A Reliable Method for Demonstrating Gram-Positive and Gram-Negative Bacteria

Combined Gram techniques have been reviewed in the interest of improving technical safety and reliability in the demonstration of bacteria, particularly the Gram-negative type. The many modifications of the technique present various difficulties (Brown and Brenn 193 1, Humberstone 1963, Taylor 1966, Luna 1968, Brown and Hopps 1973, Engbaek et al. 1979, Bancroft and Stevens 1982, Churukian and Schenk 1982).     

A Reliable Method for Demonstrating Axonal Degeneration Shortly After Axotomy

A method has been elaborated by which degenerating axons can be selectively impregnated with silver. Based on a reconsideration of the physicochemical mechanisms of the degeneration methods it takes advantage of physical developers over the chemical ones. The staining procedure is applied to frozen sections of brains fixed with formol. It consists of 6 steps: (1) pretreatment with alkaline hydroxylamine, (2) washing in acetic acid, (3) impregnation in silver nitrate in the presence of ferric ions, (4) washing in citric acid, (5) physical development, and (6) washing in acetic acid. By electron microscopy silver precipitates by this method are almost entirely restricted to the cytoplasm of dense, degenerating axons, sparing mitochondria and myelin sheaths. No special expertise is required to achieve reproducible results. Large numbers of sections treated simultaneously, and large sections, can be stained uniformly. Light microscopic criteria are described which help diagnose the source of possible failures. Low background staining allows dark field illumination and television image analysis to be applied. The method works at survival times of only 3 to 5 days after axotomy. Hence, degenerating axons and axon terminals can be stained in alternating sections from the same brain using this method and another being described separately, which, using different conditions, demonstrates degenerating axon terminals.     

A Simple, Fast and Reliable Method for Obtaining Dopamine Histofluorescence from Mesencephalic Cultures

A modified glyoxylic acid technique for obtaining dopamine histofluorescence from cultured mesencephalic cells is described. This method requires only two solutions: one contains glyoxylic acid, sucrose and monobasic potassium phosphate and is used at room temperature, the other is a Hepes buffered solution used at 37 C. Relatively high concentrations of a monoamine oxidase inhibitor and dopamine are added to the cultures to load dopaminergic neurons; the cell bodies and their processes take up and hold dopamine quickly and evenly. The cultures are dipped in a glyoxylic acid solution, dried in air, heated for 5 min and coverslipped with mineral oil. Since the cultures remain in their culture dishes, the entire procedure takes less than 2 hr. The green histofluorescence characteristic of dopamine is seen when the cultures are viewed by standard fluorescence microscopy. Various cell body types and sizes can be distinguished, as well as the complete extent of their processes and varicosities.     

A Rapid, Simple and Reliable Combined Method for G-Banding Mammalian and Human Chromosomes

A simple and reliable method for G-banding chromosomes from human and mammalian cells is described. This rapid method combines hot saline and trypsin treatments and yields high quality G-bands in both bone marrow and cultured cells.     

快速可靠的双链PCR产物直接测序法

利用T7DNA聚合酶在低温下仍具较高活性的特点,在热变性后低温下进行测序反应,使用该方法对多种PCR产物进行序列分析均取得较好的结果．     

一种保藏小单孢菌的简便方法─滤纸保藏法

用滤纸法保藏７２株小单孢菌在冰箱１０℃无干燥条件下,保藏７年５个月后检查其存活率、抗菌活力,观察其形态特征、培养特征和生化特征。结果表明,所保藏的菌种全部存活,且抗菌活力、生理特征等保持原有菌株的特征水平。我们认为,用滤纸法保藏小单孢菌效果好,方法简便,适用于在临床上、工业生产上和科研中有实用价值的庆大霉素、小诺霉素、西梭霉素、小单孢菌的保藏。     

一种保藏小单孢菌的简便方法─滤纸保藏法

用滤纸法保藏７２株小单孢菌在冰箱１０℃无干燥条件下,保藏７年５个月后检查其存活率、抗菌活力,观察其形态特征、培养特征和生化特征。结果表明,所保藏的菌种全部存活,且抗菌活力、生理特征等保持原有菌株的特征水平。我们认为,用滤纸法保藏小单孢菌效果好,方法简便,适用于在临床上、工业生产上和科研中有实用价值的庆大霉素、小诺霉素、西梭霉素、小单孢菌的保藏。     

一种简便可靠的组蛋白乙酰转移酶(HAT)抑制剂荧光检测方法

组蛋白乙酰转移酶(HAT)参与真核细胞基因转录的调节,其抑制剂由于具有调节转录并产生抗病毒、抗炎、抗氧化等作用,有希望成为一类新药.非放射性分光光度HAT测定方法虽然可替代广泛使用的放射性检测方法,但缺乏灵敏度和准确性.建立了一种简单的非放射性荧光检测方法,即测量辅酶A(CoASH)与邻苯二甲醛(OPA)和巯基乙醇反应的荧光产物.此法与分光光度法相比具有更高的准确性,可进行多种化合物HAT抑制活性的筛选.这种新方法有望成为研究转录调节和新药开发方面的一种有效工具.     

A Simple and Reliable Method for Hybridization of Homothallic Wine Strains of Saccharomyces cerevisiae

A procedure was developed for the hybridization and improvement of homothallic industrial wine yeasts. Killer cycloheximide-sensitive strains were crossed with killer-sensitive cycloheximide-resistant strains to get killer cycloheximide-resistant hybrids, thereby enabling hybrid selection and identification. This procedure also allows backcrossing of spore colonies from the hybrids with parental strains.     

A Rapid Method for Manual or Automated Purification of Fluorescently Labeled Nucleic Acids for Sequencing,Genotyping, and Microarrays

Fluorescent dyes provide specific, sensitive, and multiplexed detection of nucleic acids. To maximize sensitivity, fluorescently labeled reaction products (e.g., cycle sequencing or primer extension products) must be purified away from residual dye-labeled precursors. Successful high-throughput analyses require that this purification be reliable, rapid, and amenable to automation. Common methods for purifying reaction products involve several steps and require processes that are not easily automated. Prolinx^®, Inc. has developed RapXtract^™ superparamagnetic separation technology, affording rapid and easy-to-perform methods that yield high-quality product and are easily automated. The technology uses superparamagnetic particles that specifically remove unincorporated dye-labeled precursors. These particles are efficiently pelleted in the presence of a magnetic field, making them ideal for purification because of the rapid separations that they allow. RapXtract-purified sequencing reactions yield data with good signal and high Phred quality scores, and they work with various sequencing dye chemistries, including BigDye and near-infrared fluorescence IRDyes. RapXtract technology can also be used to purify dye primer sequencing reactions, primer extension reactions for genotyping analysis, and nucleic acid labeling reactions for microarray hybridization. The ease of use and versatility of RapXtract technology makes it a good choice for manual or automated purification of fluorescently labeled nucleic acids.     

一种实用可靠的古代 DNA 获取方法

古代DNA序列信息能够为物种演化研究提供最直接的分子证据,但获取古代DNA的技术仍存在诸多瓶颈,尤其是扩增中存在受损伤DNA模板的干扰、获取成本高和实验周期长等问题．改进了异丙醇沉淀提取法,并采用了尿嘧啶糖苷酶(UNG)去除受损伤DNA模板后进行扩增的方法,最终可以高效地获取真实的古代DNA序列．实验利用距今4 300～3 900年前的猪牙样本,将改进的古 DNA 获取方法与常规方法进行比较研究,结果表明,改进的异丙醇沉淀法提取结合UNG处理后进行PCR扩增的方法,可以在保证古代DNA获取成功率并提高获得的DNA序列可靠性的前提下,将经费投入和实验周期都各减少至常规方法的50%以下．这可以为开展大规模古代样本检测提供一种切实可行的 DNA 获取方法．     

一种高效自动化的转录组差异性表达分析方法

差异性表达分析是转录组研究的核心目标之一,对于揭示基因功能和调控规律具有重要意义。但该分析属于多步迭代且耗时较长的计算密集型过程,软件之间具有复杂的数据依赖关系,输入输出格式不尽相同。传统方式下,软件安装与使用复杂、繁琐的手动操作、分析环境的不易迁移以及无法按需集成都是有待解决的关键问题。针对上述问题,文中首次将云开源项目Docker容器技术应用于生物信息领域,提出一种高效自动化的转录组差异性表达分析方法。首先将最佳实践流程在Docker容器中内置与集成,其次多脚本联动与Web服务相结合,最后形成一个轻量级、易迁移、高效自动化的转录组差异性表达分析"黑匣子"。实验结果表明,与传统方式相比,该方法的分析时间缩短约72%,效率提升2倍多,为研究人员提供了更高效的技术支持。     

Automated Method for the Determination of Phenol in Biological Products

A method is described for the rapid and accurate determination of phenol in biological products by using automated methods.     

A Simple, Reliable, and Fast Protocol for Thraustochytrid DNA Extraction

DNA extraction of thraustochytrids, common marine unicellular organisms, is usually accomplished by either the cetyltrimethylammonium bromide (CTAB) or proteinase K protocols. A novel lysis buffer protocol for thraustochytrid total DNA extraction is described. The average isolated total DNA is 20 to 40 kb, and DNA samples are suitable for a variety of uses including 18S–ribosomal DNA polymerase chain reaction, restriction enzyme digestions, and amplified fragment length polymorphism analyses. The new protocol is also faster than the other protocols. Received July 31, 2000; accepted November 2, 2000.     

A Rapid and Reliable Method for Total Protein Extraction from Succulent Plants for Proteomic Analysis

Crassulacean acid metabolism plants have some morphological features, such as succulent and reduced leaves, thick cuticles, and sunken stomata that help them prevent excessive water loss and irradiation. As molecular constituents of these morphological adaptations to xeric environments, succulent plants produce a set of specific compounds such as complex polysaccharides, pigments, waxes, and terpenoids, to name a few, in addition to uncharacterized proteases. Since all these compounds interfere with the analysis of proteins by electrophoretic techniques, preparation of high quality samples from these sources represents a real challenge. The absence of adequate protocols for protein extraction has restrained the study of this class of plants at the molecular level. Here, we present a rapid and reliable protocol that could be accomplished in 1 h and applied to a broad range of plants with reproducible results. We were able to obtain well-resolved SDS/PAGE protein patterns in extracts from different members of the subfamilies Agavoideae (Agave, Yucca, Manfreda, and Furcraea), Nolinoideae (Dasylirion and Beucarnea), and the Cactaceae family. This method is based on the differential solubility of contaminants and proteins in the presence of acetone and pH-altered solutions. We speculate about the role of saponins and high molecular weight carbohydrates to produce electrophoretic-compatible samples. A modification of the basic protocol allowed the analysis of samples by bidimensional electrophoresis (2DE) for proteomic analysis. Furostanol glycoside 26-O-β-glucosidase (an enzyme involved in steroid saponin synthesis) was successfully identified by mass spectrometry analysis and de novo sequencing of a 2DE spot from an Agave attenuata sample.     

Simple and Reliable Method for Replica Plating Neurospora crassa

The details of a simple method for replica plating Neurospora crassa are described. The procedure has proved to be highly reliable for replica plating of colonies arising from either ascospores or conidia.