A practical method of Ludox density gradient centrifugation combined with protargol staining for extracting and estimating ciliates in marine sediments

Methodological impediments have long been the main problem in estimating the ecological role of marine benthic ciliates. Percoll density centrifugation is currently the most efficient technique for extracting ciliates from fine-grained sediments, while the high cost and low density of Percoll limit its wide application. We developed a protocol of density gradient centrifugation using the cheap sol Ludox HS 40 in combination with the quantitative protargol stain (QPS) to enumerate and identify marine benthic ciliates. The combined Ludox-QPS method involves sample collection and salt reduction, extraction with Ludox centrifugation, and preparation with the QPS technique. The recovery efficiency of Ludox was first tested with azoic sandy and muddy sediments. A 94–100% recovery rate of ciliates was reached. The method was further tested with natural sandy, muddy-sand and muddy sediments. Excellent extraction efficiencies were consistently obtained: an average of 97.6% for ciliates in sand, and 96.9–97.8% for nematodes in the three types of sediments. The high efficiencies indicate that the method allows for simultaneous enumeration of micro- and meiobenthos. Advantages of the new method include: (i) reliable and cost-efficient operation; (ii) appropriate centrifugation for both micro- and meiobenthos; and (iii) applicability to large samples and routine ecological surveys.     

Morphological and genetic characterization of a cultivated Cordyceps sinensis fungus and its polysaccharide component possessing antioxidant property in H22 tumor-bearing mice

Cordyceps sinensis, one of the most precious traditional Chinese medicines, possesses the antitumor activity, antioxidant activity and the capability of modulating the immune system. In the present study, a fungus strain G1 isolated from wild C. sinensis was identified and initially characterized. A phylogenetic tree was generated based on the sequences of the internal transcribed spacer (ITS) region of related fungi. The analysis of ITS sequence showed that fungus G1 was clustered together with C. sinensis, Tolypocladium cylindrosporum and Tolypocladium inflatum in the phylogenetic tree. Both the morphological character and the ITS sequence analysis establish that fungus G1 is one of the anamorph strains of C. sinensis and belongs to Tolypocladium genus. Furthermore, the polysaccharide (PS) extracted from fungus G1 and its antioxidant activity on H22-bearing mice was investigated. H22 cells were hypodermically injected into the right oxter of each mouse after the ICR mice were treated with PS by means of gavage for 7 days. Then the same administration process continued for 9 days. At the end of the experiments, the tumor weight of each mouse was measured. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) level in mouse liver, brain and serum, as well as glutathione peroxidase (GSH-Px) activity in mouse liver and brain were assayed. The results showed that the H22 tumor growth was significantly inhibited by PS. Moreover, PS significantly enhanced SOD activity of liver, brain and serum as well as GSH-Px activity of liver and brain in tumor-bearing mice. PS also significantly reduced the level of MDA in liver and brain of tumor-bearing mice.     

侵染莜麦的大麦黄矮病毒的分离及提纯

用液氮或低温冷冻染病组织及差速离心和葡聚糖凝胶过滤的方法,提纯了自然侵染莜麦的大麦黄矮病毒(BYDV)。电镜照片中病毒呈多面体结构,大小均一,平均直径为23.66±1.2nm,且聚集较多。将提纯的病毒以三种无毒蚜为介体,经薄膜饲毒法回接到华北二号莜麦上,表明提纯的病毒能引起类似田间病株的症状。     

云南省两株环状病毒的分离和鉴定

从云南省采集的中华(?)蚊和棕头库蚊中,分离到两株对3周鼠致死的病毒。血清学鉴定表明,该病毒与披膜病毒科(甲病毒属)、黄病毒科、布尼亚病毒科病毒的免疫腹水,以及呼肠孤病毒科环状病毒(M14)的免疫腹水均不发生反应。电镜观察病毒为球形,具双层壳体的颗粒,直径为70.35±3.07nm,毒粒常常和颗粒状包涵体及管状结构相连;核衣壳具有环状病毒特有的20面体对称结构,直径为56.06±2.42nm。在负染标本中能观察到直径为38.36±2.42nm的核心颗粒。该病毒对乙醚相对抵抗,对酸,热敏感。聚丙烯酰胺凝胶电泳分析,病毒基因组由10条RNA片段组成,分子量在0.3～2.5×10~6道尔顿之间,病毒基因组RNA带形分布(3-3-3-1)类似环状病毒,但与已知环状病毒RNA带形分布都有不同。     

1200条带阶段的人类染色体高分辨G带

在改良的850条带阶段的人类染色体高分辨显带技术基础上,对1200条带阶段的人类染色体高分辨G带进行了研究和识别,并按ISCN(1985)的规定对1200条带阶段的高分辨G带进行了命名和划分。     

临床厌氧菌的分离和鉴定

我们从口腔、胸部、腹部和盆腔等处采集了110份临床感染标本,以自制的输送培养基立即送实验室,在厌氧手套箱(霍尔玛厌氧系统1029型)或Gas-pak罐中,行厌氧菌的分离和培养,其中64份标本检出厌氧菌,阳性率为58％。临床标本中牙周炎标本厌氧菌检出率高达100％,牙髓炎标本为85％,阑尾脓肿和腹膜炎标本为83％,胆道标本为39％,脓胸标本为57％,盆腔标本为33％,早期单纯性阑尾炎和甲状腺囊肿合并感染的标本各5份,都未检出厌氧菌。从64份阳性分离的标本中共分离到厌氧菌370株,经鉴定分别属于11个菌属32个菌种(未定种的有74株),其中类杆菌最多占45.9％(类杆菌属中脆弱类杆菌占37.6％),次为梭杆菌属和消化链球菌属,各占15.1％。革兰氏阳性无芽胞厌氧菌占8％左右,而梭菌属为8.9％,其余是二氧化碳噬纤维菌属(2.9％)、韦荣氏球菌属(1.3％)、链球菌属和纤毛菌属(1.5％)等。在厌氧菌鉴定中,我们使用了微量生化直接酶测定技术和代谢产物的气相色谱分析技术,这些方法在厌氧菌鉴定中比常规方法敏感且有较大的价值。     

普通结瘤基因探针鉴定根瘤菌的研究

普通结瘤基因（ｎｏｄＡＢＣ）是所有根瘤菌所特有的、最为保守的基因,用苜蓿根瘤菌结瘤基因（ｎｏｄＡＢＣ）和豌豆根瘤菌的（ｎｏｄＣ）基因片段为探钉,与５２株包括常见土壤细菌、已知根瘤菌、根瘤未知分离物的总ＤＮＡ进行斑点杂交,探索用普通结瘤基因（ｎｏｄＡＢＣ）或（ｎｏｄＣ）探针鉴定根瘤菌的可能性。结果,未找到合适实验条件,使来自这两个种的结瘤基因只能与根瘤菌菌株杂交,而不与土壤细菌的菌株杂交。但在高温条件下,两种探针都专一性的和种内菌株杂交。此结果表明：在一定的实验条件下,普通结瘤基因探针用做根瘤菌的鉴定,只能     

Bacterial phylogeny based on 16S and 23S rRNA sequence analysis

Abstract: Molecular phylogeny increasingly supports the understanding of organismal relationships and provides the basis for the classification of microorganisms according to their natural affiliations. Comparative sequence analysis of ribosomal RNAs or the corresponding genes currently is the most widely used approach for the reconstruction of microbial phylogeny. The highly and less conserved primary and higher order structure elements of rRNAs document the history of microbial evolution and are informative for definite phylogenetic levels. An optimal alignment of the primary structures and a careful data selection are prerequisites for reliable phylogenetic conclusions. rRNA based phylogenetic trees can be reconstructed and the significance of their topologies evaluated by applying distance, maximum parsimony and maximum likelihood methods of phylogeny inference in comparison, and by fortuitous or directed resampling of the data set. Phylogenetic trees based on almost equivalent data sets of bacterial 23S and 16S rRNAs are in good agreement and their overall topologies are supported by alternative phylogenetic markers such as elongation factors and ATPase subunits. Besides their phylogenetic information content, the differently conserved primary structure regions of rRNAs provide target sites for specific hybridization probes which have been proven to be powerful tools for the identification of microbes on the basis of their phylogenetic relationships.     

Identification of apple cultivars using RAPD markers

Summary Eleven apple cultivars were differentiated using randomly amplified polymorphic DNA (RAPD) markers obtained by the polymerase chain reaction (PCR). The variability of the technique and of the origin of the DNA extract was analyzed. A set of bands consistent in their presence or absence was chosen to create a differentiating band pattern. A key is proposed by which one can differentiate apple cultivars using commercially available prime.