Escherichia coli Infection of Drosophila

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Aug 2016



Following septic insults, healthy insects, just like vertebrates, mount a complex immune response to contain and destroy pathogens. The failure to efficiently clear bacterial infections in immuno-compromised fly mutants leads to higher mortality rates which provide a powerful indicator for genes with important roles in innate immunity. The following protocol is designed to reproducibly inject a known amount of non-pathogenic E. coli into otherwise sterile flies and to measure the survival of flies after infection. The protocol can be easily adapted to different types of bacteria.

Keywords: Drosophila (果蝇), Innate immunity (天然免疫), Bacterial infections (细菌感染), Tolerance (耐受性), Survival (生存)


Classic infection experiments involve infecting Drosophila orally (Chakrabarti et al., 2016) or with a needle dipped in a concentrated bacterial solution (Romeo and Lemaitre, 2008). Unlike these protocols, our experimental procedure allows us to determine the site of infection and precisely control the dose of bacteria injected into each fly. This provides homogeneity and reproducibility, and allows us to adapt bacterial load for different experiments (Akbar et al., 2011 and 2016).

Materials and Reagents

  1. 15 ml Falcon tubes (Corning, catalog number: 352196 )
  2. 1.5 ml Eppendorf tubes (USA Scientific, catalog number: 1615-5500 )
  3. Kimwipes
  4. 26 G needle (BD, catalog number: 305111 )
  5. Spin columns (e.g., Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 69700 )
  6. Empty and clean Drosophila vials (Genesee Scientific, catalog number: 32-109 )
  7. Microloader tips (Eppendorf, catalog number: 930001007 )
  8. Vials (Genesee Scientific, catalog number: 32-109 ) with standard Drosophila food (lightly yeasted)
  9. Microslide (Corning, catalog number: 2948-75X25 )
  10. 50 Drosophila melanogaster adult virgins (Romeo and Lemaitre, 2008), aged five to seven days post eclosion (see Note 1)
  11. E. coli-DH5α containing any ampicillin-resistant plasmid (e.g., expressing GFP)
    1. pEGFP (
    2. pET-GFP-C11 (
  12. LB broth (Fisher Scientific, catalog number: BP1426-500 )
  13. Ampicillin (200 mg/ml) (Sigma-Aldrich, catalog number: A9518 )
  14. 70% ethanol–diluted from 100% ethanol (Pharmco-AAPER, catalog number: 111000200 )
  15. Mineral oil (Fisher Scientific, catalog number: O121-1 )
  16. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271 )
  17. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  18. Sodium phosphate dibasic heptahydrate (Na2HPO4·7H2O) (Sigma-Aldrich, catalog number: S9390 )
  19. Potassium phosphate monobasic (KH2PO4) (Fisher Scientific, catalog number: S397-500 )
  20. 1x PBS (see Recipes)


  1. 37 °C incubator with shaking (Eppendorf, New BrunswickTM, model: Innova® 44 )
  2. Spectrophotometer (Molecular Devices, model: SpectraMax M2 )
  3. Borosilicate glass capillaries (WPI, catalog number: TW100-4 )
  4. Centrifuge capable of spinning Falcon tubes (Eppendorf, model: 5804 R )
  5. Flaming/Brown Micropipette puller (Sutter Instrument, model: P-97 )
  6. Mini benchtop centrifuge (Fisher Scientific, model: FisherbrandTM Standard Mini Centrifuge , catalog number: 05-090-100)
  7. Pico-Injector (Nikon Instruments, catalog number: PLI-188 )–requires nitrogen gas
    Note: Should have foot pedal for injecting.
  8. 25 °C incubator (BioCold Environment, model: BC49-IN )
  9. Anesthetizing fly pad (Genesee Scientific, catalog number: 59-119 )
  10. Small brush
  11. Dissecting microscope (Leica)


  1. Prism (GraphPad) software


  1. Preparation of E. coli suspension
    1. Inoculate 50 ml LB broth with an E. coli colony from an LB agar plate under sterile conditions.
    2. Add ampicillin (200 µg/ml final).
    3. Incubate overnight at 37 °C with shaking. Use spectrophotometer to measure optical density at 600 nm (OD600).
      1. Dilute 500 µl aliquot of bacterial over-night suspension 5-fold with 2 ml of LB broth
      2. Measure OD600 of diluted bacteria with broth, and broth alone.
      3. Subtract OD600 of fresh LB broth from bacteria with broth and calculate OD600 of over-night culture.
    4. Dilute over-night 50 ml bacterial culture to OD600 = 0.1 with LB broth.
      1. Spin 50 ml of bacterial suspension 4,000 x g for 10 min in Falcon tubes at room temperature.
      2. Decant LB broth.
      3. Re-suspend in 50 ml of 1x PBS by vortexing.
      4. Adjust with PBS to OD600 = 0.1.
    5. Heat-Kill bacteria (optional; see Note 2)
      1. Aliquot 1 ml bacterial suspension into 1.5 ml Eppendorf tubes.
      2. Incubate at 65 °C for 20 min.
      3. Bacterial solution is now ready for injections.
  2. Preparation of injection needles
    1. Place glass capillary in Micropipette puller.
    2. Run program to create needles tapered like the needle pictured (Figure 1).
      1. Ramp + 5, Pull = 90, Velocity = 70, Delay = 70

        Figure 1. Tapered injection needle. Arrow indicates preferred place to break it.

    3. Stretch a Kimwipe taut over a Falcon tube and stab needle through the Kimwipe to break the very tip of the needle.
  3. Fly sterilization
    1. Use a small needle (26 G) to poke three to four holes in the paper filter of the spin columns and place back into collection tube.
    2. Add 25 anesthetized flies to one spin column (Figure 2).

      Figure 2. Spin column for Fly sterilization. 25 flies are loaded into a spin column (S) and covered with 70% ethanol. Spin columns are placed in a collection tube (C) to contain the flow through of wash solutions in the centrifuge during the quick spin. Flies are retained in the spin column by the paper filter at its bottom.

    3. Add 700 μl 70% ethanol
    4. Quick spin on mini benchtop centrifuge (~3-4 sec).
    5. Remove spin column from collection tube and pour out flow through and place spin column back into collection tube.
    6. Repeat ethanol wash steps 3c-3e.
    7. Wash with 700 μl sterile water.
    8. Quickspin on mini benchtop centrifuge (~3-4 sec).
    9. Repeat water rinse steps 3g and 3h.
    10. Fold Kimwipe and place in empty fly vial.
    11. Put flies into vial with Kimwipe, plug and place at 25 °C until flies have recovered.
  4. Calibration of injection volume (see Note 3)
    1. Load 1 μl bacterial solution into the pulled needle using Microloader tips.
    2. Attach needle to Pico-injector.
    3. Adjust Pico-injector pressures, injection time to achieve 50 nl per injection.
      1. Typical starting values for our set-up: 
        1) Balance pressure = -0.2 psi–Should be neutral .
        2) Injection pressure = 15 psi.
        3) Injection time = 2 msec.
      2. For calibration: put a drop of mineral oil on a microslide.
      3. Inject bacterial solution into oil to determine nl per injection (a needle loaded with 1 µl should yield 20 equal sized droplets of 50 nl each).
      4. Adjust injection time and pressure until desired amount is injected.
      5. Optimal Pico-injector values will vary slightly between needles.
  5. Injection of sterilized flies (see Notes 4 and 5)
    1. Sterilize fly pad with 70% ethanol, allow to dry.
    2. Place sterilized flies on fly pad.
    3. Under dissecting microscope hold a fly in place by its legs with a small brush.
    4. Hold injecting rod and needle with other hand.
    5. Insert needle into the notum of the immobilized fly (Figure 3).
    6. Inject bacterial solution into fly using foot pedal–Audible beep will confirm.
    7. Repeat until all flies have been injected.

      Figure 3. Injection of flies with bacterial suspension. Anesthetized flies are held with a brush as they are injected using a pulled glass needle (see Figure 1) filled with a bacterial suspension.

  6. Incubate flies and monitor survival
    1. Place ≤ 15 injected flies into vial containing fresh standard Drosophila media.
      Vials should be lightly yeasted and contain no more than 1.5 ml of fly food. This minimizes the danger of flies getting stuck in yeast or between the side of the vial and the food as the food tends to dry at later days and separate from the vial.
    2. Place vials with flies into 25 °C incubator and note how many flies are alive 2 h after injection. Flies that do not recover from injections should be discarded from the dataset and subsequent analysis.
    3. Count dead and living flies every day, once a day until all are dead. For bacteria more pathogenic than E. coli, shorter time intervals will be important.
    4. Flip flies into fresh vials every 5 days.

Data analysis

Recorded data are entered into Prism (GraphPad) software which is used to display survival curves (Figure 4) and analyze statistical significance of differences in survival between genotypes. Log-Rank (Mantel-Cox) test was used to assess statistical significance of differences between survival curves (Figure 4). Alternatively, multiple Log-rank analysis software packages are available online at: and Both sides offer detailed information on how to enter data.

Figure 4. Example of statistical analysis of fly survival post bacterial infection. The diagram shows the fraction of surviving wild-type flies (OreR) or Vps33B mutants after injection with heat-killed E. coli, similar to experiments previously published (Akbar et al., 2016). Statistical analysis was performed in Prism using the Log-rank (or Mantel-Cox) test to determine a P value smaller than 0.0001.


  1. A wild-type Drosophila strain (Oregon R or Canton S) should be included as a negative control group for injections. Use sibling and/or parent flies as controls when appropriate. Three replicates of fifty flies per genotype are sufficient for proper statistical analysis.
  2. It is important to note that heat inactivation can change the potential of bacteria to engage different innate immunity pathways as normally hidden epitopes may be exposed (Chung and Kocks, 2011).
  3. Most difficulties in this experimental procedure are encountered during the needle calibration and injection steps. The balance pressure is the source of most problems. If the balance pressure is set too high, the result is a constant bleeding of the bacterial solution resulting in difficulty measuring injection droplets, emptying of the needle, and dripping bacterial solution onto the fly. No liquid should escape the needle until the injection foot pedal has been pressed. Problems also arise if the balance pressure is too low. Low pressure will result in sucking up liquid that had just been injected resulting in little to no bacteria being injected (this should also be part of your calibration). Furthermore, once the needle is inserted into the fly, the negative pressure will pull fly tissues into the needle tip resulting in blockage that can often be seen through the microscope. This will prevent any further injections when the foot pedal has been pushed. Placing the needle tip into the bacterial solution and pressing the ‘clear’ button on the Pico-injector can usually solve this by high positive pressure clearing the needle. Once the blockage is cleared, leave the needle tip in the bacterial solution and hold the ‘fill’ button to refill the needle using negative pressure. Once the needle is calibrated it is also useful to inject a few droplets on or near a fly or paintbrush to get a good sense of the size of a 50 nl droplet. A 50 nl droplet is roughly the size of a fly eye.
  4. Prior to commencing bacterial injections, it is important to have a series of experiments showing flies do not die from inflammation resulting from the needle injury or from the injection media. Mock injections should be performed on wild-type and experimental flies to determine if injury alone is sufficient to kill flies. Furthermore, control injections of sterile PBS should be done to ensure flies die from infection. These mock injections should be analyzed in the same manner as injections of bacteria as described in the data analysis section.
  5. The experimental procedure described above has been optimized for DH5α E. coli. Each 50 nl injection should deliver around 2,000 CFU to each fly. If other bacterial species are to be used, bacterial loads should be optimized to ensure survival of wild-type flies when virulence allows.


  1. 1x PBS
    137 mM NaCl
    2.7 mM KCl
    10 mM Na2HPO4
    1.8 mM KH2PO4


The work herein was supported by NIH Grants EY010199, EY021922.
This protocol has been adapted and modified from our previously published work (Akbar et al., 2016; Akbar et al., 2011).
The authors declare no conflicts of interest.


  1. Akbar, M. A., Mandraju, R., Tracy, C., Hu, W., Pasare, C. and Kramer, H. (2016). ARC syndrome-linked Vps33B protein is required for inflammatory endosomal maturation and signal termination. Immunity 45(2): 267-279.
  2. Akbar, M. A., Tracy, C., Kahr, W. H. and Kramer, H. (2011). The full-of-bacteria gene is required for phagosome maturation during immune defense in Drosophila. J Cell Biol 192(3): 383-390.
  3. Chakrabarti, S., Dudzic, J. P., Li, X., Collas, E. J., Boquete, J. P. and Lemaitre, B. (2016). Remote control of intestinal stem cell activity by haemocytes in Drosophila. PLoS Genet 12(5): e1006089.
  4. Chung, Y. S. and Kocks, C. (2011). Recognition of pathogenic microbes by the Drosophila phagocytic pattern recognition receptor Eater. J Biol Chem 286(30): 26524-26532.
  5. Romeo, Y. and Lemaitre, B. (2008). Drosophila immunity: methods for monitoring the activity of Toll and Imd signaling pathways. Methods Mol Biol 415: 379-394.



背景 经典的感染实验包括口服感染果蝇(Chakrabarti等人,2016)或用浸在浓缩细菌溶液中的针(Romeo和Lemaitre,2008)。与这些方案不同,我们的实验程序允许我们确定感染部位,并精确控制注射到每只苍蝇中的细菌的剂量。这提供了均匀性和重复性,并且允许我们适应不同实验的细菌负荷(Akbar等人,2011和2016)。

关键字:果蝇, 天然免疫, 细菌感染, 耐受性, 生存


  1. 15毫升Falcon管(康宁,目录号:352196)
  2. 1.5ml Eppendorf管(USA Scientific,目录号:1615-5500)
  3. Kimwipes
  4. 26 G针(BD,目录号:305111)
  5. 旋转色谱柱(例如Thermo Fisher Scientific,Thermo Scientific TM,目录号:69700)
  6. 空的和干净的果汁小瓶(Genesee Scientific,目录号:32-109)
  7. Microloader提示(Eppendorf,目录号:930001007)
  8. 小瓶(Genesee Scientific,目录号:32-109),含有标准的果蝇食物(轻度发酵)
  9. Microslide(康宁,目录号:2948-75X25)
  10. 50果蝇黑腹果蝇成人virgins(罗密欧与Lemaitre,2008),蜕皮后五至七天(见注1)
  11. 电子。含有任何氨苄青霉素抗性质粒(例如表达GFP)的大肠杆菌-DH5α
    1. pEGFP(
    2. pET-GFP-C11(
  12. LB肉汤(Fisher Scientific,目录号:BP1426-500)
  13. 氨苄青霉素(200mg/ml)(Sigma-Aldrich,目录号:A9518)
  14. 从100%乙醇中稀释70%乙醇(Pharmco-AAPER,目录号:111000200)
  15. 矿物油(Fisher Scientific,目录号:O121-1)
  16. 氯化钠(NaCl)(Fisher Scientific,目录号:S271)
  17. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  18. 磷酸氢二钠七水合物(Na 2 HPO 4·7H 2 O)(Sigma-Aldrich,目录号:S9390)
  19. 磷酸二氢钾(KH 2 PO 4)(Fisher Scientific,目录号:S397-500)
  20. 1x PBS(见食谱)


  1. 37°C振荡孵化器(Eppendorf,New Brunswick TM,型号:Innova ® 44)
  2. 分光光度计(Molecular Devices,型号:SpectraMax M2)
  3. 硼硅玻璃毛细管(WPI,目录号:TW100-4)
  4. 离心机能够旋转Falcon管(Eppendorf,型号:5804 R)
  5. 火焰/棕色微量吸取器(Sutter Instrument,型号:P-97)
  6. 迷你台式离心机(Fisher Scientific,型号:Fisherbrand TM 标准微型离心机,目录号:05-090-100)
  7. 微型注射器(尼康仪器,目录号:PLI-188) - 需要氮气

  8. 25℃培养箱(BioCold Environment,型号:BC49-IN)
  9. 麻醉飞垫(Genesee Scientific,目录号:59-119)
  10. 小刷子
  11. 解剖显微镜(Leica)


  1. Prism(Graphpad)软件


  1. E的准备大肠杆菌悬浮液
    1. 将50毫升LB肉汤与E.在无菌条件下从LB琼脂平板上培养大肠杆菌菌落。
    2. 加氨苄青霉素(最终200μg/ml)
    3. 在37℃下振荡孵育过夜。使用分光光度计测量600nm处的光密度(OD 600)。
      1. 用2毫升LB肉汤稀释500微升等份的细菌超夜悬浮液5倍
      2. 用肉汤和肉汤单独测量稀释细菌的OD <600>。
      3. 用肉汤从细菌中减去新鲜LB肉汤的OD 600,并计算过夜培养物的OD 600。
    4. 用LB肉汤稀释超过50 ml的细菌培养物至OD 600/0.1。
      1. 在室温下,将Falcon试管中的50ml细菌悬浮液4,000 x g旋转10分钟。
      2. 滗水LB肉汤。
      3. 通过涡旋重悬于50ml PBS中。
      4. 用PBS调整至OD 600 <0.1>。
    5. 热灭菌(可选;见注2)
      1. 将1ml细菌悬浮液等分至1.5ml Eppendorf管中
      2. 在65℃孵育20分钟。
      3. 细菌溶液现在可以进行注射。
  2. 注射针的制备
    1. 将玻璃毛细管放入微量拔出器。
    2. 运行程序来创建如针图所示的针尖(图1)。
      1. 斜坡+5,拉力= 90,速度= 70,延迟= 70


    3. 将Kimwipe拉伸到Falcon管上,用针刺穿Kimwipe,以打破针尖。
  3. 飞行灭菌
    1. 使用小针(26 G)在旋转柱的纸过滤器上戳三到四个孔,然后放回收集管。
    2. 将25只麻醉的苍蝇添加到一个旋转柱(图2)


    3. 加入700μl70%乙醇
    4. 快速旋转迷你台式离心机(〜3-4秒)。
    5. 从收集管中取出旋转柱,倒出流经,并将旋转柱放回收集管中
    6. 重复乙醇洗涤步骤3c-3e。
    7. 用700μl无菌水冲洗。
    8. Quickspin在迷你台式离心机上(〜3-4秒)。
    9. 重复水冲洗步骤3g和3h。
    10. 折叠Kimwipe并放在空的飞行小瓶中。
    11. 用Kimwipe将苍蝇放入小瓶中,插入并放置在25°C,直到苍蝇恢复。
  4. 注射体积的校准(见注3)
    1. 使用Microloader提示将1μl细菌溶液装入拉针。
    2. 将针连接到微型注射器。
    3. 调整微注射器压力,注射时间达到每次注射50 nl。
      1. 我们的设置的典型起始值: 
        1)平衡压力= -0.2 psi - 应该是中立的。
        2)注射压力= 15 psi。
        3)注射时间= 2毫秒。
      2. 校准:将一滴矿物油放在微滑坡上。
      3. 将细菌溶液注入油中以确定每次注射nl(装满1μl的针头应产生20个相等大小的液滴,每个为50nl)。
      4. 调整注射时间和压力,直到注入所需的量。
      5. 针头之间的最佳微型注射器值将略有不同。
  5. 注射灭菌的苍蝇(见注释4和5)
    1. 用70%乙醇灭菌飞垫,让其干燥。
    2. 将灭菌的苍蝇放在飞垫上。
    3. 在解剖显微镜下,用小笔刷将腿部放在适当位置。
    4. 用另一只手握住注射杆和针头。
    5. 将针插入固定飞行的图形中(图3)
    6. 使用脚踏将细菌溶液注入飞行 - 可听到蜂鸣声将确认。
    7. 重复一遍,直到所有苍蝇都被注射


  6. 孵化苍蝇并监测生存
    1. 将≤15个注射的苍蝇放入含有新鲜标准果蝇的培养基的小瓶中 小瓶应轻度发酵,含有不超过1.5毫升的飞行食物。这样可以最大限度地减少苍蝇卡在酵母中或小瓶与食物侧面的危险,因为食物在稍后的日子里会变干,并与小瓶分开。
    2. 将小瓶放入25℃培养箱中,注意注射后2小时有多少苍蝇活着。不能从注射中恢复的苍蝇应该从数据集和后续分析中舍弃。
    3. 每天死亡和生活的苍蝇,每天一次,直到所有人都死亡。对于比E更致病的细菌。大肠杆菌,较短的时间间隔将很重要。
    4. 翻转每5天飞入新鲜小瓶。


记录的数据输入到用于显示存活曲线的Prism(GraphPad)软件(图4),并分析基因型之间生存差异的统计学显着性。 Log-Rank(Mantel-Cox)检验用于评估存活曲线差异的统计学显着性(图4)。或者,多个Log-rank分析软件包可以在线获取: http://bioinf.wehi。 。双方提供有关如何输入数据的详细信息

图4.细菌感染后蝇存活的统计分析实例该图显示注射后的野生型野生型(OreR)或Vps33B突变体的分数, E。大肠杆菌,类似于先前发表的实验(Akbar等人,2016)。使用Log-rank(或Mantel-Cox)测试在Prism中进行统计分析,以确定小于0.0001的P 值。


  1. 应包括野生型果蝇菌株(俄勒冈州R或Canton S)作为注射用的阴性对照组。适当时,使用兄弟和/或父母作为对照。每个基因型的五十只苍蝇的三次重复就足以进行适当的统计分析
  2. 重要的是要注意,热灭活可以改变细菌参与不同先天免疫途径的潜力,因为通常隐藏的表位可能会被暴露(Chung and Kocks,2011)。
  3. 在针头校准和注射步骤期间遇到这个实验过程中的大多数困难。平衡压力是大多数问题的根源。如果平衡压力设定得太高,结果是细菌溶液的不断渗出导致难以测量注射液滴,排空针头和将细菌溶液滴落在飞行中。注射脚踏板被压下之前,液体不应该脱落。如果平衡压力太低,也会出现问题。低压会导致刚刚注入的液体吸入,几乎没有细菌被注入(这也应该是校准的一部分)。此外,一旦将针插入飞行中,负压将将飞行组织拉入针尖,导致通常通过显微镜看到的阻塞。这将防止脚踏板被推动时进一步注射。将针尖放入细菌溶液中并按下Pico注射器上的"清除"按钮通常可以通过高正压清除针来解决此问题。一旦清除堵塞物,将针尖留在细菌溶液中,并按住"填充"按钮,用负压重新填充针头。一旦针头被校准,也可以在飞行或画笔上或附近注入几滴以获得50nl液滴的大小的良好感觉。 50毫微微滴大概是飞眼的大小。
  4. 在开始细菌注射之前,重要的是要进行一系列实验,显示苍蝇不会因针头损伤引起的炎症或从注射培养基而死亡。应对野生型和实验性蝇进行模拟注射,以确定单独的损伤是否足以杀死苍蝇。此外,应该进行无菌PBS的对照注射,以确保苍蝇死于感染。应以与数据分析部分所述细菌注射相同的方式对这些模拟注射进行分析。
  5. 上述实验程序已针对DH5αE进行了优化。大肠杆菌。每次注射50 nl,每次飞行约2,000 CFU。如果要使用其他细菌物种,应优化细菌负荷,以确保野生型苍蝇在毒力允许时的存活。


  1. 1x PBS
    137 mM NaCl
    2.7 mM KCl
    10mM Na 2 HPO 4
    1.8mM KH 2 PO 4


这项工作由NIH Grants EY010199,EY021922支持。
该协议已经从我们之前发表的作品(Akbar等人,2016; Akbar等人,2011)进行了修改和修改。


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  2. Akbar,MA,Tracy,C.,Kahr,WH和Kramer,H。(2011)。  在果蝇免疫防御期间,全细菌基因是吞噬细胞成熟所必需的。 J Cell Biol 192 3):383-390。
  3. Chakrabarti,S.,Dudzic,JP,Li,X.,Collas,EJ,Boquete,JP and Lemaitre,B。(2016)。  远程控制果蝇中血细胞的肠干细胞活性。 12(5):e1006089。
  4. Chung,YS和Kocks,C.(2011)。吞噬细胞模式识别受体Eater识别致病微生物。生物化学286(30):26524-26532。
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引用:Tracy, C. and Krämer, H. (2017). Escherichia coli Infection of Drosophila. Bio-protocol 7(9): e2256. DOI: 10.21769/BioProtoc.2256.