Liquid Serial Dilution Is Inferior to Solid Media for Isolation of Cultures Representative of the Phylum-Level Diversity of Soil Bacteria

Representatives of only four well-characterized bacterial phyla were isolated from a pasture soil by using liquid serial dilution culture. In contrast, members of Acidobacteria, Verrucomicrobia, and Gemmatimonadetes and of other poorly represented bacterial lineages were isolated in earlier experiments with solidified versions of the same media. We conclude that, contrary to expectation, liquid serial dilution culture is inferior to culturing on solid media for isolating representatives of many bacterial phyla from soil.     

Effect of bacterial satellites on Chlamydomonas reinhardtii growth in an algo-bacterial community

The growth characteristics of an algo-bacterial community (Chlamydomonas reinhardtii and bacterial satellites) were studied, as well as the mechanism and patterns of bacterial effect on algae. Four strains of predominant bacteria were isolated and partially characterized. They were assigned to the following taxa: Rhodococcus terrea, Micrococcus roseus, and Bacillus spp. A pure culture of the alga under study was obtained by plating serial dilutions on agarized media with ampicillin. Within the algo-bacterial association, the alga had a higher growth rate (0.76 day(-1)) and yield (60 microg chlorophyll/ml culture) than in pure cultures (0.4 day(-1) and 10 microg chlorophyll/ml culture, respectively). The viability of the algal cells within the association was retained longer than in pure culture. Among the isolated bacterial satellites, strains B1 and Y1, assigned to the species Rhodococcus terrae, had the highest stimulatory effect on algal growth. The culture liquid of bacteria incubated under the conditions not permitting growth stimulated algal growth; the culture liquid of actively growing bacteria had an opposite effect.     

一株产冠菌素新菌种的分离与鉴定

【目的】从不同样本中分离筛选性能稳定的产冠菌素菌株。【方法】根据冠菌素引起植物叶片产生弥散性黄萎病、块茎膨大的特性,采集各种植物病叶、病枝及感病植物的土壤,采用穿刺法与系列稀释法分离筛选菌株;液相色谱测定菌株产生的冠菌素;在电子和光学显微镜下观察菌体形态;根据生理生化试验、(G+C)mol%含量、16S rDNA序列分析等对菌株进行鉴定;对分离提纯的发酵产物进行紫外、质谱和红外分析。【结果】菌株BBC933为革兰氏阴性菌,端生鞭毛,短杆状,无芽孢。在41℃下不生长,细胞内有聚β-羟基丁酸盐颗粒积累,没有精氨酸双水解酶和氧化酶,不能水解淀粉、明胶,不进行硝酸还原及反硝化作用,过氧化氢酶反应呈阳性。菌株的(G+C)mol%含量为67.2%,根据该菌株16S rDNA序列的同源性分析,构建系统发育树。【结论】菌株BCC933鉴定为洋葱伯克霍尔德氏菌(Burkholder cepacia),具有产冠菌素性能。国内外未曾见报道洋葱伯克霍尔德氏菌产冠菌素。     

Effect of bacterial satellites on Chlamydomonas reinhardtii growth in an algo-bacterial community

The growth characteristics of an algo-bacterial community (Chlamydomonas reinhardtii and bacterial satellites) were studied, as well as the mechanism and patterns of bacterial effect on algae. Four strains of predominant bacteria were isolated and partially characterized. They were assigned to the following taxa: Rhodococcus terrea, Micrococcus roseus, and Bacillus spp. A pure culture of the alga under study was obtained by plating serial dilutions on agarized media. Within the algo-bacterial association, the alga had a higher growth rate (0.76 day^?1) and yield (60 μg chlorophyll/ml culture) than in pure cultures (0.4 day^?1 and 10 μg chlorophyll/ml culture, respectively). The viability of the algal cells within the association was retained longer than in pure culture. Among the isolated bacterial satellites, strains B1 and Y1, assigned to the species Rhodococcus terrae, had the highest stimulatory effect on algal growth. The culture liquid of bacteria incubated under the conditions not permitting growth stimulated algal growth; the culture liquid of actively growing bacteria had an opposite effect.     

Characterization and identification of numerically abundant culturable bacteria from the anoxic bulk soil of rice paddy microcosms.

Most-probable-number (liquid serial dilution culture) counts were obtained for polysaccharolytic and saccharolytic fermenting bacteria in the anoxic bulk soil of flooded microcosms containing rice plants. The highest viable counts (up to 2.5 x 10(8) cells per g [dry weight] of soil) were obtained by using xylan, pectin, or a mixture of seven mono- and disaccharides as the growth substrate. The total cell count for the soil, as determined by using 4', 6-diamidino-2-phenylindole staining, was 4.8 x 10(8) cells per g (dry weight) of soil. The nine strains isolated from the terminal positive tubes in counting experiments which yielded culturable populations that were equivalent to about 5% or more of the total microscopic count population belonged to the division Verrucomicrobia, the Cytophaga-Flavobacterium-Bacteroides division, clostridial cluster XIVa, clostridial cluster IX, Bacillus spp., and the class Actinobacteria. Isolates originating from the terminal positive tubes of liquid dilution series can be expected to be representatives of species whose populations in the soil are large. None of the isolates had 16S rRNA gene sequences identical to 16S rRNA gene sequences of previously described species for which data are available. Eight of the nine strains isolated fermented sugars to acetate and propionate (and some also fermented sugars to succinate). The closest relatives of these strains (except for the two strains of actinobacteria) were as-yet-uncultivated bacteria detected in the same soil sample by cloning PCR-amplified 16S rRNA genes (U. Hengstmann, K.-J. Chin, P. H. Janssen, and W. Liesack, Appl. Environ. Microbiol. 65:5050-5058, 1999). Twelve other isolates, which originated from most-probable-number counting series indicating that the culturable populations were smaller, were less closely related to cloned 16S rRNA genes.     

Diversity in soil fungi from undisturbed and disturbed Celtis tala and Scutia buxifolia forests in the eastern Buenos Aires province (Argentina)

The rhizospheric soil microfungi from a native forest (undisturbed and disturbed) were studied using soil dilution plate and soil washing methods. Fungi were isolated using slightly acid and alkaline culture media. 54 taxa were isolated: 49 from undisturbed forest soil and 37 from disturbed forest soil. Acremonium sp., Aspergillus ustus, Coemansia pectinata, Doratomyces stemonitis, Fusarium solani, F. oxysporum, Gliocladium roseum, Humicola fusco-atra, Mortierella sp., Penicillium lilacinum, Trichoderma harzianum, and T koningii, showed the highest frequency, in both, undisturbed and disturbed forests. In undisturbed soil forest the biodiversity index was 3.97 whereas in disturbed ones was 3.89.     

Glyphosate utilization byPseudomonas sp. andAlcaligenes sp. isolated from environmental sources

A total of 21 bacterial cultures were isolated that could utilize glyphosate (N-phosphonomethyl glycine) as a sole source of phosphorus in a mineral salts medium. Sources of inocula for enrichment cultures included aerobic digester liquid, raw sewage, trickling filter effluent, pesticide disposal pit liquid, and soil. Eleven cultures were identified asPseudomonas sp., one asPseudomonas stutzeri, and nine asAlcaligenes sp. Aminomethylphosphonic acid, the major metabolic intermediate of glyphosate degradation in soil, could also serve as a sole phosphorus source for all 21 isolates. Neither glyphosate nor aminomethylphosphonic acid could serve as carbon sources in mineral salts media. Experiments withPseudomonas sp. SG-1 (isolated from aerobic digester liquid) suggested that enzymatic activity responsible for glyphosate degradation was intracellular, inducible, and required the cofactors pyruvate and pyridoxal phosphate. The degradation pathway for glyphosate in this culture may be similar to that previously reported for aminoethylphosphonic acid.     

Recovery of somatic embryos and plantlets from protoplast cultures of Larix × eurolepis

Summary Somatic embryos and plantlets were regenerated from protoplasts of hybrid larch (Larix × eurolepis) isolated from two embryogenic callus and cell suspension culture lines (L1 and L2). L2, which was highly embryogenic, consistently yielded protoplasts that gave rise to somatic embryos. Centrifugation on a discontinuous medium/Percoll density gradient resulted in accumulation of embryogenic protoplasts in one of the Percoll interfaces. First division frequencies were in the range of 28–39% in line 1 and 18–20% in line 2 in both liquid and agarose-solidified culture media. The critical factor in maintaining high viability of cultures was lowering of osmotic pressure by dilution of the initial medium. The first somatic embryos were detected in 23- to 28-day-old cultures. Some of these developed into plants that were transferred to soil.     

Growth of an Ethane-utilizing Mixed Culture in a Chemostat

Bacteria capable of utilizing ethane as their sole source of carbon and energy at 42° were readily isolated from soil samples obtained from Brunei with batch culture enrichment techniques. No ethane utilizers capable of growth at 42° were isolated, however, from soil samples obtained from Britain. An ethane-utilizing mixed culture was grown under ammonia limitation in chemostat culture. The lipid content of the culture was inversely proportional to the dilution rate. Similarly, the amount of organic carbon excreted into the culture supernatant was inversely proportional to the growth rate and could be accounted for mainly as carbohydrate. The specific rates of ethane and oxygen consumption, carbon dioxide production and yield from ethane were directly proportional to the dilution rate.     

Milk-Clotting Enzymes From Microorganisms

A total of 230 cultures of fungi and 43 cultures of bacteria, isolated from such sources as soil, butter, and milk, were screened for their milk-clotting activity. The fungi were cultivated on semisolid media, and the bacteria were grown in milk media in shake culture. Phytic acid, added as calcium phytate, was found to stimulate production of the enzyme in most of the bacterial isolates. Proteolytic activity was invariably found to be associated with the milk-clotting enzyme in bacterial isolates. There was considerable variation in the ratio of the two enzymes from strain to strain.     

Optimization of culture media for enhanced chitinase production from a novel strain of Stenotrophomonas maltophilia using response surface methodology

Chitinase is one of the most important mycolytic enzymes with industrial significance. This enzyme is produced by a number of organisms including bacteria. In this study we describe optimization of media components with increased production of chitinase for selected bacteria Stenotrophomonas maltophilia isolated from the soil. Different components of the defined media responsible for influencing chitinase secretion by the bacterial isolate were screened using Plackett-Burman experimental design and were further optimized by Box-Behnken factorial design of response surface methodology (RSM) in liquid culture. Maximum chitinase production was predicted in medium containing chitin 4.94 g/l, maltose 5.56 g/l, yeast extract 0.62 g/l, KH2PO4 1.33 g/l and MgSO4.7H2O 0.65 g/l using Response surface plots and point prediction tool of DESIGN EXPERT 7.1.6 (Statease, USA) software.     

盐度对稀释平板法研究红树林区土壤微生物数量的影响

在使用稀释平板法分离潮间带红树林及其对照光滩土壤微生物以及计数时,多数情况下使用陈海水制作培养基和稀释水,很少考虑培养基和稀释水的盐度对最终计数结果的影响.使用稀释平板法研究了盐度对福建九龙江口红树林区与深圳福田红树林保护区土壤微生物平板计数的影响,结果表明培养基与稀释水盐度对微生物数量有明显的影响.统计分析显示细菌的海水稀释效果优于淡水,而放线菌与真菌则刚好相反(P<0.05,一个例外).海水不适合配制红树林区土壤微生物平板计数的培养基,从0～35,高盐度的平板培养基会降低微生物的数量,尤其是放线菌的数量,尽管培养基的盐度对真菌影响无规律,但细菌数量在低盐度时比在高盐度和不加氯化钠时要多.根据盐度效应,提出了稀释平板技术应用于潮间带的红树林及其相应光滩时的优化方法,认为细菌应该用海水作无菌稀释水,而放线菌和真菌则应用淡水作稀释水;包括光滩在内的红树林区土壤微生物分离与计数的培养基宜控制较低盐度范围.     

Bacterial competition in serial transfer culture

A mathematical model of bacterial competition for a single growth-limiting substrate in serial transfer culture is formulated. Each bacterial strain is characterized by a growth response function, e.g. Monod function determined by a maximum growth rate and half-saturation nutrient concentration, and the length of its lag phase following the dilution event. The goal of our study is to understand what factors determine an organisms fitness or competitive ability in serial transfer culture. A motivating question is: how many strains can coexist in serial transfer culture? Unlike competition in the chemostat, coexistence of two strains can occur in serial transfer culture. Numerical simulations suggest that more than two may coexist.     

Two unidentified aerobic bacterial strains that transform 2,4,6-trinitrotoluene

Several strains of aerobic bacteria tolerant to some chemical toxins were isolated from the water of man-made Yerevan Lake and from the soil and air of different regions of the city of Yerevan. Some of these bacteria were capable of degrading 2,4,6-trinitrotoluene (TNT) during shake-flask fermentation in liquid medium. Two bacterial strains with good ability to degrade TNT were isolated. These strains showed visible morphological and physiological differences during growth on numerous elective media. The comparative ability of these strains to transform TNT was investigated.     

High frequency production of embryos inDatura innoxia from isolated pollen grains by combined cold treatment and serial culture of anthers in liquid medium

Summary This study concerns the development of pollen embryos as affected by various physical conditions of culture in media devoid of hormones. Freshly isolated pollen, from anthers ofDatura, failed to form embryos regardless of whether they were cultured on liquid or solid medium. In contrast, pollen isolated from anthers precultured on solid medium did form embryos and the response could be increased by prior cold treatment of anthers at 4 °C for 4 days. However, the best results were obtained when anthers were cultured from the very beginning in liquid medium and transferred serially to fresh medium. Under such conditions, the anthers dehisced, allowing spontaneous shedding of pollen grains. It was thus possible to have several fractions of shed pollen continuing their development into embryos. When serial culture was started with anthers from cold-treated buds not only were embryos formed in all the fractions of shed pollen but the frequency was also considerably higher than in any mode of culturing.     

Bacterial Community Composition in Brazilian Anthrosols and Adjacent Soils Characterized Using Culturing and Molecular Identification

Microbial community composition was examined in two soil types, Anthrosols and adjacent soils, sampled from three locations in the Brazilian Amazon. The Anthrosols, also known as Amazonian dark earths, are highly fertile soils that are a legacy of pre-Columbian settlement. Both Anthrosols and adjacent soils are derived from the same parent material and subject to the same environmental conditions, including rainfall and temperature; however, the Anthrosols contain high levels of charcoal-like black carbon from which they derive their dark color. The Anthrosols typically have higher cation exchange capacity, higher pH, and higher phosphorus and calcium contents. We used culture media prepared from soil extracts to isolate bacteria unique to the two soil types and then sequenced their 16S rRNA genes to determine their phylogenetic placement. Higher numbers of culturable bacteria, by over two orders of magnitude at the deepest sampling depths, were counted in the Anthrosols. Sequences of bacteria isolated on soil extract media yielded five possible new bacterial families. Also, a higher number of families in the bacteria were represented by isolates from the deeper soil depths in the Anthrosols. Higher bacterial populations and a greater diversity of isolates were found in all of the Anthrosols, to a depth of up to 1 m, compared to adjacent soils located within 50–500 m of their associated Anthrosols. Compared to standard culture media, soil extract media revealed diverse soil microbial populations adapted to the unique biochemistry and physiological ecology of these Anthrosols. The author J. Peterson is already deceased.     

Microorganisms--degraders of polychlorinated biphenyls

Four strains belonging to the genus Bacillus, capable of degrading polychlorinated biphenyls (PCB), were isolated by screening the collection strains of soil bacteria, degrading a organochlorine pesticide, hexachlorocyclohexane (HCCH). A method for production of tritium-labeled PCB was developed. Consumption and degradation of PCB by the soil bacterial strains selected were studied using tritium-labeled PCB and GLC. It was demonstrated that PCB are degradable both in culture media and under in model soil samples.     

不同培养基组合提高土壤细菌可培养性的研究

为选择性采用多培养基组合以提高土壤细菌可培养性,利用变性梯度凝胶电泳(DGGE)技术研究了贫营养、富营养和自然营养培养基在3种培养方式下获得细菌种群的差异。结果表明:平板培养条件下,细菌在贫营养培养基上生长较慢,菌落连续稳定形成。培养5d后,富营养的LB培养基和贫营养的R2A培养基获得菌落数最多,分别是贫营养的0.1×LB培养基获得菌落数的5.1倍和5.3倍。7种培养基中,LB培养基获得细菌种群数目最多,营养成分适当稀释后,培养物中有新的种群出现。贫营养培养基和富营养培养基培养物DGGE图谱相似性低,条带互补性强。三角瓶静置培养时,R2A和LB培养基获得细菌种群数目较多,其它几种培养基获得的细菌类群都能在这2种培养基中找到。试管静置培养条件下,LB培养基获得细菌种群数目最多,某些种群也只出现在R2A培养基和TSB培养基上,R2A及LB培养基与TSB培养基获得的细菌种群差异较为明显。研究结果为特殊培养基设计及选用合适培养基分离土壤细菌提供参考。     

陕西定边盐湖嗜盐菌A393的分离及其种属鉴定

采用高盐选择性培养基和稀释平板法,从陕西定边盐湖土壤样本中,分离筛选获得嗜盐菌株A393,通过形态学观察、生理生化特征和系统发育学16S rDNA序列分析鉴定嗜盐菌株A393分类学地位。获得的A393最适生产盐浓度在8%～20%。表型特征和16S rDNA序列分析结果初步鉴定其为中度嗜盐菌,属于海球菌属(Marinococcus sp.)菌株。     

Distribution of Burkholderia cepacia phenotypes by niche, method of isolaiton and pathogenicity of onion

Several proposals have been made for the subdivision of the bacterial species Burkholderia cepacia. Data applying these schemes to a collection of 188 strains of B. cepacia, isolated using a variety of methods, from soils, decayed onions, and nosocomial sources are presented. None of the schemes could be shown to be associated with the method of isolation, niche from which a strain was isolated, pectolytic activity, or pathogenicity to onion. Strains isolated by serial dilution on onion slices were much more uniform than were strains isolated using semiselective media. The nutritionally stringent medium of Lumsden and Sasser (US Patent No. 4 588 584) was judged better for the collection of a phenotypically broad sample of B. cepacia than those of Hagedorn, Gould, Bardinelli & Gustavson (1987) and Wu & Thompson (1984), which are based on antibiotic insensitivity. Pectolytic activity and pathogenicity to onion were shown to be highly correlated characters. None of the strains of clinical origin was capable of macerating onion tissue.