我们要感谢安东尼. Weinhold 和多年来与我们合作的众多学生的技术支持, 以及改进我们的昆虫饲养程序的建议。

1. 收集卵子 (图 1)
<ol><li>准备好怂恿杯, 把布的一端放在500毫升的烧杯里, 里面装满了温暖的 (~ 40 °c) 的水。重叠的杯子的两侧, 并修复与橡胶带。将布料松散的一端折回杯顶。</li>
	<li>将怂恿杯放在8–10天的老苍蝇笼中 2 h. 妊娠雌性稳定的苍蝇会在布料上排卵。</li>
	<li>从笼中取出怂恿杯, 将怂恿布上的鸡蛋冲洗成一个小平底锅, 用挤压洗瓶。</li>
</ol>2. 准备幼虫饲养盘
<ol><li>
准备培养基 (1 饲养盘的数量)
	<ol>
<li>将麦麸 (500 克)、木屑 (200 克) 和鱼粉 (115 克) 组合在10升塑料碟盘中, 混合均匀。</li>
		<li>加入碳酸氢铵 (50 克) 到水 (~ 25 °c, 1600 毫升) 和混合, 直到溶解。</li>
		<li>添加水溶液的干燥配料和混合, 以确保没有 unwetted 材料。在平底锅中的水平介质, 但不压缩。</li>
	</ol>
</li>
	<li>
添加稳定的飞卵
	<ol>
<li>在中等长度的平底锅上作浅沟。</li>
		<li>将1毫升稳定的飞卵 (8000 卵) 在沟中用蛋吸管 (图 2) 存入。 注: 通过在所需点切割一个塑料刻度吸管, 插入100网格屏幕, 再将吸管粘在一起, 制成的蛋吸管。</li>
		<li>用一层薄薄的培养基盖上鸡蛋以防止脱水。</li>
		<li>盖平底锅与枕头盒, 关闭与橡胶带和标签。在幼虫室放置平底锅 (23 2 °c, 30–50% RH 和 12:12 [L: D] h 光周期)。 注意: 幼虫发育需要从产卵时10–14天。</li>
	</ol>
</li>
</ol>3. 蛹处理后, 幼虫在产卵和 Pupariate 13 天或14后移到培养基7–9天的边缘。
<ol><li>铲蛹, 主要位于锅的边缘周围的外壳, 形成在表面上的介质, 出和放置在一个干净的平底锅。</li>
	<li>装满平底锅, 用蛹, ½¾满水。分解蛹和培养基的团簇。蛹 > 1 天老浮19, 中间水槽的麦麸和蛋白质组分。</li>
	<li>去除浮动材料, 蛹和一些中等组分与漏勺并且洗涤通过一系列筛子 (#5, #7, #12, #20) 去除剩余的媒介。#12 筛收集蛹, #20 使固体废弃物进入排水管。用水槽喷雾器在筛子之间洗蛹。</li>
	<li>冲洗蛹, 在 #12 筛, 成一个干净的锅。把锅装满水½。将漂浮的蛹倒入漏勺中, 允许多余的水排出。</li>
	<li>将抽干的蛹放到烘干屏上, 直到干燥 (图 3)。</li>
</ol>4. 蛹货架, 另一种收集蛹的方法是与蛹货架18
注: 货架是由一个从10升碟盘 (10.2 厘米高 x 10.2 厘米宽 x 31.9 厘米长) 的一端的塑料切割。
<ol><li>准备介质如2.1 所述。锥形介质在平底锅从2.5 厘米深的一端到7.5 厘米深。</li>
	<li>
将架子放在饲养盘的浅端和胶带上, 以防幼虫爬行在架子上 (图 4)。
	<ol>
<li>饱和海绵 (~ 14.5 x 9.0 x 4.5 cm3, 再生纤维素) 并且包装它在水被浸泡的布料 (30.5 x 42 cm2, 棉花) 和地方在架子大约一个半英寸从媒介。</li>
	</ol>
</li>
	<li>
添加鸡蛋, 如2.2 所述, 并继续像以前一样。
	<ol>
<li>每天检查平底锅以确保海绵保持湿润。</li>
	</ol>
</li>
	<li>将蛹从货架上冲洗出来, 海绵和布料放入一个干净的盘子里, 产卵后14天倒入漏勺。</li>
</ol>5. 质量控制
<ol><li>称量所有在平底锅中生产的蛹 (总重量)。</li>
	<li>隔离100蛹, 称重并放置在9厘米培养皿中。</li>
	<li>伯爵和性成人 ~ 5–8天后确定出现率和性别比例。储存培养皿与出现的成年人在一个冰箱, 以计数在以后的日期, 如果需要的话。苍蝇是奸的形状, 他们的眼睛和宽度的额轨道板20或生殖器在低放大倍数。</li>
	<li>记录总重量, 重量100蛹, 并且出现成年男性和女性的数量。</li>
</ol>6. 准备血液
<ol><li>从当地屠宰场收集新鲜牛血19升桶, 含70克柠檬酸钠三元二水合物500毫升水。大力搅拌血液5–10分钟, 返回实验室。</li>
	<li>通过漏勺的血液来去除血块并分成2升的容器, 标签与收集的日期和储存在一个冰箱 (-20 °c)。</li>
	<li>在需要前2天, 从冰箱中取出容器并放入冰箱。血液可用于2周, 一旦解冻, 并储存在冰箱里长达1年。</li>
</ol>7. 成人保养
<ol><li>准备笼子, 线笼底部用屠夫纸方便清洗。</li>
	<li>在每个笼子里放置50克干燥稳定的飞蛹 (~ 3500)。成人 eclose 几个小时后, 蛹处理后几天。</li>
	<li>在每个笼子的顶部放一个新鲜的血液浸泡女性餐巾喂养。开始喂养24小时内成人羽化和重复每天直到产卵前的一天 (9–10天后出现)。</li>
	<li>产卵后 (1 节), 把笼子放在冰箱里 4–8 h, 然后清除纸张, 用热水冲洗两侧和地板上的取食量, 用洗衣粉和氯漂白剂擦洗所有表面。用热水冲洗笼子, 让干燥。</li>
</ol>

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作者没有什么可透露的。

稳定的苍蝇可以发现发展在广泛的各种基质的性质, 可以饲养在许多类型的媒体在实验室。我们用木屑和蛭石作膨胀剂。蛭石工作良好, 但使分离蛹从饲养培养基困难和昂贵 ($ 0.60/平底锅)。可能是由于增加了从培养基中分离蛹的严谨性, 羽化也降低了蛭石, 57% vs 75% 用于木屑。棉刨花可媲美蛭石, 但有时难以采购。最重要的成分似乎是植物或纤维素材料发酵和维持一个 "开放" 或良好的充气培养基和?...

稳定的苍蝇, Stomoxys calcitrans (L), 是 hematophagous 苍蝇, 其痛苦的叮咬扰乱牲畜的放牧行为, 造成痛苦和痛苦的同伴动物和扰乱人类娱乐活动的世界各地。不成熟的稳定蝇生长在发酵的植物物质, 经常被动物粪便污染。改变农艺做法和农作物造成了作物残留物的稳定蝇的严重暴发, 澳大利亚的蔬菜1, 巴西的甘蔗2, 哥斯达黎加的菠萝3。虽然每只动物的14只稳定苍蝇被认为是经济阈值4, 但在最近的疫情中, 每只动物的2000多只苍蝇的观测结果已经达到5。这种侵扰程度将主机生产率降低到接近零, 并可能导致死亡率6。由于 agronomically 相关的暴发, 稳定的苍蝇正在接受新的兴趣和对实验室殖民地的需求急剧增加。
对于所有 holometamorphic 昆虫, 稳定的苍蝇在未成熟或幼体阶段获得生长所需的所有养分。因此, 饲养系统的一个重要组成部分是幼虫饮食或基质。稳定的蝇幼虫已经观察到在田间7的广泛基质中发展, 它们依赖于基底8,9的微生物群落。天然幼虫基质主要由分解或发酵经常被含氮废物污染的营养物质组成。
在实验室饲养中, 稳定的蝇幼虫基质通常由营养物质和添加的氮源组成。大量的材料已用于稳定的飞行幼虫饮食。第一种幼虫饮食模仿天然基质, 包括发酵燕麦秸秆和马或牛粪10,11。碳水化合物来源包括麦麸12、13、14、苜蓿膳食12、13、14和由化学专业开发的商业配方。制造商协会 (CSMA, 33% 麦麸, 27% 苜蓿膳食, 40% 啤酒酵母颗粒)13,14,15,16。氮源包括酵母悬浮液12、鱼粉和碳酸氢铵17。惰性膨胀材料通常包括燕麦壳12, 蔗渣13, 蛭石16, 木片13,18和颗粒花生壳14。
实验室饲养的一个主要目标是生产一种与 "野生型" 相似的产品, 以便实验室实验能产生反映田间人群的结果。这就要求最大限度地减少育种和选择, 以维持遗传多样性, 并使营养资源与实地的情况相媲美。次要目标是尽量减少劳动和开支。减少开支的一个主要组成部分是利用当地现有的饮食成分。为实现这些目标, 研制了稳定的飞养系统。

<table>
	<tbody>
		<tr>
			<td>Diamalt</td>
			<td>Premier Malt Products, Inc., Saddle Brook, NJ</td>
			<td>2540</td>
			<td></td>
		</tr>
		<tr>
			<td>CSMA Fly media</td>
			<td>Purina Animal Nutrition, Arden Hills, MN</td>
			<td>5S6Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin Maxi Pad</td>
			<td>The Tranzonic Co., Cleveland, OH, USA</td>
			<td>5001M</td>
			<td></td>
		</tr>
		<tr>
			<td>Calf Manna</td>
			<td>MannaPro, Chesterfield, MO, USA</td>
			<td>Manna Pro</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Bicarbonate</td>
			<td>Spectrum Chemical Manufacturing Corp, Gardena, CA</td>
			<td>A1125</td>
			<td></td>
		</tr>
		<tr>
			<td>Wheat bran, Coarse</td>
			<td>Siemer Milling Company, Teutopolis, IL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wood shavings</td>
			<td>Tractor Supply Company, Brentwood, TN</td>
			<td>502770699</td>
			<td></td>
		</tr>
		<tr>
			<td>Fishmeal</td>
			<td>Consumer Supply Distributing, North Sioux City, SD</td>
			<td>F1550</td>
			<td></td>
		</tr>
		<tr>
			<td>Adult cages</td>
			<td>All Aluminum Window Company, Lincoln, Ne</td>
			<td>Custom</td>
			<td>45 &#215; 45 &#215; 45 cm, 18 &#215; 16 mesh aluminum screen, stockinette access</td>
		</tr>
		<tr>
			<td>9 &#215; 28 cm black cotton cloth</td>
			<td>Robert Kaufman Fabrics, Los Angeles, CA</td>
			<td>K040-114</td>
			<td>Egging cloth</td>
		</tr>
		<tr>
			<td>10 liter plastic dish pans</td>
			<td>Rubbermaid, Saratoga Springs, NY</td>
			<td>FG2951ARWHT</td>
			<td>Larval pans</td>
		</tr>
		<tr>
			<td>Stockinette, Cotton, 12&#34; X 25 yd roll</td>
			<td>Tex-Care Medical Company, Burlington, NC</td>
			<td>91311-225</td>
			<td></td>
		</tr>
	</tbody>
</table>

laboratory rearing of stable flies and other muscoid diptera

稳定的苍蝇, Stomoxys calcitrans, 是家畜, 人类, 同伴动物和野生动物的严重害虫。在过去的20年中, 农艺做法的变化导致几个国家的稳定蝇的严重暴发。这些暴发扰乱了牲畜生产和人类的娱乐活动, 导致公众要求为这种害虫增加研究和管理工作。提出了一种简单、廉价的用于实验室研究的稳定蝇的饲养方法。该程序使用当地现有的饮食成分, 设备和用品。该程序可适用于饲养其他 muscoid 苍蝇, 包括面部苍蝇 (蝇 autumnalis), 喇叭飞行 (Haematobia irritans), 和房子苍蝇 (家蝇)。该程序生产稳定的飞行蛹平均12.5 毫克和35% 鸡蛋的成年生存。大约3000只苍蝇在每个平底锅被生产。

幼虫 pupariate 10–14天和成人在产卵后14–16天出现。世代时间, 蛋到蛋, 是 ~ 24 天。2013年5月至2017年1月的饲养数据有三种不同的膨胀剂和两个菌落,如图 5所示。白杨提供了最好的产量, 3867 ±1442<img alt="Equation" src="/files/ftp_upload/57341/57341eq1.jpg"> </img> (标准偏差) 蛹重12.5 ±1.6 毫克74±19% 羽化生产2872±1294成年蝇为建立的实验室殖民地。性别比例略有偏向女性 0...

Watch this Scientific Journal Video about Laboratory Rearing of Stable Flies and Other Muscoid Diptera at JoVE.com

Laboratory Rearing of Stable Flies and Other Muscoid Diptera

提出了一种饲养稳定蝇 (Stomoxys calcitrans) 的方法。该程序使用当地现有的材料, 为膳食成分, 设备和用品。

稳定蝇及其他 Muscoid 双翅目的实验室饲养

Stable flies, Stomoxys calcitrans, are serious pests of livestock, humans, companion animals and wildlife worldwide. During the last 20+ years, changes in ...

laboratory-rearing-of-stable-flies-and-other-muscoid-diptera

Research

JoVE Journal

Biology

8.5K 次观看.  ARS-USDA. 提出了一种饲养稳定蝇 (Stomoxys calcitrans) 的方法。该程序使用当地现有的材料, 为膳食成分, 设备和用品。

视频: Laboratory Rearing of Stable Flies and Other Muscoid Diptera

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

Protocol for Mosquito Rearing (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night cycle. For this procedure, you'll need mosquito cages, 10% sterile sucrose solution, paper towels, beaker, whatman filter paper, glass feeders, human blood and serum, water bath, parafilm, distilled water, clean plastic trays, mosquito food (described below), mosquito net to cover the trays, vacuum, and a collection chamber to collect adults.

Protocol for Mosquito Rearing A. gambiae

This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.

蚊子养育的议定书“（冈比亚按蚊）

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

This technique exposes the Drosophila embryonic neuromusculature for immunohistochemistry or electrophysiological recording. It is useful for studying early events in neuromuscular development or performing electrophysiology in mutants that cannot hatch.

收获和准备果蝇胚胎的电生理记录和其他程序

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

DNA Stable-Isotope Probing (DNA-SIP)

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and nutrients into cellular biomass. As such, this cultivation-independent technique has been an important methodology for assigning metabolic function to the diverse communities inhabiting a wide range of terrestrial and aquatic environments. Following the incubation of an environmental sample with stable-isotope labelled compounds, extracted nucleic acid is subjected to density gradient ultracentrifugation and subsequent gradient fractionation to separate nucleic acids of differing densities. Purification of DNA from cesium chloride retrieves labelled and unlabelled DNA for subsequent molecular characterization (e.g. fingerprinting, microarrays, clone libraries, metagenomics). This JoVE video protocol provides visual step-by-step explanations of the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labelled DNA. The protocol also includes sample SIP data and highlights important tips and cautions that must be considered to ensure a successful DNA-SIP analysis.

DNA Stable-Isotope Probing DNA-SIP

DNA stable-isotope probing is a cultivation-independent method to identify and characterize active communities of microorganisms that are capable of utilizing specific substrates. Assimilation of substrate enriched in heavy isotope leads to incorporation of labelled atoms into microbial biomass. Density gradient ultracentrifugation retrieves labelled DNA for downstream molecular analyses.

DNA的稳定同位素探测（DNA的SIP）

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and ...

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine parasitoids known to infect the host during the egg stage1. Second, it has a wide range of potential fruit fly hosts. Perhaps due to its life history, F. arisanus has been a successfully used for biological control of fruit flies in multiple tropical regions2-4. One impediment to the wide use of F. arisanus for fruit fly control is that it is difficult to establish a stable laboratory colony5-9. Despite this difficulty, in the 1990s USDA researchers developed a reliable method to maintain laboratory populations of F. arisanus10-12. There is significant interest in F. arisanus biology13,14, especially regarding its ability to colonize a wide variety of Tephritid hosts14-17; interest is especially driven by the alarming spread of Bactrocera fruit fly pests to new continents in the last decade18. Further research on F. arisanus and additional deployments of this species as a biological control agent will benefit from optimizations and improvements of rearing methods. In this protocol and associated video article we describe an optimized method for rearing F. arisanus based on a previously described approach12. The method we describe here allows rearing of F. arisanus in a small scale without the use of fruit, using materials available in tropical regions around the world and with relatively low manual labor requirements.

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus is an egg-larval parasitoid of Tephritid fruit flies that is successfully used in biological control of these important tropical pests. We describe here an optimized protocol for rearing F. arisanus on a small scale using readily available materials.

一个优化的协议饲养 Fopius arisanus Tephritid果寄生蝇

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine ...

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 ...

Simple Homemade Tools to Handle Fruit Flies&#8212;Drosophila melanogaster

The fruit fly, Drosophila melanogaster, is widely used both in biological research and biology education. Handling adult flies is common but difficult in practice, as adult flies fly. Demonstrated here is how to make some simple and cost-effective tools to address difficult issues in the handling of Drosophila. Holes in foam stoppers are made and pipette tips or funnels are inserted into the holes. Flies then move only in one direction into the pipette tip/funnel assemblage, allowing efficient control of the transfer of adult Drosophila into or out of a vial. Existing protocols have been modified for cool-anesthetizing flies by chilling in crushed ice and transferring them onto a cold, hard icepack surface. The icepack is covered with a piece of medical gauze that keeps immobilized flies from the condensed water when examined under a stereomicroscope. The flies are finally euthanized for counting and sorting or discarded by microwaving. A bottle-shaped cage has also been developed for collecting eggs, as well as a labor-saving device and accompanying protocol for cleaning glass culture vials.

Described here is the use of several homemade tools to transfer, chill, and kill adult Drosophila, as well as to clean glass culture vials and collect eggs. These tools are easy to make and are rather efficient in handling Drosophila.

简单的自制工具处理果蝇*果蝇黑色素

The fruit fly, Drosophila melanogaster, is widely used both in biological research and biology education. Handling adult flies is common but difficult in ...

Dissection of the Endolymphatic Sac from Mice

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the human phenotypes. Although important progress has been done in the understanding of the development and function of the neurosensory epithelia of the cochlea and vestibula, limited knowledge is available regarding the development, cellular composition, molecular pathways and functional characteristics of the endolymphatic sac. This is, in large part, due to the difficulty of visualizing and microdissecting this tissue, which is an epithelium comprised of only one cell layer. The study presented here describes an approach to access and microdissect the endolymphatic sac from the wild-type mouse inner ear at different ages. The result of a similar dissection is shown in a pendrin-deficient mouse model of enlargement of the vestibular aqueduct. A transgenic mouse with a fluorescent endolymphatic sac is presented. This reporter mouse can be used to readily visualize the endolymphatic sac with limited dissection and determine its size. It can also be used as an educational tool to teach how to dissect the endolymphatic sac. These dissection procedures should facilitate further characterization of this understudied part of the inner ear.

<meta charset="utf8"></head><body>小鼠内淋巴囊解剖

This study describes how to dissect the endolymphatic sac from the inner ear of the mouse at different ages. The result of a similar dissection is shown in an Slc26a4-null mouse model of enlargement of the endolymphatic sac. A transgenic mouse with a fluorescent reporter expressed in the endolymphatic sac is presented as a model to readily visualize the endolymphatic sac, and educational tool.

小鼠内淋巴囊解剖

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the ...

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules derived from the parental cells. EVs function as intra- and inter-species communication vectors while also contributing to the interaction between bacteria and host organisms in the context of infection and colonization. Given the multitude of functions attributed to EVs in health and disease, there is a growing interest in isolating EVs for in vitro and in vivo studies. It was hypothesized that the separation of EVs based on physical properties, namely size, would facilitate the isolation of vesicles from diverse bacterial cultures. 
The isolation workflow consists of centrifugation, filtration, ultrafiltration, and size-exclusion chromatography (SEC) for the isolation of EVs from bacterial cultures. A pump-driven tangential flow filtration (TFF) step was incorporated to enhance scalability, enabling the isolation of material from liters of starting cell culture. Escherichia coli was used as a model system expressing EV-associated nanoluciferase and non-EV-associated mCherry as reporter proteins. The nanoluciferase was targeted to the EVs by fusing its N-terminus with cytolysin A. Early chromatography fractions containing 20-100 nm EVs with associated cytolysin A - nanoLuc were distinct from the later fractions containing the free proteins. The presence of EV-associated nanoluciferase was confirmed by immunogold labeling and transmission electron microscopy. This EV isolation workflow is applicable to other human gut-associated gram-negative and gram-positive bacterial species. In conclusion, combining centrifugation, filtration, ultrafiltration/TFF, and SEC enables scalable isolation of EVs from diverse bacterial species. Employing a standardized isolation workflow will facilitate comparative studies of microbial EVs across species.

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Bacteria secrete nanometer-sized extracellular vesicles (EVs) carrying bioactive biological molecules. EV research focuses on understanding their biogenesis, role in microbe-microbe and host-microbe interactions and disease, as well as their potential therapeutic applications. A workflow for scalable isolation of EVs from various bacteria is presented to facilitate standardization of EV research.

从 大肠杆菌 和其他细菌中可扩展地分离和纯化细胞外囊泡

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules ...

稳定蝇及其他 Muscoid 双翅目的实验室饲养

摘要

探索更多视频

此视频中的章节

微生物群落，在自然和实验室 - 面试

蚊子养育的议定书“（冈比亚按蚊）

收获和准备果蝇胚胎的电生理记录和其他程序

DNA的稳定同位素探测（DNA的SIP）

一个优化的协议饲养 Fopius arisanus Tephritid果寄生蝇

无菌实验室技术:电镀方法

简单的自制工具处理果蝇**果蝇黑色素*

小鼠内淋巴囊解剖

小鼠内淋巴囊解剖

从大肠杆菌和其他细菌中可扩展地分离和纯化细胞外囊泡

稳定蝇及其他 Muscoid 双翅目的实验室饲养

摘要

探索更多视频

此视频中的章节

微生物群落，在自然和实验室 - 面试

蚊子养育的议定书“（冈比亚按蚊）

收获和准备果蝇胚胎的电生理记录和其他程序

DNA的稳定同位素探测（DNA的SIP）

一个优化的协议饲养 Fopius arisanus Tephritid果寄生蝇

无菌实验室技术:电镀方法

简单的自制工具处理果蝇*果蝇黑色素

小鼠内淋巴囊解剖

小鼠内淋巴囊解剖

从 大肠杆菌 和其他细菌中可扩展地分离和纯化细胞外囊泡

简单的自制工具处理果蝇**果蝇黑色素*

从大肠杆菌和其他细菌中可扩展地分离和纯化细胞外囊泡