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Microfluidic-based Time-kill Kinetic Assay
基于微流体的时间与杀菌关系的动力学分析   

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Abstract

In many environments, bacteria favor a sessile, surface-attached community lifestyle. These communities, termed biofilms, are ubiquitous among many species of bacteria. In some cases, biofilms form under flow conditions. Flow chambers, and in particular microfluidic channels, can be used to observe biofilm development and physiological effects while varying nutrient conditions, flow velocities, or introducing antimicrobials to the biofilm in real time. Here, we describe a microfluidic-based kill-kinetics assay for the observation of antimicrobial effects on biofilms under flowing conditions.

Materials and Reagents

  1. Glycerol stock of Pseudomonas aeruginosa (P. aeruginosa) (acquired from a single colony, stored at -80 °C)
  2. Bacto-tryptone (BD Biosciences, catalog number: 211705 )
  3. Sodium chloride (Sigma-Aldrich, catalog number: S7653 )
  4. Colistin sodium sulfate (Sigma-Aldrich, catalog number: C4461 )
  5. Polydimethylsiloxane (PDMS) (Sylgard 184 Silicone Elastomer Kit) (Dow Corning Corporation)
  6. Petri dishes (150 mm x 15 mm) (VWR International, catalog number: 25384-326 )
  7. 100% Ethanol
  8. Sterile dH2O
  9. LIVE/DEAD BacLight Bacterial Viability Kit with Syto9/Propidium Iodide (Life Technologies, catalog number: L-7012 )
  10. 1% TB liquid medium (see Recipes)

Equipment

  1. Sterile 14 ml polystyrene culture tubes (VWR International, catalog number: 60818-725 )
  2. Sterile 1 μl plastic loops (Santa Cruz, catalog number: SC-200266 )
  3. Spectrophotometer for absorbance readings
  4. 37 °C shaking incubator
  5. Microfluidic channel molds (Figure 1)


    Figure 1. Microfluidic channels. Each channel is 100 μm deep and 500 μm wide. The 10 channels were fabricated by depositing SU-8 2150 on silicon wafers using standard soft lithography techniques (Xia and Whitesides, 1998). This device allows for 10 parallel experiments.

  6. SU-8 2150 (MicroChem Corp.)
  7. Glass Slides (75 x 50 mm) (Ted Pella, catalog number: 26005 ) or cover glass (75 x 50 mm) (Ted Pella, catalog number: 260462 )
  8. Lens paper
  9. Desiccator with vacuum line
  10. Exacto-knife or scalpel
  11. 5 ml syringes (BD, catalog number: 309647 )
  12. Polyethylene tubing (ID 0.58 mm, OD 0.965) (BD, IntramedicTM, Clay Adams®, catalog number: 427411 )
  13. Harris Unicore hole punch (1.20 mm) (Ted Pella, catalog number: 15074 )
  14. Hand-held corona generator (Laboratory Corona Treater, Electro-Technic Products, Model: BD-20AC )
  15. Syringe pump with 10-syringe holder adaptor (Harvard Apparatus)
  16. Fluorescence microscope with automated focus/stage system (e.g., Nikon TE2000-E equipped with an Andor iXon-885 and a 40x long working distance objective)

Software

  1. ImageJ

Procedure

  1. Microchannel molds (Figure 1) were prepared by depositing SU-8 2150 on silicon wafers using standard soft lithography techniques (Xia and Whitesides, 1998).
    Note: Stanford Microfluidics Foundry (http://www.stanford.edu/group/foundry/index.html) or the SIMTech Microfluidics Foundry in Singapore (http://www.simtech.a-star.edu.sg/smf/) provide services for fabrication. The mask pattern is included as a pdf supplemental file.
  2. Microfluidic device assembly:
    1. Place the mold into a 150 mm x 15 mm petri dish with the patterned side facing up.
    2. Prepare 25 ml of the PDMS mixture. In a plastic weigh-boat, weigh 10 parts silicon elastomer then add 1 part curing agent. Mix thoroughly for at least one minute, and then pour over the silicon master.
    3. Place the dish in a desiccator and degas under vacuum for 1 h. It is important to remove all air bubbles from the PDMS before curing.
    4. Cure the PDMS for 24 h in a 37 °C incubator, or, alternatively, for 2 h at 65 °C.
      Note: Read the manufacturer’s suggestions for curing PDMS.
    5. The cured PDMS should be firm to the touch. Using a sharp blade (Exacto-knife or scalpel) carefully cut out all of the lanes in a single, rectangular piece. The patterned microchannels will be visible in the PDMS. Cover channels with cellophane tape to protect from dust particles and debris.
    6. Punch holes at the channel inlets and outlets with the 1.2 mm Harris Unicore hole punch. Take care that the PDMS plugs are removed. Remove cellophane tape. Thoroughly clean the PDMS device and a glass slide with 100% ethanol and lens paper to remove any debris.
    7. Place the glass slide and PDMS channel side up on a clean flat surface. Hold the hand-held corona generator approximately 1-2 cm above the surface. Treat for 30 sec uniformly over each surface.
    8. Carefully place the PDMS device on the treated glass slide, with the microchannel pattern facing the glass slide. Apply gentle, uniform pressure by hand to ensure a uniform seal. Care must be taken not to apply too much pressure, which can lead to channel collapse. After a final bonding step at 65 °C for 15 min, the device is ready to use.
  3. Prepare up to five 14 ml polystyrene culture tubes with 5 ml of 1% TB. Inoculate by carefully scraping the surface of the bacterial strain glycerol stock. For this device, 5 separate strains can be tested using the 10 microchannels. For each strain, one microchannel will serve as a control and the other will be used for an antimicrobial challenge.
  4. Place the cultures in a 37 °C incubator, shaking at 250 rpm overnight.
  5. Remove the culture from the incubator and prepare another 14 ml polystyrene culture tube with 5 ml of 1% TB. Inoculate the culture with 50 μl from the overnight culture.  
  6. Incubate in the 37 °C incubator, shaking at 250 rpm for approximately 4 h, monitoring until an OD600 of 0.5 is reached indicating mid-exponential phase. Use this time to sterilize the device.
  7. Dilute the culture to an OD600 of 0.0025 in 5 ml of 1% TB.
  8. Insert tubing in the inputs and outputs of each microchannel, applying enough pressure to force the tubing roughly half-way from the PDMS surface to the glass slide but carefully to avoid cracks in the PDMS inlet and outlets.  
  9. Attach the output tubing to a 5 ml syringe equipped with a BD 23G1 gauge needle for each microchannel. Place the syringes on to the syringe pump and mount the device onto the microscope stage (Figure 2). Set the diameter of the syringe on the syringe pump.


    Figure 2. Microfluidic device setup. The microfluidic device is mounted onto the microscope stage for the duration of the experiment. A syringe pump equipped with a 10-syringe holder withdraws fluid from the culture tube reservoirs, flowing it through each of the 10 channels run in parallel.

  10. Place the input tubing into a reservoir of 100% ethanol. Set the syringe pump to withdrawal. Sterilize the tubing and microchannels by flowing 100% ethanol into the channels for a minimum of 15 min and rinse with sterilized water.
    Note: Fluid is replaced in the reservoir by adding a separate line of tubing attached to a free syringe into the bottom of the reservoir. After stopping the flow, the fluid is manually removed and replaced by pipetting in new volume of fluid. This step facilitates exchange between fluids without introduction of air bubbles.
  11. Once the device is sterilized and rinsed, place the input tubing into each of the prepared cultures and start the syringe pump to withdraw the medium from the cultures at a flow rate of 25 μl/min. Withdraw at this rate for 15 min or until the culture medium has reached the microchannels.
  12. Set the flow rate to 0.5 μl/min for 18 h. This will facilitate attachment of P. aeruginosa, yielding a monolayer of cells.  
    Note: For longer time points, a more mature, thicker biofilm requires a confocal microscope for 3 dimensional quantification via z-stack reconstruction.
  13. After 18 h, replace the culture medium in the reservoir tubes with 3 ml of 1% TB. Wash each microchannel at a flow rate of 25 μl/min. Using the microscope stage/focus positioning software, program 3 fields of view for each microchannel. Include image capture every 5 min for 2 h using phase contrast and filter cubes corresponding to the excitation/emission wavelengths of Syto 9 and Propidium Iodide.
  14. Prepare the Syto 9/Propidium Iodide viability dye at a 1:1 ratio. Mix 6 μl of dye solution for 1 ml of dH2O to achieve the final working concentration of dye.  
    Note: After attempting several solvents, dH2O proved to be the most efficient. Other solvents such as 1% TB medium, 1x PBS, and nanopure H2O resulted in decreased binding of both dyes.
  15. Introduce the dye into the microchannel at a flow rate of 25 μl/min for approximately 15 min. Stop flow. Let the dye remain in the channel for 15-30 min.
    Note: For the next step, it is important to know when the antibiotic solution reaches the microchannel since time zero is a critical data point for the kill kinetics curve. This is simple to calculate by monitoring the time that it takes for the dye to reach the microchannel at the suggested flow rate. Make note of this time for the next step in the protocol.
  16. Prepare a solution of 20 μg/ml of colistin in 1% TB. Remove residual dye from the reservoir tubes for the microchannel inputs. For each strain, add 1 ml of the colistin solution to the first microchannel and 1 ml of dH2O to the second microchannel to serve as a control. Initiate flow at 25 μl/min and reduce flow rate to 0.5 μl/min once the antibiotic has reached the microchannel. Start the imaging software at time zero (point when the antibiotic reaches the microchannels) to record images at the designated fields of view.
  17. As cell death progresses, an increase in red cells will be observed for antibiotic susceptible cells and a decrease in green cells as propidium iodide quenches the Syto 9.
  18. The total number of dead cells in the biofilms for each time point can be quantified in ImageJ (Schneider et al., 2012) by converting the fluorescence image to an 8-bit binary image. Apply a constant threshold to each image to subtract background fluorescence. The final data can be expressed as a percentage of the total biofilm area (phase-contrast images) for each time point.

Recipes

  1. 1% TB liquid medium (1 L)
    10 g of Bacto-tryptone
    2.5 g Sodium chloride
    Add dH2O to 1 L
    Sterilize by autoclaving on liquid cycle

Acknowledgments

The development of this protocol was funded by the following: NIH Grant R01 EB017755, NIH-NIEHS Training Grant in Toxicology T32 ES7020-37, NSF OCE-0744641-CAREER, NIH 1R01GM100473, NSF CBET-0966000.

References

  1. Billings, N., Millan, M., Caldara, M., Rusconi, R., Tarasova, Y., Stocker, R. and Ribbeck, K. (2013). The extracellular matrix component Psl provides fast-acting antibiotic defense in Pseudomonas aeruginosa biofilms. PLoS Pathog 9(8): e1003526.
  2. Schneider, C. A., Rasband, W. S. and Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9(7): 671-675.
  3. Xia, Y. and Whitesides G. M. (1998). Soft lithography. Angewandte Chemie-International Edition 37(5): 551-575.

简介

在许多环境中,细菌喜欢固着的,表面附着的社区生活方式。 这些社区,称为生物膜,在许多物种的细菌中是普遍存在的。 在一些情况下,在流动条件下形成生物膜。 流动室,特别是微流体通道,可以用于观察生物膜的发展和生理效应,同时改变营养物条件,流速,或实时地将抗微生物剂引入生物膜。 在这里,我们描述了一种基于微流体的杀菌动力学测定法,观察在流动条件下对生物膜的抗微生物效应。

材料和试剂

  1. (从单个菌落获得,储存在-80℃)的铜绿假单胞菌(<绿脓杆菌
  2. 细菌胰蛋白胨(BD Biosciences,目录号:211705)
  3. 氯化钠(Sigma-Aldrich,目录号:S7653)
  4. 粘菌素硫酸钠(Sigma-Aldrich,目录号:C4461)
  5. 聚二甲基硅氧烷(PDMS)(Sylgard 184硅氧烷弹性体试剂盒)(Dow Corning Corporation)
  6. 培养皿(150mm×15mm)(VWR International,目录号:25384-326)
  7. 100%乙醇
  8. 无菌dH 2 O 2/b
  9. LIVE/DEAD BacLight细菌活力试剂盒(含Syto9 /碘化丙啶)(Life Technologies,目录号:L-7012)
  10. 1%TB液体培养基(参见配方)

设备

  1. 无菌14ml聚苯乙烯培养管(VWR International,目录号:60818-725)
  2. 无菌1μl塑料环(Santa Cruz,目录号:SC-200266)
  3. 用于吸光度读数的分光光度计
  4. 37℃振荡培养箱
  5. 微流体通道模具(图1)


    图1.微流体通道。每个通道的深度为100μm,宽度为500μm。 通过使用标准软光刻技术在硅晶片上沉积SU-8 2150来制造10个通道(Xia和Whitesides,1998)。 此设备允许10次并行实验。

  6. SU-8 2150(MicroChem Corp.)
  7. 玻璃片(75×50mm)(Ted Pella,目录号:26005)或盖玻片(75×50mm)(Ted Pella,目录号:260462)
  8. 镜头纸
  9. 干燥器带真空管道
  10. 外刀或手术刀
  11. 5ml注射器(BD,目录号:309647)
  12. 聚乙烯管(ID 0.58mm,OD 0.965)(BD,Intramedic TM ,Clay Adams ,目录号:427411)
  13. Harris Unicore打孔器(1.20mm)(Ted Pella,目录号:15074)
  14. 手持电晕发生器(实验室电晕处理器,电子产品,型号:BD-20AC)
  15. 带有10针注射器支架适配器(Harvard装置)的注射泵
  16. 具有自动聚焦/平台系统的荧光显微镜(例如,配备有Andor iXon-885和40x长工作距离物镜的Nikon TE2000-E)

软件

  1. ImageJ

程序

  1. 通过使用标准软光刻技术(Xia和Whitesides,1998)将SU-8 2150沉积在硅晶片上制备微通道模具(图1)。
    注意:Stanford Microfluidics Foundry( http://www .stanford.edu/group/foundry/index.html )或新加坡的SIMTech Microfluidics铸造厂( http://www.simtech.a-star.edu.sg/smf/ )提供 制造服务。 掩模图案作为pdf补充文件包含。
  2. 微流体装置组件:
    1. 将模具放入一个150毫米×15毫米的培养皿,图案的一面朝上。
    2. 制备25ml的PDMS混合物。 在塑料称重船,重10 然后加入1份固化剂。 彻底混合   至少一分钟,然后倒在硅主板上。
    3. 将盘放在干燥器中,在真空下脱气1小时。 它是 重要的是在固化之前从PDMS中除去所有气泡
    4. 在37℃培养箱中固化PDMS 24小时,或者在65℃固化2小时。
      注意:请阅读制造商关于固化PDMS的建议。
    5. 固化的PDMS应该是坚硬的触摸。 使用锋利的刀片 (切割刀或手术刀)小心地切出了所有的车道 单个,矩形件。 图案化微通道将是可见的 在PDMS中。 用玻璃纸胶带覆盖通道,以防灰尘 颗粒和碎片
    6. 在通道入口和冲孔处打孔 出口与1.2毫米哈里斯Unicore打孔器。 注意 移除PDMS插塞。 取下玻璃纸胶带。 彻底清洁 PDMS装置和具有100%乙醇和透镜纸的载玻片以除去   任何碎片。
    7. 将载玻片和PDMS通道侧向上放置   一个干净的平面。 大约握住手持式电晕发生器   在表面上1-2厘米。 在每个表面上均匀地处理30秒。
    8. 小心地将PDMS设备放在处理过的载玻片上, 其中微通道图案面向载玻片。 应用温和, 手动均匀压力,确保均匀密封。 必须小心 不要施加太大的压力,这可能导致通道崩溃。 在65℃的最终结合步骤15分钟后,装置准备好 使用。
  3. 准备五个14毫升聚苯乙烯培养管与5毫升1%的TB。通过仔细刮细菌菌株甘油原液的表面接种。对于该装置,可以使用10个微通道测试5个单独的菌株。对于每个菌株,一个微通道将用作对照,而另一个将用于抗微生物攻击。
  4. 将培养物置于37℃培养箱中,以250rpm振荡过夜
  5. 从培养箱中取出培养物,并准备另一个14毫升聚苯乙烯培养管与5毫升1%的TB。接种培养物与50微升从过夜培养。  
  6. 在37℃培养箱中孵育,在250rpm摇动约4小时,监测直至达到0.5的OD 600,表明指数中期。使用此时间对设备进行消毒。
  7. 将培养物稀释至在5ml 1%TB中0.0025的OD 600。
  8. 在每个微通道的输入和输出中插入管道,施加足够的压力以迫使管道从PDMS表面到玻璃载片大约一半,但小心地避免PDMS入口和出口中的裂纹。  
  9. 将输出管连接到配备有用于每个微通道的BD 23G1量规针的5ml注射器。将注射器放在注射泵上,将设备安装到显微镜载物台上(图2)。设置注射器泵上的注射器直径。


    图2.微流体装置设置。在实验期间将微流体装置安装到显微镜载物台上。装有10注射器支架的注射泵从培养管储器中抽出流体,使其流过平行延伸的10个通道中的每个通道。

  10. 将输入管道放入100%乙醇的储器中。将注射泵设置为取出。通过流动100%乙醇至通道至少15分钟,用无菌水冲洗消毒管和微通道。
    注意:通过将附加到自由注射器的单独管线连接到储存器的底部,在储存器中更换流体。在停止流动之后,手动移除流体并通过在新体积的流体中移液来替换。此步骤有助于在不引入气泡的情况下进行流体交换。
  11. 一旦装置被灭菌和漂洗,将输入管放入每个制备的培养物中,并启动注射泵以25μl/min的流速从培养物中取出培养基。以此速度取出15分钟或直到培养基达到微通道
  12. 将流速设定为0.5μl/min,持续18小时。这将便于附加 P。绿脓杆菌,产生单层细胞。  
    注意:对于较长的时间点,更成熟,更厚的生物膜需要共聚焦显微镜通过z-堆叠重建进行3维量化。
  13. 18小时后,用3ml的1%TB替换储存管中的培养基。以25μl/min的流速洗涤每个微通道。使用显微镜载物台/聚焦定位软件,为每个微通道编程3个视野。包括使用相应于Syto 9和碘化丙啶的激发/发射波长的相差和滤波器立方体,每5分钟采集图像2小时。
  14. 以1:1的比例制备Syto 9 /碘化丙锭活性染料。混合6μl染料溶液1ml的dH 2 O以达到染料的最终工作浓度。  
    注意:尝试几种溶剂后,dH 2 O证明是最有效的。其它溶剂,例如1%TB培养基,1×PBS和纳米级H 2 O导致两种染料的结合降低。
  15. 将染料以25μl/min的流速引入微通道约15分钟。停止流。让染料保留在通道中15-30分钟。
    注意:对于下一步,重要的是知道抗生素溶液何时到达微通道,因为时间零点是杀死动力学曲线的关键数据点。这可以通过监测染料以建议的流速到达微通道所花费的时间来计算。请记下此时间,以便执行协议中的下一步。
  16. 准备20μg/ml粘菌素的1%TB溶液。从微通道输入的储存管中移除残留染料。对于每个菌株,向第一微通道中加入1ml粘菌素溶液,向第二微通道中加入1ml dH 2 O以作为对照。一旦抗生素到达微通道,启动25μl/min的流速,并将流速降低至0.5μl/min。在时间零点(当抗生素到达微通道时的点)开始成像软件以在指定的视野处记录图像。
  17. 随着细胞死亡的进展,对于抗生素敏感细胞将观察到红细胞的增加,并且当碘化丙啶淬灭Syto 9时,绿细胞减少。
  18. 通过将荧光图像转换为8位二进制图像,可以在ImageJ(Schneider等人,2012)中量化每个时间点的生物膜中的死细胞总数。对每个图像应用恒定阈值以减去背景荧光。最终数据可以表示为每个时间点的总生物膜面积(相位对比图像)的百分比。

食谱

  1. 1%TB液体培养基(1 L) 10g细菌用胰蛋白胨 2.5克氯化钠
    将dH <2> O添加到1 L

    通过高压灭菌在液体循环中灭菌

致谢

本方案的开发由以下资金资助:NIH Grant R01 EB017755,NIH-NIEHS毒理学训练基金T32 ES7020-37,NSF OCE-0744641-CAREER,NIH 1R01GM100473,NSF CBET-0966000。

参考文献

  1. Billings,N.,Millan,M.,Caldara,M.,Rusconi,R.,Tarasova,Y.,Stocker,R。和Ribbeck,K。 细胞外基质成分Psl在绿脓杆菌中提供快速作用的抗生素防御 biofilms。 PLoS Pathog 9(8):e1003526。
  2. Schneider,C.A.,Rasband,W. S.和Eliceiri,K.W。(2012)。 NIH Image to ImageJ:25 years of image analysis。 Nat Methods 9(7):671-675。
  3. Xia,Y。和Whitesides G.M.(1998)。 Soft lithography。 Angewandte Chemie-International Edition 37(5):551-575。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Billings, N., Rusconi, R., Stocker, R. and Ribbeck, K. (2014). Microfluidic-based Time-kill Kinetic Assay. Bio-protocol 4(9): e1116. DOI: 10.21769/BioProtoc.1116.
  2. Billings, N., Millan, M., Caldara, M., Rusconi, R., Tarasova, Y., Stocker, R. and Ribbeck, K. (2013). The extracellular matrix component Psl provides fast-acting antibiotic defense in Pseudomonas aeruginosa biofilms. PLoS Pathog 9(8): e1003526.
提问与回复

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当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。