特别感谢IROC设计CORI桑德斯准备插图和克里斯Reddi在加州大学洛杉矶分校，为建立数字样品文化。霍华德休斯医学研究所（HHMI的批准号:52006944），为这个项目提供了资金。

<ol>
	<li>Barker, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> At the Bench: A Laboratory Navigator. , (1998).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biosafety in Microbiological And Biomedical Laboratories (BMBL). , (2009).</li><li>Bykowski, T., Stevenson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aseptic+Technique.">Aseptic Technique.</a> Current Protocols in Microbiology. Appendix 4, Appendix 4D (2008).</li><li>Cot&#233;, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aseptic+Technique+for+Cell+Culture.">Aseptic Technique for Cell Culture.</a> Current Protocols in Cell Biology. Chapter 1, Unit 1.3 (2001).</li><li>Grimes, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+basic+laboratory+manual+for+the+small-scale+production+and+testing+of+I-2+Newcastle+disease+vaccine.">A basic laboratory manual for the small-scale production and testing of I-2 Newcastle disease vaccine.</a> RAP Publication. AC802/E, 12-13 (2002).</li><li>Guzman, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pipetting:+A+Practical+Guide.">Pipetting: A Practical Guide.</a> The American Biology Teacher. 63 (2), 128-131 (2001).</li><li>Jordan, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> RESEARCH: National Genomics Research Initiative Phage Resource Laboratory Manual. , (2008).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning - A Laboratory Manual. , 978-087969576 (2001).</li><li>Seidman, L. A., Moore, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Basic Laboratory Methods for Biotechnology: Textbook and Laboratory Reference. , (2000).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> PIPETMAN P User's Guide. , (2012).</li></ol>

我什么都没有透露。

无菌技术指的是一套做是为了防止无菌解决方案和文化，成为在实验室中的有害微生物污染的例行程序。这种技术是必不可少的，需要生长的细胞的实验。虽然1工作设置完全无菌能不能得到实现，如实验室的表面进行消毒，创建1无菌领域使用本生灯，限制封顶文化和媒体曝光的空气消毒，如瓶，管及玻璃吸管材料的程序，避免接触表面非无菌的无菌文书，减少污染实验中的解决方案和文化的可能性。我们的目标是成为第二自然这些预防程序;培训和实践，同时在实验室工作。 用无菌解决方案和培养，使用，如血清移液器和麦克风文书传输量ropipettors是在实验室进行的常规技术的种类很多。仪器能够转移不同，但精准，体积不同实验应用程序调用。血清移液器用于微生物实验室准备需要媒体的筹备工作涉及毫升体积的细胞培养，而micropipettors是必不可少的分子生物学实验需要的解决方案，仅微升。当这些文书实行无菌技术，污染最小化无论在体积转让金额液体或类型的实验。 虽然没有在这个协议的讨论，通常用于防止污染等手段之一是工作在层流罩（ 图10）。此设备是组织文化归类为BLS-2或更高的微生物实验的关键。层流罩，包含了HEPA（高效率微粒空气）过滤器，除去空气中的污染物，从引擎盖到流动的空气，同时防止未经过滤的空气渗透到工作区的房间。值得注意的是，不能用于本生灯内的层流罩，因为从火焰的热量会破坏空气流罩的功能至关重要。 检查您的无菌技术质量，进行实验时，它往往是有益的。以确认解决方案和文化传媒不要在实验操作污染，随时准备阴性对照 。例如，如果准备细菌培养生长的肉汤管，不要只留下无菌媒体接种一管。孵育然后检查未接种的迹象，如无意引入到管的有害细胞的生长浊度污染控制管一起接种管的介质。如果被污染的控制管，管的实验很可能被污染的同时，将不得不重复实验。这些预防措施应做到每一个实验。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> LB培养基</td><td> DIFCO </td><td> 244620 </td><td>配方也可以参考6 </td></tr><tr><td colspan="4"> TE缓冲液: </td></tr><tr><td> EDTA二钠盐二水</td><td>西格玛</td><td> E5134 </td><td></td></tr><tr><td> trizma-盐酸</td><td>西格玛</td><td>的T-3253 </td><td></td></tr><tr><td> CiDecon </td><td> DECON实验室，公司</td><td> 8504 </td><td>消毒剂</td></tr><tr><td>乙醇</td><td> Fisher Scientific则</td><td> A406 </td><td>作为消毒剂使用，准备用蒸馏水70％（V / V） </td></tr></tbody></table>

aseptic laboratory techniques: volume transfers with serological pipettes and micropipettors

微生物无处不在 - 在空气，土壤，人体以及实验室长凳和电脑键盘一样的老成表面上。微生物无处不在实验室建立一个供应丰富的潜在污染物。为了确保实验的成功，设备和工作表面上的污染物数量必须减少。许多微生物学实验中常见的是涉及文化含有细菌细胞或病毒颗粒的测量和传输的技术。要做到没有非无菌表面接触或污染无菌媒体要求:（1）准备无菌工作区，（2）精确设置和准确读取液体无菌转让文书，（3）正确操作仪器，文化瓶，瓶管内无菌领域。学习这些程序的培训和实践要求。起初，行动应该是缓慢的，故意的，和无菌ţ目标控制echnique成为第二本性在替补席上时。在这里，我们目前测量血清移液器和micropipettors的本生灯创造一个无菌的领域内使用的卷的步骤。卷的范围从微升（μL），毫升（ml）根据使用的仪器。通常转移的液体，包括无菌肉汤或化学溶液以及细菌培养和噬菌体股票。通过这些程序后，学生应该能够: <ul><li>本生灯火焰所造成的不育领域内的工作。 </li><li>血清移液器使用而不影响仪器育。 </li><li>血清移液器吸液体，正是读通过调整液体由移液器毕业商标的形成半月板的校正量。 </li><li>保持培养瓶，烧瓶，试管和无菌液体转移期间各自的上限。 </li><li>塑料与玻璃血清PIP识别不同的应用ettes。 </li><li>国家micropipettors精度的限制。 </li><li>精确和准确的上micropipettors设置卷。 </li><li>知道如何正确使用移液器的第一个和第二站抽吸和传输正确的卷。 </li></ul>

Watch this Scientific Journal Video about Aseptic Laboratory Techniques: Volume Transfers with Serological Pipettes and Micropipettors at JoVE.com

Aseptic Laboratory Techniques: Volume Transfers with Serological Pipettes and Micropipettors

在实验室工作时，当务之急是减少污染源。无菌技术是指允许转让，同时避免与非无菌表面的接触文化和试剂的程序。血清移液器和micropipettors，用于测量精确量不妥协的解决方案，在实验中使用的无菌。

无菌实验室技术:量与血清学移液器和Micropipettors的转移

Microorganisms are everywhere - in the air, soil, and human body as well as on inanimate surfaces like laboratory benches and computer keyboards. The ...

aseptic-laboratory-techniques-volume-transfers-with-serological

Nanyang Technological University

Research

JoVE Journal

Biology

162.6K 次观看.  University of California, Los Angeles. 在实验室工作时，当务之急是减少污染源。无菌技术是指允许转让，同时避免与非无菌表面的接触文化和试剂的程序。血清移液器和micropipettors，用于测量精确量不妥协的解决方案，在实验中使用的无菌。

视频: Aseptic Laboratory Techniques: Volume Transfers with Serological Pipettes and Micropipettors

Microbial Communities in Nature and Laboratory - Interview

微生物群落，在自然和实验室 - 面试

科研

生物学

Making Patch-pipettes and Sharp Electrodes with a Programmable Puller

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades. Today, such pipettes are made from glass capillaries using a programmable puller. Such instruments heat the capillary using either a metal filament or a laser and draw out the glass using gravity, a motor or both. Pipettes for patch-clamp recording are formed using only heat and gravity, while sharp electrodes for intracellular recording use a combination of heat, gravity, and a motor. The procedure used to make intracellular recording pipettes is similar to that used to make injection needles for a variety of applications, including cRNA injection into Xenopus oocytes. In general, capillary glass &lt;1.2 mm in diameter is used to make pipettes for patch clamp recording, while narrower glass is used for intracellular recording (outer diameter = 1.0 mm). For each tool, the puller is programmed slightly differently. This video shows how to make both kinds of recording pipettes using pre-established puller programs.

This video shows how to use a programmable puller to make patch pipettes and sharp electrodes for electrophysiology. The same procedure can be used to make a variety of glass tools, including injection needles.

膜片吸液管和夏普电极与一个可编程的拖轮

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades.  Today, such pipettes are made from glass capillaries ...

Pressure-polishing Pipettes for Improved Patch-clamp Recording

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording. We first developed this method for fabricating pipettes suitable for recording from small (&lt;3 m) neuronal cell bodies. The basic principal is similar to glass-blowing and combines air pressure and heat to modify the shape of patch pipettes prepared by a conventional micropipette puller. It can be applied to so-called soft (soda lime) and hard (borosilicate) glasses. Generally speaking, pressure polishing can reduce pipette resistance by 25% without decreasing the diameter of the tip opening (Goodman and Lockery, 2000). It can be applied to virtually any type of glass and requires only the addition of a high-pressure valve and fitting to a microforge. This technique is essential for recording from ultrasmall cells (&lt;5 m) and can also improve single-channel recording by minimizing pipette resistance. The blunt shape is also useful for perforated-patch clamp recording since this tip shape results in a larger membrane bleb available for perforation.

This is a guide to modifying the shape of glass micropipettes. Specifically, by using heat and air pressure the taper is widened without increasing the tip opening, leading to lower pipette resistance. This is critical to obtain low noise recordings of small cells but is useful in many applications.

改进的膜片钳记录的压力抛光移液器

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording.  We first developed this method for fabricating pipettes suitable for ...

Large Volume (20L+) Filtration of Coastal Seawater Samples

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For this method, samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. This video documents large volume (&ge;20 L) filtration of microbial biomass, ranging between 0.22&mu;m and 2.7&mu;m in diameter, from the water column. Two 20L samples can be filtered simultaneously using a single pump unit equipped with four rotating heads. Filtration is done in the field on extended trips, or immediately upon return for day trips. It is important to record the amount of water passing through each sterivex filter unit. To prevent biofilm formation between sampling trips, all filtration equipment must be rinsed with dilute HCl and deionized water and autoclaved immediately after use. This procedure will take approximately 5 hours plus an additional hour for clean up.

Large Volume 20L+ Filtration of Coastal Seawater Samples

This video documents large volume (&ge;20 L) filtration of microbial biomass, ranging between 0.22&mu;m and 2.7&mu;m in diameter, from the water column.

大容量（20L）过滤沿海海水样品

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For this method, ...

Modeling Biological Membranes with Circuit Boards and Measuring Electrical Signals in Axons: Student Laboratory Exercises

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology used in electrophysiology. The same equipment is used in the membrane model as on live preparations. Some properties of an isolated nerve cord are investigated: nerve action potentials, recruitment of neurons, and responsiveness of the nerve cord to environmental factors.

This is a demonstration of how biological membranes can be understood using electrical models. We also demonstrate procedures for recording action potentials from the ventral nerve cord of the crayfish for student orientated laboratories.

建模生物膜与电路板，并在轴突测量电气信号:学生实验室练习

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology ...

Aseptic Laboratory Techniques: Plating Methods

Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories (BMBL) as well as Material Safety Data Sheets (MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection (ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to: 
 
&#9679; Perform plating procedures without contaminating media. 
&#9679; Isolate single bacterial colonies by the streak-plating method. 
&#9679; Use pour-plating and spread-plating methods to determine the concentration of bacteria. 
&#9679; Perform soft agar overlays when working with phage. 
&#9679; Transfer bacterial cells from one plate to another using the replica-plating procedure. 
&#9679; Given an experimental task, select the appropriate plating method.

When working with media and reagents used to culture microorganisms, aseptic technique must be practiced to ensure contamination is minimized. A variety of plating methods are routinely used to isolate, propagate, or enumerate bacteria and phage, all of which incorporate procedures that maintain the sterility of experimental materials.

无菌实验室技术:电镀方法

Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies ...

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can also be used to investigate and validate novel techniques and algorithms in volumetric medical imaging and therapies. Creating 3D high-resolution atlases of different organs1,2,3 is another application of histology volume reconstruction. This provides a resource for investigating tissue structures and the spatial relationship between various cellular features. We present an image registration approach for histology volume reconstruction, which uses a set of optical blockface images. The reconstructed histology volume represents a reliable shape of the processed specimen with no propagated post-processing registration error. The Hematoxylin and Eosin (H&#38;E) stained sections of two mouse mammary glands were registered to their corresponding blockface images using boundary points extracted from the edges of the specimen in histology and blockface images. The accuracy of the registration was visually evaluated. The alignment of the macrostructures of the mammary glands was also visually assessed at high resolution.
This study delineates the different steps of this image registration pipeline, ranging from excision of the mammary gland through to 3D histology volume reconstruction. While 2D histology images reveal the structural differences between pairs of sections, 3D histology volume provides the ability to visualize the differences in shape and volume of the mammary glands.

We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands.

3维组织学音量和其研究小鼠乳腺腺体的应用改造

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

对于外小泡的使用流式细胞仪分析技术

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Techniques to Induce and Quantify Cellular Senescence

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed cellular senescence. Accumulation of senescent cells occurs with organismal aging and through continual culturing in vitro. Senescent cells influence many biological processes, including embryonic development, tissue repair and regeneration, tumor suppression, and aging. Hallmarks of senescent cells include, but are not limited to, increased senescence-associated &#946;-galactosidase activity (SA-&#946;-gal); p16INK4A, p53, and p21 levels; higher levels of DNA damage, including &#947;-H2AX; the formation of Senescence-associated Heterochromatin Foci (SAHF); and the acquisition of a Senescence-associated Secretory Phenotype (SASP), a phenomenon characterized by the secretion of a number of pro-inflammatory cytokines and signaling molecules. Here, we describe protocols for both replicative and DNA damage-induced senescence in cultured cells. In addition, we highlight techniques to monitor the senescent phenotype using several senescence-associated markers, including SA-&#946;-gal, &#947;-H2AX and SAHF staining, and to quantify protein and mRNA levels of cell cycle regulators and SASP factors. These methods can be applied to the assessment of senescence in various models and tissues.

Cellular senescence, the irreversible state of cell-cycle arrest, can be induced by various cellular stresses. Here, we describe protocols to induce cellular senescence and methods to assess markers of senescence.

技术诱导和量化细胞衰老

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed ...

Volume Segmentation and Analysis of Biological Materials Using SuRVoS (Super-region Volume Segmentation) Workbench

Segmentation is the process of isolating specific regions or objects within an imaged volume, so that further study can be undertaken on these areas of interest. When considering the analysis of complex biological systems, the segmentation of three-dimensional image data is a time consuming and labor intensive step. With the increased availability of many imaging modalities and with automated data collection schemes, this poses an increased challenge for the modern experimental biologist to move from data to knowledge. This publication describes the use of SuRVoS Workbench, a program designed to address these issues by providing methods to semi-automatically segment complex biological volumetric data. Three datasets of differing magnification and imaging modalities are presented here, each highlighting different strategies of segmenting with SuRVoS. Phase contrast X-ray tomography (microCT) of the fruiting body of a plant is used to demonstrate segmentation using model training, cryo electron tomography (cryoET) of human platelets is used to demonstrate segmentation using super- and megavoxels, and cryo soft X-ray tomography (cryoSXT) of a mammalian cell line is used to demonstrate the label splitting tools. Strategies and parameters for each datatype are also presented. By blending a selection of semi-automatic processes into a single interactive tool, SuRVoS provides several benefits. Overall time to segment volumetric data is reduced by a factor of five when compared to manual segmentation, a mainstay in many image processing fields. This is a significant savings when full manual segmentation can take weeks of effort. Additionally, subjectivity is addressed through the use of computationally identified boundaries, and splitting complex collections of objects by their calculated properties rather than on a case-by-case basis.

Volume Segmentation and Analysis of Biological Materials Using SuRVoS Super-region Volume Segmentation Workbench

Segmentation of three-dimensional data from many imaging techniques is a major bottleneck in analysis of complex biological systems. Here, we describe the use of SuRVoS Workbench to semi-automatically segment volumetric data at various length-scales using example datasets from cryo-electron tomography, cryo soft X-ray tomography, and phase contrast X-ray tomography techniques.

利用 SuRVoS (超区域体积分割) 工作台进行生物材料的体积分割与分析

Segmentation is the process of isolating specific regions or objects within an imaged volume, so that further study can be undertaken on these areas of ...