线菌株数分别占拮抗性放线菌总株数的70.0%, 90.0%和81.8%.     

Fatty acids of actinomycete phospholipids

The fatty acid composition of phospholipids was studied in different actinomycetes growing in two media in order to detect their biological activity. The total phospholipids of the actinomycetes did not differ qualitatively and their composition was represented by the same series of fatty acids (C13--C19). The qualitative composition and the quantitative content of fatty acids in phospholipids depended on the composition of the growth medium. When the actinomycetes were cultivated in a complex medium, the proportion between fatty acids (C14:0, C14:1, C16:0, C17:0, C17:1, C18:1, C18:2) changed. The qualitative composition of fatty acids in phospholipids varied among the cultures. However, the content of palmitoleic, palmitic and oleic acids was elevated in all of the cultures. Under the given experimental conditions, the actinomycetes were found to synthesize phospholipids containing fatty acids with a high degree of unsaturation (mainly at the account of C16:1 and C18:1 acids).     

Isolierung und Zählung von Bodenactinomyceten auf Erdplatten mit Membranfiltern

Summary A technique is described to separate actinomycetes from other micro-organisms with membrane filters on soil plates. A measured quantity of soil dilution was passed through a membrane filter and the filter with the adhering organisms was placed upside down on a medium provided with soil. After an incubation period of two weeks all actinomycetes had penetrated the filter developing colonies on the sterile surface, whereas the other organisms were retained by the filter. By this procedure the counting and isolation of actinomycetes has been facilitated. The selection of bacteriostatic active forms is practicable by stamping the crowded filter on a medium that contains test bacteria.

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Various principles of detecting the producers of beta-lactamase inhibitors among soil Actinomycetes

Principles of detecting organisms producing beta-lactamase inhibitors among soil actinomycetes were developed. For detecting such cultures it was recommended to use the Gauze agarized medium No. 1 supplemented with beta-lactam antibiotics. Benzylpenicillin proved to be the most efficient. Various liquid fermentation media for detecting the inhibitory activity of soil actinomycetes were compared. Two media were the most favourable i.e. the glucose-yeast medium No. 18/3 and the soybean-glucose medium with Na2SO4 and CoCl2 No. 20/3. The use of test cultures with relatively low resistance to benzylpenicillin was shown expedient in screening cultures producing beta-lactamase inhibitors. Test cultures with high resistance should be used in more detailed characterization of the selected cultures.     

In vitro infection of Cryptosporidium parvum to four different cell lines

To determine a suitable condition for in vitro infection model of Cryptosporidium parvum, four different cell lines, AGS, MDCK, HCT-8 and Caco-2, were used as host cell lines which were cultured at various concentrations of added supplements. These supplement include fetal bovine serum (FBS), sodium choleate, ascorbic acid, folic acid, calcium pantothenate, para-aminobenzoic acid and pyruvate and their effects on the cell lines which were infected with C. parvum were evaluated. The results of this study showed that the AGS cell line was most susceptible to C. parvum whereas the Caco-2 cells appeared to be least susceptible to C. parvum. In regards to the serum condition, 10% FBS was suitable for the growth of AGS and HCT-8 cells, and 1% FBS was good for the growth of the MDCK cells when they were inoculated with C. parvum. Vitamins had a positive effect on the AGS cells, and pyruvate also showed positive effects on all of the cell lines except for Caco-2. Modified medium for each cell line was prepared by adding appropriate amounts of each supplement which resulted in the highest parasite infection number. Modified media increased the number of parasites infected on AGS cells to 2.3-fold higher when compared to the control media. In this study, we found that the AGS cell line was a suitable host model for evaluating C. parvum in vitro study and the media contents for the optimal infection conditions were suggested.     

Novel method for selective isolation of actinomycetes

A new technique for the selective isolation of actinomycetes from natural mixed microbial populations is described. A nutrient agar medium was overlaid with a 0.22- to 0.45-microns-pore cellulose ester membrane filter, and the surface of the filter was inoculated. During incubation, the branched mycelia of the actinomycetes penetrated the filter pores to the underlying agar medium, whereas growth of nonactinomycete bacteria was restricted to the filter surface. The membrane filter was removed, and the agar medium was reincubated to allow the development of the isolated actinomycete colonies. This procedure selects actinomycetes on the basis of their characteristic mycelial mode of growth, offers a general method for their selective isolation, and does not rely on the use of special nutrient media or of antibacterial antibiotics.     

Novel method for selective isolation of actinomycetes.

A new technique for the selective isolation of actinomycetes from natural mixed microbial populations is described. A nutrient agar medium was overlaid with a 0.22- to 0.45-microns-pore cellulose ester membrane filter, and the surface of the filter was inoculated. During incubation, the branched mycelia of the actinomycetes penetrated the filter pores to the underlying agar medium, whereas growth of nonactinomycete bacteria was restricted to the filter surface. The membrane filter was removed, and the agar medium was reincubated to allow the development of the isolated actinomycete colonies. This procedure selects actinomycetes on the basis of their characteristic mycelial mode of growth, offers a general method for their selective isolation, and does not rely on the use of special nutrient media or of antibacterial antibiotics.     

Oligotrophy is Helpful for the Isolation of Bioactive Actinomycetes

It is necessary to develop new methods for the isolation of unknown actinomycetes from soils. To evaluate the effects of oligotrophic medium on the isolation of soil actinomycetes and develop a new isolation method, the Gause’s synthetic medium was diluted to one tenth the recommended concentration in the present study. Soil dilution plate technique was used to isolate actinomycetes from the soil samples. Oligotrophy decreased actinomycete and streptomycete counts, as well as the number of antagonistic actinomycete species. Oligotrophy also decreased the number of actinomycete species in five samples. Some actinomycete species were cultured only on the oligotrophic medium, whereas other species could not be cultured. Oligotrophy decreased actinomycete counts more significantly for soils with organic matter content >40 g/kg. We used 16S rRNA sequence analysis to identify 22 actinomycete species that were only cultured on the oligotrophic medium. Oligotrophic medium was helpful for the isolation of Streptomyces spp., Micromonospora spp. and Streptosporangium spp. Slightly more than 80 % of the identified actinomycete species were biologically active. Therefore, we could draw a conclusion that oligotrophic medium could be helpful for the discovery of new antibiotic producers and the exploitation and utilization of new, biologically active compounds.     

The production of auxins by the endophytic bacteria of winter rye

The ability of the actinomycetes and coryneform bacteria isolated from the root tissues of winter rye to produce auxin in a liquid culture was studied. The isolates of coryneform bacteria produced indolyl-3-acetic acid (IAA) into the medium in the amount of 9.0–95.0 μg/ml and the isolates of actinomycetes in the amount of 39.5–83.0 μg/ml. The maximal IAA accumulation in culture liquid of actinomycetes coincided, in general, with the beginning of the stationary growth phase. The dependences of IAA synthesis by actino-mycetes on the composition and pH of nutrient medium, tryptophan concentration, and aeration conditions were determined. Biological activity of the bacterial IAA was assessed. Treatment of winter rye seeds with coryneform auxin-producing bacteria increased the germination capacity and enhanced an intensive seedling growth in vitro.     

邳州银杏内生放线菌分离、筛选及活性菌株鉴定

【目的】从银杏中分离、筛选得到具有抑菌作用的内生放线菌,为放线菌在生物防治上的应用提供新的菌种资源。【方法】采用组织贴片培养法进行分离,生长对峙法进行筛选。【结果】从银杏的根、茎、叶中分离得到98株、50株、8株内生放线菌(共计156株),47株放线菌具有拮抗植物病原真菌活性。菌株KLBMP 5501抗菌活性最好且具有广谱性,基于形态特征、培养特征、生理生化特征和16S rRNA基因序列的相似性分析等多项分类特征表明,菌株KLBMP 5501是一株浅紫链霉菌(Streptomyces violascens)。【结论】筛选得到了具有应用潜力的高活性菌株,并进行了菌种鉴定。     

Screening of marine actinomycetes isolated from the Bay of Bengal,India for antimicrobial activity and industrial enzymes

A total of 288 marine samples were collected from different locations of the Bay of Bengal starting from Pulicat lake to Kanyakumari, and 208 isolates of marine actinomycetes were isolated using starch casein agar medium. The growth pattern, mycelial coloration, production of exopolysaccharides and diffusible pigment and abundance of Streptomyces spp. were documented. Among marine actinomycetes, Streptomyces spp. were present in large proportion (88%). Among 208 marine actinomycetes, 111 isolates exhibited antimicrobial activity against human pathogens, and 151 showed antifungal activity against two plant pathogens. Among 208 isolates, 183, 157, 116, 72 and 68 isolates produced lipase, caseinase, gelatinase, cellulase and amylase, respectively. The results of diversity, antimicrobial activity and enzymes production have increased the scope of finding industrially important marine actinomycetes from the Bay of Bengal and these organisms could be vital sources for the discovery of industrially useful molecules/enzymes.     

Isolation of Endophytic Streptomyces Strains from Surface-Sterilized Roots

When the roots of 28 plant species were surface sterilized and incubated on agar medium, endophytic actinomycetes in the root cortex were observed by direct microscopic observation and pure culture techniques.     

Growth and morphological differentiation of acidophilic and neutrophilic soil streptomyces

Acidophilic actinomycetes are shown to possess a special mechanism of adaptation to low pH conditions, which shows up in their ability to grow faster on acidified than on neutral media and to adjust the pH of the medium to a level favorable for the formation of aerial mycelium and spores. On nutrient media with pH below 5, neutrophilic actinomycetes either fail to grow or grow much slower than on neutral media; they do not alkalize the medium and do not form aerial mycelium.