Name: rearing the fruit fly drosophila melanogaster under axenic and gnotobiotic conditions
Uploaded: 2016-07-30T14:00:00.000+00:00
Duration: 9 min 34 s
Description: Watch this Scientific Journal Video about Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions at JoVE.com

该协议的一些细节与亚当博士布森，谁也对稿件提供了有益的意见的协助下进行了优化。这项工作是由该基金会为美国国立卫生研究院的支持（FNIH）授权号R01GM095372（JMC，A（CN）W，AJD和AED）。 FNIH授权号码1F32GM099374-01（PDN），和杨百翰大学的启动资金（江铃汽车，MLK，MV）。出版费用由生命科学杨百翰大学学院和植物和野生动物科学系的支持。

1.细菌培养（开始采摘〜鸡蛋前1周） <ol><li>制备改良的MRS 20（MMRS）板和肉汤管（ 表1）。倾20毫升MMRS琼脂到每个百毫米培养皿并冷却/干燥过夜，和5ml MMRS肉汤分为18 mm试管。 </li><li>条纹醋pomorum，A. 热带 ， 短乳杆菌 ，和L.植物上MMRS琼脂平板上。孵育醋杆菌在30℃下过夜。通过在密封前用二氧化碳将所述板在密封容器中，并驱孵育乳杆菌厌氧。在30℃下孵育过夜。 注:L。短菌落可能是不可见的，直到24-48小时。板可贮存于4℃，菌落可以用于长达3周。 </li><li> dechorionated蛋转移到无菌的饮食（部分5）前两至三天，挑从MMRS板单菌落置于试管CON泰宁MMRS肉汤。成长醋振荡和静态成长乳杆菌 24小时或直至混浊，都在30℃。 </li></ol> 2.准备无菌饮食<ol><li>在2升Erlenmeyer烧瓶制备无菌饲料（ 见表2）。微波饮食，直到它已煮3顺序倍;每间熬混合英寸</li><li>广场上的轰动板烧瓶保持在传输饮食，锥形离心管中搅拌。转移7.5毫升饮食至50ml离心管中。松散帽管，并放入有盖，高压灭菌聚丙烯架。 </li><li>消毒用在121℃的高压釜中，15磅25分钟飞饮食。从反应釜中取出衣架，立刻水平摇晃每个机架，保证饮食冷却过程中不分离。小心不可撼动的机架垂直，以防止食物转移到盖子或管的边缘。允许饮食，在振动台上冷却正好45分钟，再用手水平摇晃饮食。 注意:饮食可以储存在4-15°C至一个星期。 </li><li>作为替代使用覆盖高压灭菌机架（步骤2.1-2.3），或在含有酸防腐剂饮食提高苍蝇，执行以下步骤: <ol><li>与在烧瓶中搅拌棒的2L烧瓶中制备1升液体的饮食。高压灭菌饮食。 </li><li>高压灭菌后，加入10 mL防腐剂在加热搅拌盘不断搅拌。当琼脂已冷却至50-60℃，将该烧瓶加热至50-60℃移动到加热的搅拌板上在生物安全柜并保持烧瓶的温度。 </li><li>在生物安全柜，吸管〜7.5毫升到单独锥形管。 </li></ol></li></ol> 3.准备产蛋笼<ol><li>通过微波100ml水中，将10g啤酒酵母，10g的葡萄糖，和1g琼脂使葡萄果汁琼脂平板上。煮沸3次，在步骤1.1，并添加10克冷冻葡萄汁的合作ncentrate加大对琼脂平板鸡蛋的可见性。 </li><li>当琼脂已冷却至55℃，倾20毫升入100 25mm培养皿，并允许固化。 </li><li>通过混合1克啤酒酵母用15g水覆盖的酵母膏的琼脂平板的表面上。倒入酵母粘贴到琼脂平板上，确保表面覆盖，然后倒出多余的，留下薄薄的酵母残留落后。如果使用多个板，浆料可以顺序倒入到多个板。 </li><li>转移琼脂平板成笼的底部。 注:32盎司熟食容器是丙烯酸飞笼很好的替代品。 <ol><li>通过切割在顶部的孔并用无毒胶水空穴胶合一个透气网作出32盎司熟食容器盖。如果胶是水溶性防止水接触到盖子。 </li></ol></li><li>转让200-300飞入容器中，并盖上盖子。盖一个空的培养皿的盖子网状保护孔防止措施从琼脂面T蒸发和根据需要添加湿润薄纸在盖的内部。孵育在25℃下过夜苍蝇为16-20小时。 </li></ol> 4.收集鸡蛋<ol><li>通过将尼龙网到塑料套管准备鸡蛋收集筛子。 </li><li>以检索与在其上的蛋的板，通过转移到一个空的容器从笼中移除苍蝇。如果相同蝇将要使用的第二天，立即转移到含有新鲜yeasted葡萄果汁琼脂平板新笼子。果蝇可用于3-5顺序天</li><li>取下琼脂死苍蝇用干净的画笔，小心不要分手琼脂。 </li><li>通过用蒸馏水冲洗琼脂平板上，轻轻地从琼脂表面涂刷鸡蛋，并浇在网状浆料收集鸡蛋。重复3-4次，直到所有或​​大部分卵已经从琼脂平板中移除。 </li></ol> 5. Dechorionate鸡蛋，并转移到无菌ðIET <ol><li>通过用70％乙醇喷雾内（包括边）制备的生物安全柜中。擦拭用实验室组织的底部，并消毒用UV光对〜15分钟的引擎盖。通过用乙醇喷涂和在生物安全柜立即放置消毒所有非生物用品（样品杯，画笔，镊子，废物容器，加入400ml无菌水和100ml 0.6％次氯酸钠）。用UV光杀菌15分钟。 </li><li>通过将与鸡蛋衬套到120ml的样品杯或其它无菌容器开始第一次的2次氯酸钠洗涤。慢慢倒入〜90毫升0.6％的次氯酸钠溶液注入衬套直到刚好低于边缘。 </li><li>冲洗鸡蛋2.5分钟。通过使用镊子上下在次氯酸盐溶液移动至衬套定期重新悬浮卵。 </li><li>直接传输衬套成第二试样杯，预填充用90毫升漂白剂，生物安全柜的内部。 </li><li>重复步骤5.3生物安全柜内。在第二漂白处理结束时，鸡蛋应开始粘附到套管的侧面。 </li><li>执行步骤在生物安全柜5.7-5.8。 </li><li>丢弃漂白，并用无菌水冲洗套管3次。通过移动与钳套管重悬卵每次洗涤过程中数次。由第三洗涤的端部最蛋应连接到套管的侧。 </li><li>使用在乙醇灭菌画笔，从套管的侧转移蛋无菌饮食。单独或小批量传输鸡蛋。瞄准每瓶30-50鸡蛋。离开松瓶盖，让氧气进入管。如果小瓶保持无菌，转移到昆虫孵化器;否则，如下补充细菌。 </li></ol> 6.进行悉生蝇使用4种细菌<ol><li>准备细菌<ol><li>准备好必要的用品消毒生物安全柜（Pipettes，枪头盒，灭菌离心管，MRS肉汤，和试管架）如在步骤4.1。擦拭试管的外面用乙醇浸泡过的实验室生物安全柜放置前擦拭。 </li><li>通过第一转印500微升过夜生长至无菌微量离心管沉淀细菌。如果细菌密度低，最多1.5毫升添加到每个管或依次除去上清液，加入额外培养到相同的管中。从生物安全柜，离心在10000克10分钟取出样品。使用过滤嘴，以避免样品间污染。 </li><li>通过测量OD 600确定每种培养的密度。如果使用多孔板读取器，转移200μl的每种培养到96孔板中的1-，2-，和4-倍稀释液。 </li><li>确定MMRS在其中稀释使用平板读数分光光度计每个细胞沉淀（5.2.2）和下列公式的量。计划添加的肉汤接种50＃956; l每个飞瓶。 <ol><li>收集的OD 600读数为1:1，1:2和1:在平板读数分光光度计4稀释各细菌培养。选择稀释产生0.1和0.2，并使用这个值和相应的稀释因素，因为在6.1.4.2 6.1.4.3或给出的公式&#39;O&#39;和&#39;D&#39;之间的OD值600各种菌株。 </li><li>如果使用4种这里描述的，使用此方程正常化细胞等效集落形成单位（CFU）/ ml的密度（OD 600〜CFU转换确定先前20）: E =（（OB）x垂直x深）/ C 其中E =体积重悬在（微升）颗粒，O = OD 600细菌，B = 600 OD空白介质，D =倍稀释，V =微升细菌培养离心前，预定常数C = OD 600。见补充代码文件使用这些公式计算的例子。对于分光光度计S中的自动空白，代替"OB"使用"O"。 注意:预定常数（单位OD 600，归一化至10 7 CFU毫升-1，在20导出常数）如下:A.热带 （0.052），A。 pomorum（0.038），L。 短 （0.056），L。杆菌 （0.077）。 </li><li>如果使用其他的细菌种类（无CFU / OD 600恒定可用），密度正常化使用这个公式OD 600 = 0.1: E =（（OB）x垂直x深）/0.1 OD 600 注:单位中的相同步骤6.1.4.2。见补充代码文件使用这些公式计算的例子。 </li></ol></li><li>在生物安全柜，除去上清液用枪头重悬新鲜MMRS或PBS颗粒作为步骤6.1.4.2计算。 </li></ol></li><li>细菌接种<ol><li>转移50微升细菌到锥形管中，用无菌饮食的d。在生物安全柜dechorionated鸡蛋。加入鸡蛋的细菌转移后，以防止小瓶间污染。 </li><li>放置接种管在培养箱中在25℃ </li></ol></li></ol>对于无菌7.测量负荷CFU /测试<ol><li>为了测量整个身体飞匀浆，转移5苍蝇（5-7天后羽化）含有的陶瓷珠125微升和125微升MMRS肉汤1.7 ml离心管中CFU负荷。以4.0米/秒均化使用30秒的组织匀浆苍蝇。 <ol><li>或者，省略珠和手在微量离心管均质用塑料杵1分钟。 </li><li>如果量化肠道菌群，表面消毒苍蝇删除外源性微生物22。转印蝇至含有100μl的70％乙醇1分钟，吸出乙醇，并转移到为homogenizations一个新的离心管离心管。如果肠的DNA含量进行测定，漂洗˚F或1分钟用乙醇洗涤前0.6％次氯酸钠。 </li></ol></li><li>稀释用875微升MMRS，涡旋5秒匀浆，和吸管120微升匀浆到微量滴定板的第一井。 </li><li>执行两个连续的1:8使用10微升匀浆和70微升MRS在接下来的两个井稀释。 <ol><li>从第一井去除10微升，并将其添加到第二阱含有70微升的MRS。调匀第二阱中的内容，传输从第二井10微升到第三孔含有70微升的MRS，调匀。这导致3总浓度原始1000微升匀浆:未稀释的，1:8，和1:64。 </li></ol></li><li>转移10微升每种稀释到MMRS板（如果需要使用多通道移液管）。略微倾斜的菜扩散稀释几毫米下来琼脂表面，并允许液体移动板之前干燥。液体晒干日e盘若快速板2天，减少了两个相邻液滴混合。 </li><li>孵育在30℃下1-2天。从培养箱一次不同，单个菌落是可见的除去板，以及从与10-100分离的菌落稀释计数。 </li><li>用公式:E = C x深/ P X [V / F，其中每飞E = CFU，C =计数的菌落数，D =稀释，P =微升镀金，V =掺量匀浆计算每飞CFU和F =匀浆蝇数。 </li></ol>

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作者什么都没有透露。

<html>此处所描述的方法是几种方法用于胚胎dechorionation 8,11,18,25,26,27之一，饲养无菌蝇，包括无菌的成年人18,27的串行传输或抗生素治疗13,18的替代方法在一起。其他dechorionation方法包括乙醇洗涤，减少11,25,26或延长8次氯酸盐治疗。不同的洗涤步骤可以帮助饲养不同蝇基因型:在先前的研究中最〜的饲养时如这里概述（无乙醇洗涤）100 果蝇基因型无菌的...

An erratum was issued for: <a href="https://www.jove.com/t/54219/rearing-fruit-fly-drosophila-melanogaster-under-axenic-gnotobiotic">Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions</a>. The Protocol was updated.
Step 6.1.4.2 of the Protocol was updated from:
If using the 4 species described here, normalize cells to equivalent colony forming unit (CFU)/ml densities (OD600 to CFU conversion determined previously20) using this equation: 
E = ((O-B) x V x D)/C 
where E = volume to resuspend pellet in (&#956;l), O = OD600 bacteria, B = OD600 blank media, D = fold-dilution, V = &#181;l bacterial culture prior to centrifugation, C = OD600 of predetermined constant. See Supplemental Code File for examples of calculations using these equations. For spectrophotometers that automatically blank, use &#34;O&#34; in place of &#34;O-B&#34;. 
Note: The predetermined constants (units OD600, normalized to 107 CFU ml-1, constants derived in20) are as follows: A. tropicalis (0.052), A. pomorum (0.038), L. brevis (0.056), L. plantarum (0.077).
to:
If using the 4 species described here, normalize cells to equivalent colony forming unit (CFU)/ml densities (OD600 to CFU conversion determined previously20) using this equation: 
E = ((O-B) x V x D)/C 
where E = volume to resuspend pellet in (&#956;l), O = OD600 bacteria, B = OD600 blank media, D = fold-dilution, V = &#181;l bacterial culture prior to centrifugation, C = OD600 of predetermined constant. See Supplemental Code File for examples of calculations using these equations. For spectrophotometers that automatically blank, use &#34;O&#34; in place of &#34;O-B&#34;. 
Note: The predetermined constants (units OD600, normalized to 107 CFU ml-1, constants derived in20) are as follows: A. tropicalis (0.053), A. pomorum (0.038), L. brevis (0.077), L. plantarum (0.056).

An erratum was issued for: <a href="https://www.jove.com/t/54219/rearing-fruit-fly-drosophila-melanogaster-under-axenic-gnotobiotic">Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions</a>. The Protocol was updated.

Erratum: Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

大多数动物密切与细菌（&#39;微生物&#39;）从出生到死亡1有关。免费微生物（&#39;无菌&#39;）和微生物相关的（"传统"）的动物的比较表明微生物影响动物健康的各个方面，包括代谢，营养，血管，肝脏，呼吸系统，免疫，内分泌和神经功能2。果蝇果蝇是用于理解许多微生物3,4的存在下这些方法的并为研究对动物健康5,6-菌群影响的关键模式。没有细菌种类存在于每一个个体（"核心"），但醋酸和乳酸杆菌数字都占据主导地位实验室饲养和野生捕捞D的微生物果蝇 。其他Acetobacteraceae（包括Komagataeibacter和葡萄糖酸 ）， 菲尔米cutes（如肠球菌和明串珠菌 ）和肠杆菌科细菌无论是在低丰度经常出现在果蝇中的个人，或不定期出现在高丰度7-12。 果蝇和哺乳动物的微生物是内和跨世代14,19见异思迁。测量微生物相关的特征时，菌群世态炎凉会导致表型的噪音。例如，Acetobacteraceae影响脂质（甘油三酯）存储在果蝇 15-18。如果Acetobacteraceae是比另外19一小瓶的苍蝇更丰富，同基因苍蝇可以有不同的表型20。自1960年代以来，一种用于小鼠14菌群世态炎凉的问题的解决方案已经在实践中，通过引入定义8优势微生物物种社会的乳鼠每一代新产品（改变Schaedler菌群）确保每个小狗暴露于小鼠菌群的同一主要成员。即使当菌群不研究32的主要目标，并设置先例，以确保密钥的微生物在各种实验条件下的存在于微生物群组成这种做法控制。 上定义果蝇营养微生物的影响下，用于导出无菌飞线几个协议已被开发，包括胚胎次氯酸钠dechorionation（无论是衍生从头每一代或通过转移到无菌的饮食的世代保持）和抗生素治疗13。有益处的不同方法，如方便与快捷为抗生素治疗和串行传输，相对于与从头 dechorionation混杂变量（ 例如 ，卵密度，残留污染微生物，脱靶抗生素效果）的更大的控制。不管该方法的准备，出台规定微生物物种对无菌胚胎允许果蝇的文化与自定义（"悉生&#39;）社区。可替代地，模仿使用Schaedler菌群，该社区可以接种到常规的鸡蛋（以下步骤6-7只），以确保在每个小瓶特质影响微生物的存在，并避免菌群反复无常的并发症。在这里，我们描述了协议用 ​​于通过胚胎从头 dechorionation提高无菌和限菌果蝇 ，并确认导入或污染微生物类群的存在。

<table><tbody><tr><td>Brewer&#039;s Yeast</td><td>MP Biomedicals, LLC.</td><td>903312</td><td>http://www.mpbio.com/product.php?pid=02903312</td></tr><tr><td>Glucose</td><td>Sigma Aldrich</td><td>158968-3KG</td><td>http://www.sigmaaldrich.com/catalog/product/aldrich/158968?lang=en&amp;amp;region=US</td></tr><tr><td>Agar</td><td>Fisher--Lab Scientific</td><td>fly802010</td><td>https://www.fishersci.com/shop/products/drosophila-agar-8-100mesh-10kg/nc9349177</td></tr><tr><td>Welch&#039;s 100% Grape Juice Concentrate</td><td>Walmart or other grocery store</td><td>9116196</td><td>http://www.walmart.com/ip/Welch-s-Frozen-100-Grape-Juice-Concentrate-11.5-oz/10804406</td></tr><tr><td>Cage: 32 oz. Translucent Round Deli Container</td><td>Webstaurant Store</td><td>999L5032Y</td><td>http://www.webstaurantstore.com/newspring-delitainer-sd5032y-32-oz-translucent-round-deli-container-24-pack/999L5032Y.html</td></tr><tr><td>Translucent Round Deli Container Lid</td><td>Webstaurant Store</td><td>999YNL500</td><td>http://www.webstaurantstore.com/newspring-delitainer-ynl500-translucent-round-deli-container-lid-60-pack/999YNL500.html</td></tr><tr><td>Stock Bottles</td><td>Genesee Scientific</td><td>32-130</td><td>https://geneseesci.com/shop-online/product-details/?product=32-130</td></tr><tr><td>Droso-Plugs</td><td>Genesee Scientific</td><td>49-101</td><td>https://geneseesci.com/shop-online/product-details/?product=49-101</td></tr><tr><td>Nylon Mesh</td><td>Genesee Scientific</td><td>57-102&amp;nbsp;</td><td>https://geneseesci.com/shop-online/product-details/715/?product=57-102</td></tr><tr><td>Plastic Bushing</td><td>Home Depot</td><td>100404002</td><td>http://www.homedepot.com/p/Carlon-1-in-Non-Metallic-Terminal-Adapter-E943FR-CTN/100404002</td></tr><tr><td>Plastic Bushing Cap</td><td>Home Depot</td><td>100153897</td><td>http://www.homedepot.com/p/1-1-4- in-Rigid-Plastic-Insulated-Bushing-2-Pack-27529/100153897</td></tr><tr><td>Specimen Cup</td><td>MedSupply Partners</td><td>K01-207067</td><td>http://www.medsupplypartners.com/covidien-specimen-containers.html</td></tr><tr><td>Repeater M4</td><td>Eppendorf</td><td>4982000322</td><td>https://online-shop.eppendorf.us/US-en/Manual-Liquid-Handling-44563/Dispensers--Burettes-44566/Repeater-M4-PF-44619.html</td></tr><tr><td>50 ml Centrifuge Tubes</td><td>TrueLine Centrifuge Tubes</td><td>TR2003</td><td>https://www.lpsinc.com/Catalog4.asp?catalog_nu=TR2003</td></tr><tr><td>Food Boxes</td><td>USA Scientific</td><td>2316-5001</td><td>http://www.usascientific.com/search.aspx?find=2316-5001</td></tr><tr><td>Lysing Matrix D Bulk</td><td>MP Biomedicals, LLC.</td><td>116540434</td><td>http://www.mpbio.com/search.php?q=6540-434&amp;amp;s=Search</td></tr><tr><td>Filter Pipette Tips, 300 &amp;mu;l</td><td>USA Scientific</td><td>1120-9810</td><td>http://www.usascientific.com/search.aspx?find=1120-9810</td></tr><tr><td>Petri Dishes</td><td>Laboratory Product Sales</td><td>M089303</td><td>https://www.lpsinc.com/Catalog4.asp?catalog_nu=M089303</td></tr><tr><td>Ethanol</td><td>Decon Laboratories, INC.</td><td>2701</td><td>http://www.deconlabs.com/products.php?ID=88</td></tr><tr><td>Paintbrush</td><td>Walmart</td><td>5133</td><td>http://www.walmart.com/ip/Chenille-Kraft-5133-Acrylic-Handled-Brush-Set-Assorted-Sizes-colors-8-Brushes-set/41446005</td></tr><tr><td>Forceps</td><td>Fisher</td><td>08-882</td><td>https://www.fishersci.com/shop/products/fisherbrand-medium-pointed-forceps-3/p-128693</td></tr><tr><td>Household Bleach (6-8% Hypochlorite)</td><td>Walmart</td><td>550646751</td><td>http://www.walmart.com/ip/Clorox-Concentrated-Regular-Bleach-121-fl-oz/21618295</td></tr><tr><td>Universal Peptone</td><td>Genesee Scientific</td><td>20-260</td><td>https://geneseesci.com/shop-online/product-details/?product=20-260</td></tr><tr><td>Yeast Extract</td><td>&amp;nbsp;Fisher Scientific</td><td>BP1422-500</td><td>https://www.fishersci.com/shop/products/fisher-bioreagents-microbiology-media-additives-yeast-extract-3/bp1422500?matchedCatNo=BP1422500</td></tr><tr><td>Dipotassium Phosphate</td><td>Sigma Aldrich</td><td>P3786-1KG</td><td>http://www.sigmaaldrich.com/catalog/search?term=P3786-1KG&amp;amp;interface=All&amp;amp;&lt;br/&gt;
N=0&amp;amp;mode=match%20partialmax&amp;amp;lang=&lt;br/&gt;
en&amp;amp;region=US&amp;amp;focus=product</td></tr><tr><td>Ammonium Citrate</td><td>Sigma Aldrich</td><td>25102-500g</td><td>http://www.sigmaaldrich.com/catalog/search?term=25102-500g&amp;amp;interface=All&amp;amp;N&lt;br/&gt;
=0&amp;amp;mode=match%20partialmax&amp;amp;lang=&lt;br/&gt;
en&amp;amp;region=US&amp;amp;focus=product</td></tr><tr><td>Sodium Acetate</td><td>VWR</td><td>97061-994</td><td>https://us.vwr.com/store/catalog/product.jsp?catalog_number=97061-994</td></tr><tr><td>Magnesium Sulfate</td><td>Fisher Scientific</td><td>M63-500</td><td>https://www.fishersci.com/shop/products/magnesium-sulfate-heptahydrate-crystalline-certified-acs-fisher-chemical-3/m63500?matchedCatNo=M63500</td></tr><tr><td>Manganese Sulfate</td><td>Sigma Aldrich</td><td>10034-96-5</td><td>http://www.sigmaaldrich.com/catalog/search?term=10034-96-5&amp;amp;interface=CAS%20No.&amp;amp;N=0&amp;amp;mode=match&lt;br/&gt;
%20partialmax&amp;amp;lang=en&amp;amp;region&lt;br/&gt;=US&amp;amp;focus=product</td></tr><tr><td>MRS Powder</td><td>Sigma Aldrich</td><td>69966-500G</td><td>http://www.sigmaaldrich.com/catalog/product/sial/69966?lang=en&amp;amp;region=US</td></tr><tr><td>96 Well Plate Reader</td><td>BioTek (Epoch)&amp;nbsp;</td><td>NA</td><td>http://www.biotek.com/products/microplate_detection/epoch_microplate_&lt;br/&gt;spectrophotometer.html</td></tr><tr><td>1.7 ml Centrifuge Tubes</td><td>USA Scientific</td><td>1615-5500</td><td>http://www.usascientific.com/search.aspx?find=1615-5500</td></tr><tr><td>Filter Pipette Tips, 1,000 &amp;mu;l</td><td>USA Scientific</td><td>1122-1830</td><td>http://www.usascientific.com/search.aspx?find=1122-1830</td></tr><tr><td>96 Well Plates</td><td>Greiner Bio-One</td><td>655101</td><td>https://shop.gbo.com/en/usa/articles/catalogue/article/0110_0040_0120_0010/&lt;br/&gt;13243/</td></tr><tr><td>Ceramic Beads</td><td>MP Biomedicals, LLC.</td><td>6540-434</td><td>http://www.mpbio.com/product.php?pid=116540434</td></tr><tr><td>Tissue Homogenizer</td><td>MP Biomedicals, LLC.</td><td>116004500</td><td>http://www.mpbio.com/product.php?pid=116004500</td></tr><tr><td>Class 1 BioSafety Cabinet</td><td>Thermo Scientific&amp;nbsp;</td><td>Model 1395</td><td>http://www.thermoscientific.com/en/product/1300-series-class-ii-type-a2-biological-safety-cabinet-packages.html</td></tr></tbody></table>

rearing the fruit fly drosophila melanogaster under axenic and gnotobiotic conditions

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is a model for understanding microbial interactions with animal hosts, facilitated by approaches to rear large sample sizes of Drosophila under microorganism-free (axenic) conditions, or with defined microbial communities (gnotobiotic). This work outlines a method for collection of Drosophila embryos, hypochlorite dechorionation and sterilization, and transfer to sterile diet. Sterilized embryos are transferred to sterile diet in 50 ml centrifuge tubes, and developing larvae and adults remain free of any exogenous microbes until the vials are opened. Alternatively, flies with a defined microbiota can be reared by inoculating sterile diet and embryos with microbial species of interest. We describe the introduction of 4 bacterial species to establish a representative gnotobiotic microbiota in Drosophila. Finally, we describe approaches for confirming bacterial community composition, including testing if axenic Drosophila remain bacteria-free into adulthood.

无菌蝇饲养成功是没有从D的全身homogenizations CFU的隔离证实果蝇成虫（ 图1）。或者，如果镀覆匀浆得到的菌落，小瓶被污染并应该被丢弃。为限菌苍蝇，四个细菌菌株是从5成年男性池隔离，以表明其与成蝇（ 图1）相关联的总活菌的CFU的差异。每个细菌物种具有不同的形态，并可以在视觉上加以区分（ 图2）。如果没有?...

Watch this Scientific Journal Video about Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions at JoVE.com

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

提出了一种用于无菌和限菌条件下饲养果蝇的方法。飞胚dechorionated在次氯酸钠，无菌转移到无菌的饮食，并在密闭容器中饲养。接种饮食和胚胎与细菌导致限菌协会，和细菌的存在是通过电镀全身果蝇匀浆证实。

饲养果蝇<em&gt;果蝇</em&gt;在无菌和悉生条件

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is ...

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Developmental Biology

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

21.0K 次观看.  Brigham Young University. 提出了一种用于无菌和限菌条件下饲养果蝇的方法。飞胚dechorionated在次氯酸钠，无菌转移到无菌的饮食，并在密闭容器中饲养。接种饮食和胚胎与细菌导致限菌协会，和细菌的存在是通过电镀全身果蝇匀浆证实。 

视频: Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron &#8211; dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

一种研究外周和中枢神经系统 Neuroregeneration 的果蝇体内损伤模型

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various ...

科研

医学

Drosophila melanogaster Larva Injection Protocol

The use of unconventional models to study innate immunity and pathogen virulence provides a valuable alternative to mammalian models, which can be costly and raise ethical issues. Unconventional models are notoriously cheap, easy to handle and culture, and do not take much space. They are genetically amenable and possess complete genome sequences, and their use presents no ethical considerations. The fruit fly Drosophila melanogaster, for instance, has provided great insights into a variety of behavior, development, metabolism, and immunity research. More specifically, D. melanogaster adult flies and larvae possess several innate defense reactions that are shared with vertebrate animals. The mechanisms regulating immune responses have been mostly revealed through genetic and molecular studies in the D. melanogaster model. Here a novel larval injection technique is provided, which will further promote investigations of innate immune processes in D. melanogaster larvae and explore the pathogenesis of a wide range of microbial infections.

Drosophila melanogaster Larva Injection Protocol

Drosophila melanogaster adult flies have been extensively utilized as model organisms to investigate the molecular mechanisms underlying host antimicrobial innate immune responses and microbial infection strategies. To promote the D. melanogaster larva stage as an additional or alternative model system, a larval injection technique is described.

果蝇 幼虫注射方案

The use of unconventional models to study innate immunity and pathogen virulence provides a valuable alternative to mammalian models, which can be costly ...

免疫与感染

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

Foraging and feeding behaviors allow animals to access sources of energy and nutrients essential for their development, health, and fitness. Investigating the neuronal regulation of these behaviors is essential for the understanding of the physiological and molecular mechanisms underlying nutritional homeostasis. The use of genetically tractable animal models such as worms, flies, and fish greatly facilitates these types of studies. In the last decade, the fruit fly Drosophila melanogaster has been used as a powerful animal model by neurobiologists investigating the neuronal control of feeding and foraging behaviors. While undoubtedly valuable, most studies examine adult flies. Here, we describe a protocol that takes advantage of the simpler larval nervous system to investigate neuronal substrates controlling feeding behaviors when larvae are exposed to diets differing in their protein and carbohydrates content. Our methods are based on a quantitative colorimetric no-choice feeding assay, performed in the context of a neuronal thermogenetic-activation screen. As a read-out, the amount of food eaten by larvae over a 1 h interval was used when exposed to one of the three dye-labeled diets that differ in their protein to carbohydrates (P:C) ratios. The efficacy of this protocol is demonstrated in the context of a neurogenetic screen in larval Drosophila, by identifying candidate neuronal populations regulating the amount of food eaten in diets of different macronutrient quality. We were also able to classify and group the genotypes tested into phenotypic classes. Besides a brief review of the currently available methods in the literature, the advantages and limitations of these methods are discussed and, also, some suggestions are provided about how this protocol might be adapted to other specific experiments.

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

Described here is a protocol that enables the colorimetric quantification of the amount of food eaten within a defined interval of time by Drosophila melanogaster larvae exposed to diets of different macronutrient quality. These assays are conducted in the context of a neuronal thermogenetic screen.

使用 德罗索菲拉· 拉瓦在恒温遗传神经元屏幕上摄入的宏量营养素的量化

Foraging and feeding behaviors allow animals to access sources of energy and nutrients essential for their development, health, and fitness. Investigating ...

神经科学

Neural Stem Cell Reactivation in Cultured Drosophila Brain Explants

Neural stem cells (NSCs) have the ability to proliferate, differentiate, undergo apoptosis, and even enter and exit quiescence. Many of these processes are controlled by the complex interplay between NSC intrinsic genetic programs with NSC extrinsic factors, local and systemic. In the genetic model organism, Drosophila melanogaster, NSCs, known as neuroblasts (NBs), switch from quiescence to proliferation during the embryonic to larval transition. During this time, larvae emerge from their eggshells and begin crawling, seeking out dietary nutrients. In response to animal feeding, the fat body, an endocrine organ with lipid storage capacity, produces a signal, which is released systemically into the circulating hemolymph. In response to the fat body-derived signal (FBDS), Drosophila insulin-like peptides (Dilps) are produced and released from brain neurosecretory neurons and glia, leading to downstream activation of PI3-kinase growth signaling in NBs and their glial and tracheal niche. Although this is the current model for how NBs switch from quiescence to proliferation, the nature of the FBDS extrinsic cue remains elusive. To better understand how NB extrinsic systemic cues regulate exit from quiescence, a method was developed to culture early larval brains in vitro before animal feeding. With this method, exogenous factors can be supplied to the culture media and NB exit from quiescence assayed. We found that exogenous insulin is sufficient to reactivate NBs from quiescence in whole-brain explants. Because this method is well-suited for large-scale screens, we aim to identify additional extrinsic cues that regulate NB quiescence versus proliferation decisions. Because the genes and pathways that regulate NSC proliferation decisions are evolutionarily conserved, results from this assay could provide insight into improving regenerative therapies in the clinic.

Neural Stem Cell Reactivation in Cultured Drosophila Brain Explants

A method to reactivate quiescent neural stem cells in cultured Drosophila brain explants has been established. Using this method, the role of systemic signals can be uncoupled from tissue-intrinsic signals in the regulation of neural stem cell quiescence, entry and exit.

培养的 果蝇 脑外植体中的神经干细胞再激活

Neural stem cells (NSCs) have the ability to proliferate, differentiate, undergo apoptosis, and even enter and exit quiescence. Many of these processes ...

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ecologically-important endpoints, though they are rarely considered in toxicological studies. D. melanogaster is emerging as a toxicology model for the behavioral, neurological, and genetic impacts of toxicants, to name a few. More importantly, species in the genus Drosophila can be utilized as a model system for an integrative framework approach to incorporate emergent properties and answer ecologically-relevant questions in&#160;toxicology research. The aim of this paper is to provide a protocol for exposing species in the genus Drosophila to pollutants to be used as a model system for a range of phenotypic outputs and ecologically-relevant questions. More specifically, this protocol can be used to 1) link multiple biological levels of organization and understand the impact of toxicants on both individual- and population-level fitness; 2) test the impact of toxicants at different stages of developmental exposure; 3) test multigenerational and evolutionary implications of pollutants; and 4) test multiple contaminants and stressors simultaneously.

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

In this paper, we provide a detailed protocol for exposing species in the genus Drosophila to pollutants with the goal of studying the impact of exposure on a range of phenotypic outputs at different developmental stages and for more than one generation.

利用果蝇作为无脊椎动物毒性试验模型系统的实验协议

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ...

生物学

Oral Bacterial Infection and Shedding in Drosophila melanogaster

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic infections, there has been a recent increase of interest in the mechanisms of gut immunocompetence to pathogens, which require methods to orally infect flies. Here we present a protocol to orally expose individual flies to an opportunistic bacterial pathogen (Pseudomonas aeruginosa) and a natural bacterial pathogen of D. melanogaster (Pseudomonas entomophila). The goal of this protocol is to provide a robust method to expose male and female flies to these pathogens. We provide representative results showing survival phenotypes, microbe loads, and bacterial shedding, which is relevant for the study of heterogeneity in pathogen transmission. Finally, we confirm that Dcy mutants (lacking the protective peritrophic matrix in the gut epithelium) and Relish mutants (lacking a functional immune deficiency (IMD) pathway), show increased susceptibility to bacterial oral infection. This protocol, therefore, describes a robust method to infect flies using the oral route of infection, which can be extended to the study of a variety genetic and environmental sources of variation in gut infection outcomes and bacterial transmission.

Oral Bacterial Infection and Shedding in Drosophila melanogaster

This protocol describes methods to orally expose and infect the fruit fly Drosophila melanogaster with bacterial pathogens, and to measure the number of infectious bacteria shed following gut infection. We further describe the effect of immune mutants on fly survival following oral bacterial infection.

果蝇的口腔细菌感染和脱落

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic ...

Establishment and Maintenance of Gnotobiotic American Cockroaches (Periplaneta americana)

Gnotobiotic animals are a powerful tool for the study of controls on microbiome structure and function. Presented here is a protocol for the establishment and maintenance of gnotobiotic American cockroaches (Periplaneta americana). This approach includes built-in sterility checks for ongoing quality control. Gnotobiotic insects are defined here as cockroaches that still contain their vertically transmitted endosymbiont (Blattabacterium) but lack other microbes that normally reside on their surface and in their digestive tract. For this protocol, egg cases (oothecae) are removed from a (nonsterile) stock colony and surface sterilized. Once collected and sterilized, the oothecae are incubated at 30 &#176;C for approximately 4&#8722;6 weeks on brain-heart infusion (BHI) agar until they hatch or are removed due to contamination. Hatched nymphs are transferred to an Erlenmeyer flask containing a BHI floor, sterile water, and sterile rat food. To ensure that the nymphs are not housing microbes that are unable to grow on BHI in the given conditions, an additional quality control measure uses restriction fragment-length polymorphism (RFLP) to test for nonendosymbiotic microbes. Gnotobiotic nymphs generated using this approach can be inoculated with simple or complex microbial communities and used as a tool in gut microbiome studies.

Establishment and Maintenance of Gnotobiotic American Cockroaches Periplaneta americana

This protocol is used in establishing and maintaining gnotobiotic American cockroaches (Periplaneta americana) by surface sterilizing the egg cases (oothecae) prior to hatching. These gnotobiotic insects contain their vertically transmitted Blattabacterium endosymbionts but have axenic guts.

建立和维护侏罗育美国蟑螂 （佩里普拉内塔美洲）

Gnotobiotic animals are a powerful tool for the study of controls on microbiome structure and function. Presented here is a protocol for the establishment ...

Drosophila Maintenance

Drosophila melanogaster, commonly known as fruit flies, are a frequently used model organism for life science research. Although starting a collection of these critters may seem as easy as leaving a banana on your kitchen counter for too long, a productive fly colony in the lab requires careful husbandry and maintenance.
This video demonstrates the necessary steps for maintaining a healthy fly stock. The overview begins with the preparation and storage of the yeast and sugar-containing media on which flies feed. Next, the vessels most commonly used for housing Drosophila are shown, as well as how and when to move flies between these containers. Finally, the presentation also includes examples of the ways in which housing and feeding conditions are manipulated for biological experiments.

Maintenance and Husbandry of Drosophila melanogaster

果蝇的饲养与保存

Drosophila melanogaster, commonly known as fruit flies, are a frequently used model organism for life science research. Although starting a collection of ...

教育

JoVE Science Education

基础生物学

模式生物I:酵母，果蝇和秀丽线虫

Drosophila melanogaster Embryo and Larva Harvesting and Preparation

Drosophila melanogaster embryos and larvae are easy to manipulate and develop rapidly by mechanisms that are analogous to other organisms, including mammals. For these reasons, many researchers utilize fly embryos and larvae to answer questions in diverse fields ranging from behavioral to developmental biology. Prior to experimentation, however, the embryos and larvae must first be collected.
This video will first demonstrate how "egg-laying cups" are used to collect Drosophila embryos on agar plates. The harvest and dechorionation of embryos will then be described. Next, the video will demonstrate how to identify and manipulate Drosophila in one of the three larval stages that follow the embryo stage. Finally, examples of some of the ways in which fly embryos and larvae are used in biological research are provided.

Drosophila melanogaster Embryo and Larva Harvesting and Preparation

黑腹果蝇卵和幼虫采集及准备

Drosophila melanogaster embryos and larvae are easy to manipulate and develop rapidly by mechanisms that are analogous to other organisms, including ...

Drosophila Egg Collection and Dechorionation: A Method to Remove the Outermost Egg Layer

Source: Koyle, M. L., et al. <a href="https://www.jove.com/video/54219/" target="_blank">Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions</a>. J. Vis. Exp. (2016).
The Drosophila embryo is surrounded by protective outer membranes. To gain access to the embryo, the chorion&#8211;the outermost of the membranes&#8211;is often removed. This video describes a commonly used method of dechorionation using sodium hypochlorite. The example protocol demonstrates the procedure while employing sterile techniques necessary for axenic or gnotobiotic animal rearing.

果蝇鸡蛋收集和去绒毛膜:一种去除最外层鸡蛋层的方法

Source: Koyle, M. L., et al. Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions. J. Vis. Exp. (2016).
The Drosophila ...

饲养果蝇

摘要

探索更多视频

此视频中的章节

一种研究外周和中枢神经系统 Neuroregeneration 的果蝇体内损伤模型

果蝇幼虫注射方案

使用德罗索菲拉· 拉瓦在恒温遗传神经元屏幕上摄入的宏量营养素的量化

培养的果蝇脑外植体中的神经干细胞再激活

利用果蝇作为无脊椎动物毒性试验模型系统的实验协议

果蝇的口腔细菌感染和脱落

建立和维护侏罗育美国蟑螂（佩里普拉内塔美洲）

果蝇的饲养与保存

黑腹果蝇卵和幼虫采集及准备

果蝇鸡蛋收集和去绒毛膜:一种去除最外层鸡蛋层的方法

饲养果蝇

摘要

探索更多视频

此视频中的章节

一种研究外周和中枢神经系统 Neuroregeneration 的果蝇体内损伤模型

果蝇 幼虫注射方案

使用 德罗索菲拉· 拉瓦在恒温遗传神经元屏幕上摄入的宏量营养素的量化

培养的 果蝇 脑外植体中的神经干细胞再激活

利用果蝇作为无脊椎动物毒性试验模型系统的实验协议

果蝇的口腔细菌感染和脱落

建立和维护侏罗育美国蟑螂 （佩里普拉内塔美洲）

果蝇的饲养与保存

黑腹果蝇卵和幼虫采集及准备

果蝇鸡蛋收集和去绒毛膜:一种去除最外层鸡蛋层的方法

果蝇幼虫注射方案

使用德罗索菲拉· 拉瓦在恒温遗传神经元屏幕上摄入的宏量营养素的量化

培养的果蝇脑外植体中的神经干细胞再激活

建立和维护侏罗育美国蟑螂（佩里普拉内塔美洲）