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May 2019
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Rapid Detection of Proliferative Bacteria by Electrical Stimulation
利用电刺激快速检测细菌增殖   

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Abstract

Detecting live bacteria is an important task for antimicrobial susceptibility testing (AST) in the medical sector and for quality-monitoring in biological industries. Current methods for live-bacteria detection suffer limitations in speed or sensitivity. In a recent paper, we reported that electrical response dynamics in membrane potential enable single-cell rapid detection of live bacteria. The electrical response can be observed within a minute after electrical stimulation. Thus, it has potential in accelerating AST and the monitoring of biological samples. This method also enables experiments for biophysical and microbiological investigations into bacterial electrophysiology. With the hope that more researchers, scientists and engineers will use electrical stimulation for their assays, here we detail each step of the electrical stimulation experiment.

Keywords: Bacterial electrophysiology (细菌电生理学), Membrane potential (细胞膜电势), Bacterial detection (细菌检测), Anti-microbial resistance (AMR) (细菌耐药), Cell vitality assay (细胞活力检测), Cell biophysics (细胞生物物理学)

Background

From fundamental microbiology research to antimicrobial susceptibility testing (AST), quantifying the number of live bacteria is an important task. When it comes to detecting live bacteria, the colony forming unit (CFU) assay, dating from the 19th century, is still the gold standard due to its reliability and sensitivity. However, the CFU assay has a major drawback; it requires 1-3 days of incubation. To overcome this limitation, developments in rapid bacterial detection technology has flourished in recent years (Iqbal et al., 2000; Ahmed et al., 2014). Examples of recently developed technologies are based on impedance spectroscopy, Raman spectroscopy or detection of biomolecules (e.g., ATP, DNA). Nevertheless, overcoming the tradeoff between speed, accuracy and robustness remains a challenge.

Membrane potential indicators are proven useful for distinguishing live and dead bacteria by flow cytometry and fluorescence microscopy (Sträuber and Müller, 2010). However, this method is technically challenging because it requires a careful calibration for bacterial species/strains, media conditions, indicator concentrations, light sources and detectors. In a recent study, we showed that such limitations can be overcome using optical measurements of the electrical response dynamics of membrane potential under stimulation by an external electrical field (Stratford et al., 2019). The responses of proliferative and inhibited cells were distinguishable within a minute; hence, it substantially shortens the time required for a live bacterial assay. Simulations of a phenomenological mathematical model and biophysical understanding suggested that this technology is applicable for different microbial species and antimicrobial treatments. This means that by comparing the electrical dynamics of unperturbed and antimicrobial-treated cells, it may be possible to accelerate AST. An important next step is examining how widely this technology can be used with environmental and pathogenic microbes. This protocol will assist conducting electrical stimulation experiments for AST and live bacteria detection assays with various bacterial species and strains as well as with various antimicrobial treatments.

In addition to its fundamental role in cell proliferation, membrane potential mediates bacterial electrical signaling in biofilms and during the processes of sporulation, mechano-sensation and cell division (Strahl and Hamoen, 2010; Prindle et al., 2015; Bruni et al., 2017; Sirec et al., 2019, Benarroch and Asally, 2020). Membrane potential is also associated with antibiotic resistance (Damper and Epstein, 2010). While these recent studies are beginning to garner attention, membrane potential remains largely overlooked in the field of microbiology. We suspect this oversight is partially due to the technical difficulties associated with experimentally controlling membrane potential. To this end, this protocol will also assist biophysical and microbiological investigations into bacterial electrophysiology.

Materials and Reagents

Notes:

  1. The following chemicals are interchangeable with appropriate replacements, per the investigator’s discretion.
  2. All these ingredients can be stored in dry conditions at room temperature.

  1. Stainless steel mounting wafer
  2. Copper wires
  3. Parafilm
  4. 22 x 22 mm glass slip
  5. Microscope Coverslips
  6. Bacterial samples
  7. Titanium-Gold Alloy
  8. DifcoTM Agar, Granulated (VWR, BD Biosciences, catalog number: 90000-786)
  9. L-Glutamic acid monosodium hydrate (Sigma-Aldrich, catalog number: G1626)
  10. Glycerol 99% purity (Fisher Chemical, catalog number: G/0650/17)
  11. 3-(N-morpholino)propanesulfonic acid (MOPS) (Sigma-Aldrich, catalog number: M9381)
  12. Potassium Phosphate dibasic Trihydrate, K2HPO4·7H2O (Sigma-Aldrich, catalog number: 431478)
  13. Potassium Dihydrogen Orthophosphate, KH2PO4 (Fisher Chemical, catalog number: 7778-77-0)
  14. D-Glucose (Sigma-Aldrich, catalog number: D9434)
  15. Ammonium Chloride, NH4Cl (Fisher Chemical, catalog number: 12125-02-9)
  16. Calcium Chloride, CaCl2·2H2O (Fisher Chemical, catalog number: 10035-04-8)
  17. Magnesium Chloride, MgCl2 (Fisher Chemical, catalog number: 7791-18-6)
  18. Iron (III) Chloride, FeCl3·6H2O (Fisher Chemical, catalog number: 10025-77-1)
  19. Manganese Chloride, MnCl2 (Sigma-Aldrich, catalog number: 244589)
  20. Zinc Chloride, ZnCl2 (Fisher Chemical, catalog number: 7646-85-7)
  21. Thiamine Hydrochloride (Sigma-Aldrich, catalog number: T4625)
  22. Thioflavin T (Sigma Aldrich, catalog number: T3516)
  23. NaOH
  24. LB Broth (Fisher Scientific, catalog number: BP1426-2)
  25. 0.5 M potassium phosphate buffer (pH 7) (see Recipes)
  26. MOPS buffer (see Recipes)
  27. Modified MSgg (see Recipes)

Equipment

Note: The list of the specific equipment used in Stratford et al., 2019 can be found in Tables 1-2.

Essential Equipment

  1. Inverted Fluorescence Microscope
  2. Microscope Incubation Chamber
  3. Temperature Controller
  4. Fluorescent Light Source
  5. Arbitrary Function Generator
  6. Bespoke electrode dishes (The bill of materials is in Table 3)
  7. Relay Circuit (The diagram is in Figure 4)
  8. Electron Beam Vapor Deposition System or equivalent alloy deposition method
Note: Items #5-8 are not necessary with CytePulse method.

Supplementary Equipment
  1. pH meter or testing strips
  2. Weighing scales
  3. Shaking incubator
  4. Adjustable volume pipettes ranging from 0.1 μl to 1,000 μl
  5. Measuring cylinder or serological pipettes ranging from 5-100 ml
  6. Scalpel
  7. Tweezers
  8. Bunsen Burner

    Table 1. List of equipment for Example Essential Equipment


    Table 2. List of equipment for Example Supplementary Equipment


    Table 3. Bespoke electrode dish bill of materials

Software

  1. Microscope Control Software (e.g., MetaMorph, micro-manager [micro-manager.org/])
  2. Arduino IDE (arduino.cc) (not necessary with Cytepulse method)
  3. Image processing software (e.g., Fiji/ImageJ (fiji.sc) (Schindelin et al., 2012))
  4. Data processing software (e.g., Matlab, Anaconda Python/R Distribution [anaconda.com]).

Procedure

For those using the bespoke method, as in Stratford et al. (2019) all steps must be followed. For those using the CytePulse system (Cytecom Ltd), skip to Procedure C “Sample preparation” section. Also, of note, those steps demarcated with an asterisk (*) in the procedure only pertain to those following the bespoke method. If using the CytePulse product and consumables, disregard these steps.


  1. Bespoke electrode dish construction
    The following methods are based on the use of an electron beam vapor deposition system. Different methods may vary between different constructions. Figure 2 provides an illustrative instruction.
    1. Construct a negative stainless-steel mask (Figure 1). This is done by a Laser Manufacturing Company.


      Figure 1. Negative electrode mask. Diagram of the negative electrode mask for construction of bespoke electrode dishes.

    2. Construct the mounting wafer (Figure 2).


      Figure 2. Mounting wafer construction process. i. Materials required for mounting wafer construction (1x stainless steel mounting wafer; 4x stainless steel washer; double-sided adhesive tape). ii. Apply double-sided adhesive tape to the bottom of each washer. iii. Secure each washer to the mounting wafer.

    3. Place the mask into the glass-bottomed microscope dish (Figure 3).


      Figure 3. Bespoke Electrode Dish Construction; Steps A3-A6. All numbers correspond to their respective steps within the bespoke electrode construction process.

    4. Using non-magnetic tweezers place 12 nickel magnets in positions parallel to each electrode. This ensures that the mask is in contact with the glass.
    5. Mount the dish with mask, magnets and wire onto the stainless-steel platform on the mounting wafer. Place into the electron vapor deposition system.
    6. Deposit the Titanium-Gold Alloy onto the dish using Electron Vapor Deposition (20 nm Ti; 80 nm Au).
    7. Remove the dish from the vapor deposition machine. Check the electrode gap is uninterrupted for each position using an optical microscope.
    8. Solder 7 copper wires of approximately 5 cm to the corresponding pins of a male IDC connector.
    9. Using the soldering iron, create 7 small holes in the plastic rim of the now patterned glass-bottom electrode dish. Feed each of the copper wires through a hole. Secure the male IDC connector to the rim of the dish with Araldite two-part epoxy.
    10. Trim the copper wires to appropriate lengths in order to contact each electrode terminal (including ground) as denoted in Figure 3. Bond the copper wire with conductive graphite epoxy. Once graphite epoxy has solidified, further coat the graphite bond with Araldite two-part epoxy for the purpose of waterproofing.

  2. Relay circuit construction
    The following circuit is for controlling electrical stimulation across electrodes. Individual electrodes are gated until triggered either manually or after a predetermined number of microscope images, dependent on user microscope configuration. The bill of materials may be found in Table 2. Code to run the circuit is deposited at github (github.com/ConorEd/ElectrodeStimulation).
    1. Solder two female BNC connectors; 6 Relays; 6 1MΩ resistors and two 10-way male IDC connectors to the prototyping board, as shown in Figure 4.
    2. Mount the Arduino UNO microcontroller to the prototyping board and connect the Arduino GPIO pins to the circuit as in Figure 4. This may be accomplished by either extruding breadboard jumper wires from the circuit and plugging them into the pins, or creating a ‘shield’ by soldering 2.54 mm pitch header pins to the circuit board.
    3. Cut ribbon cable to appropriate lengths to reach the microscope and a signal recording device.
    4. Connect two 10-way female IDC connectors to either end of the microscope cable.
    5. Connect the Arduino UNO microcontroler to a computer with the USB B cable and upload the code found at www.github.com/ConorEd/ElectrodeStimulation.


      Figure 4. Relay Circuit Diagram. Detailed schematic for construction of the relay circuit.

  3. Sample preparation (Figures 5 and 6)
    The method may be applied to many bacterial strains or species, subject to appropriate choice of liquid and solid growth media. Wildtype Bacillus subtilis (NCIB3610) or Escherichia coli (K12), at a density of OD600 = 1.5, serves as a good model organism for the method and was used in Stratford et al., 2019.
      The following protocol details the procedure for a culture of wildtype B. subtilis (NCIB3610), OD600 ~1.5. Using cultures treated with or without antibiotic vancomycin, the procedure determines between antibiotic-susceptible and -resistant cells. In Stratford et al. (2019), we showed that cells treated with UV, ethanol or CCCP show the response distinct from untreated proliferative cells. Cells treated with different antimicrobial agents or procedures can be tested for their electrical responses for rapid AST.

    Note: While this method should apply to a broad range of microbes, adaptation may be required for use with species and/or strains with different growth requirements.

    LB culture
    1. Place a single colony of B. subtilis into 5 ml aliquots of LB liquid media. Use the standard aseptic technique of microbiology.
    2. Place in overnight in a shaking incubator (37 °C; 200 rpm). Monitor the optical density of cell cultures using a spectrophotometer, the benchmark OD600 for this procedure at the time of experimentation is approximately 1.5.

    MSgg liquid culture
    1. Centrifuge B. subtilis LB cultures at 3,220 x g (4,000 rpm) for 5 min; retain the pellet and discard the liquid supernatant.
    2. Resuspend the pellets in the equal volume of liquid MSgg media supplemented with or without vancomycin (or other antimicrobial agents).
    3. Place the culture in a shaking incubator (30 °C; 200 rpm) for 1 h.

    Creating agar pads *
    1. ~30 min prior to retrieving the cultures, create the agar pads
      Place a microscope cover glass on Parafilm, this ensures the liquid MSgg agar solution remains on the cover glass. Add 1 ml of liquid MSgg agar to a single 22 x 22 mm glass slip, carefully covering the whole slip.
      1. Immediately place another coverslip on top of the agar, then wait for the agar to solidify. Avoid bubbles to be formed.
      2. Using tweezers and scalpel, prize the top glass slide off the solid agar, then cut the agar into ~50 mm2 square pads.

  4. Loading samples into electrode dish or Cytecom sample holder (Figures 5 and 6)
    1. Add 1.5 μl of cell suspension to each of the agar pads, ensuring the droplet is central and not touching edges of the pad, to prevent lateral pad drifts due to bacterial growth.
    2. Wait 10 min for the drop to dry into the agar pad at room temperature.
    3. Using a scalpel and tweezers, slowly lift the agar pad from the bottom and flip onto the electrode, sample side down, so as the sample is in direct contact with the electrode.*
      Note: The agar pads can be very fragile, take care not to break or scratch agar pads. Make sure to place the inoculated side facing the electrode.
    4. Close the lid on the dish or Cytecom sample holder to prevent sample drying.
      Note: If using the Bespoke electrode dish, ensure Parafilm seals the dish shut, to prevent excessive moisture loss from the samples.

  5. Preparing sample for microscopy
    1. Secure the dish in the microscope stage insert. If using an oil immersion lens, add immersion oil onto the bottom of the dish or Cytecom sample holder, over each electrode.
    2. Place the stage insert onto the microscope stage.
    3. Open MetaMorph (or equivalent microscope operating software).
    4. Locate the position of an electrode and find the correct focus under phase contrast, DIC or brightfield illumination. Save the position of electrode using the microscope software. Repeat this for all 6 electrodes.
      Note: Add a ‘rest’ position well away from the electrodes, this should be a place for the objective to remain away from the electrodes between imaging sequences. This helps prevent the pads from drying out.
    5. Leave the sample to settle in the microscope chamber for 1 h at 30 °C to prevent focus drift due to thermal shift.

  6. Time-lapse microscopy settings
    1. Set the microscope parameters as per Table 4. Time-lapse is taken for each position separately.

      Table 4. Time-lapse Microscopy Parameters


    2. Upload code (https://github.com/ConorEd/ElectrodeStimulation) to the Arduino UNO.* microcontroller, ensuring all relays are set to be closed. Either manual or microscope-triggered relay control may be set prior to upload within the Arduino IDE.
    3. Verify that the electrical stimulation settings are correct (as per Table 5).
    4. Turn on the signal generator.*
    5. Connect the electrode dish to the device via IDC connector (Figure 7).

      Table 5. Electrical stimuli parameters



      Figure 5. Overview of the bespoke protocol, from cell culture to microscope. Illustrative diagram for creating cell culture, MSgg liquid culture, creating agar pads and loading samples into Cytecom sample holder.


      Figure 6. Overview of the cytecom protocol; from cell culture to microscope. Si. Creating Cell Cultures (Method found in supplementary information). Ci. MSgg Liquid Culture. D. Loading samples into the Cytecom sample holder.


      Figure 7. Connecting dish to device. Connect the signal output IDC connector to i) the bespoke dish or ii) CytePulse stage insert, respectively.

    6. Start image acquisition through the microscope software.

Data analysis

  1. Load the time-lapse image series in Fiji/ImageJ (Schindelin et al., 2012) (or, equilibrant image analysis software)
    Note: It is advisable to play the dataset as a movie to ensure there is no focus drifting, image drifts or bubble formation by electrolysis.
  2. Add regions of interest (ROIs) around individual cells using ROI manager in ImageJ.
  3. Use multi-measures function in ROI manager to measure the intensity values over time. In the founding study, we observed that fluorescence intensity increases in proliferative cells while decreases in inhibited cells.

Recipes

Note: All Media need to be autoclaved and treated with standard microbiology aseptic techniques.

  1. LB
    Mix 25 g/L as directed on the bottle
  2. 0.5 M potassium phosphate buffer (pH 7)
    1. Mix 68.5 ml 1 M K2HPO4 and 31.5 ml 1 M KH2PO4
    2. Add 100 ml of water to dilute down to 0.5 M
  3. 1 M MOPS buffer
    1. Make up 1 mole MOPS salt into 900 ml water
    2. Titrate the pH to 7.0 using NaOH
    3. Mass up to 1 L
  4. Modified MSgg
    1. 20 ml sterile MillQ water
    2. 200 μl 1 M MOPS buffer
    3. 200 μl 0.5 M potassium phosphate buffer
    4. For MSgg Agar only: 0.3 g agar
    Note: Autoclave after adding ingredients a-c [a-d in the case of solid MSgg agar]. Add ingredients e-n [e-o in the case of solid MSgg agar] just prior to use of the media in the procedure.
    1. 200 μl of 50% glycerol (v/v)
    2. 200 μl of 25% glucose (w/v)
    3. 400 μl of 25% L-Glutamic acid (w/v)
    4. 200 μl of 0.1 M Ammonium chloride
    5. 20 μl of 0.7 M CaCl2
    6. 20 μl of 2 M MgCl2
    7. 20 μl of 0.1 M FeCl3
    8. 20 μl of 0.05 M MnCl2
    9. 0.4 μl of 0.1 M Thiamine
    10. 0.2 μl of 0.1 M ZnCl2
    11. For MSgg Agar only: 20 μl of 10 mM ThT
    12. For AST, supplement with antimicrobial agents as needed

Acknowledgments

This protocol is adapted from Stratford et al., 2019. The work is supported by Biotechnology and Biological Sciences Research Council (BBSRC)/Engineering and Physical Sciences Research Council (EPSRC) grant to the Warwick Integrative Synthetic Biology Centre (Grant BB/M017982/1).

Competing interests

JPS and MA are the founder and shareholders of Cytecom Ltd (Cytecom.co.uk). JPS and CLAE are employees of Cytecom Ltd. JPS and MA have filed a patent for the reported technology.

References

  1. Ahmed, A., Rushworth, J. V., Hirst, N. A. and Millner, P. A. (2014). Biosensors for whole-cell bacterial detection. Clin Microbiol Rev 27(3): 631-646.
  2. Benarroch, J. M. and Asally, M. (2020). The microbiologist’s guide to membrane potential dynamics. Trends in Microbiology.
  3. Bruni, G. N., Weekley, R. A., Dodd, B. J. T. and Kralj, J. M. (2017). Voltage-gated calcium flux mediates Escherichia coli mechanosensation. Proc Natl Acad Sci U S A 114(35): 9445-9450.
  4. Damper, P. D. and Epstein, W. (1981). Role of the membrane potential in bacterial resistance to aminoglycoside antibiotics. Antimicrob Agents Chemother 20(6): 803-808.
  5. Iqbal, S. S., Mayo, M. W., Bruno, J. G., Bronk, B. V., Batt, C. A. and Chambers, J. P. (2000). A review of molecular recognition technologies for detection of biological threat agents. Biosens Bioelectron 15(11-12): 549-578.
  6. Prindle, A., Liu, J., Asally, M., Ly, S., Garcia-Ojalvo, J. and Süel, G. M. (2015). Ion channels enable electrical communication in bacterial communities. Nature 527(7576): 59-63.
  7. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J. Y., White, D. J., Hartenstein, V., Eliceiri, K., Tomancak, P. and Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7): 676-682.
  8. Sirec, T., Benarroch, J. M., Buffard, P., Garcia-Ojalvo, J. and Asally, M. (2019). Electrical polarization enables integrative quality control during bacterial differentiation into spores. iScience 16: 378-389. 
  9. Strahl, H. and Hamoen, L. W. (2010). Membrane potential is important for bacterial cell division. Proc Natl Acad Sci U S A 107(27): 12281-12286.
  10. Stratford, J. P., Edwards, C. L. A., Ghanshyam, M. J., Malyshev, D., Delise, M. A., Hayashi, Y. and Asally, M. (2019). Electrically induced bacterial membrane-potential dynamics correspond to cellular proliferation capacity. Proc Natl Acad Sci U S A 116(19): 9552-9557.
  11. Sträuber, H. and Müller, S. (2010). Viability states of bacteria-specific mechanisms of selected probes. Cytometry A 77(7): 623-634.

简介

[摘要 ] 检测活细菌是医学领域抗菌药敏测试(AST)和生物行业质量监测的重要任务。Ç 为活细菌urrent方法检测遭受速度或灵敏度的限制。在最近的一篇论文中,我们报道了膜电势中的电响应动力学能够实现单细胞快速检测活细菌。电刺激后一分钟内即可观察到电响应。因此,它具有加速AST和监测生物样品的潜力。这种方法还可以进行生物物理和微生物研究实验 进入细菌电生理。希望有更多的研究人员,科学家和工程师将电刺激用于他们的测定,这里我们详细介绍电刺激实验的每个步骤。

[背景 ] 从基础微生物学研究药敏试验(AST),量化活的细菌的数量是一个重要的任务。当涉及到检测活细菌,菌落形成单位(CFU)测定,从19约会个世纪,仍然是金标准,由于其可靠性和灵敏度。但是,CFU测定法有一个主要缺点。需要1-3天的孵育时间。为了克服该限制,近年来快速细菌检测技术的发展蓬勃发展(Iqbal 等,2000; Ahmed 等,2014)。最近开发的技术的实例为基于阻抗光谱,拉曼光谱或生物分子的检测(例如,ATP,DNA)。然而,克服速度,准确性和鲁棒性之间的权衡仍然是一个挑战。

膜电位指示剂通过流式细胞仪和荧光显微镜已被证明可用于区分活细菌和死细菌(Sträuber 和Müller,2010年)。但是,此方法在技术上具有挑战性,因为它需要对细菌种类/菌株,培养基条件,指示剂浓度,光源和检测器进行仔细校准。在最近的研究中,我们表明可以通过光学测量外部电场刺激下膜电位的电响应动力学来克服这些局限性(Stratford 等人,2019)。一分钟内就可以区分出增殖细胞和抑制细胞的反应。因此,它大大缩短了活细菌测定所需的时间。现象学数学模型和生物物理理解的模拟表明,该技术适用于不同的微生物种类和抗菌治疗。这意味着,通过比较不受干扰和经过抗菌处理的细胞的电动力学,可能会加速AST。下一步的重要步骤是研究该技术可与环境和病原微生物一起使用的范围。该协议将有助于针对各种细菌种类和菌株以及各种抗菌治疗进行AST和活菌检测测定的电刺激实验。

除了其在细胞增殖中的基本作用外,膜电位还可以介导生物膜中以及在孢子形成,机械感觉和细胞分裂过程中的细菌电信号传导(Strahl 和Hamoen ,2010; Prindle 等,2015; Bruni 等, 2017; Sirec 等人,2019 ; Benarroch 和Asally ,2020 )。膜电位也与抗生素耐药性有关(Damper和Epstein,2010)。尽管这些最近的研究开始引起人们的注意,但在微生物学领域中膜的潜力仍然被大大忽略。我们怀疑这种疏忽部分是由于与实验控制膜电位相关的技术困难。为此,该协议还将协助对细菌电生理进行生物物理和微生物学研究。

关键字:细菌电生理学, 细胞膜电势, 细菌检测, 细菌耐药, 细胞活力检测, 细胞生物物理学

材料和试剂


 


注意小号:


根据研究人员的判断,以下化学物质可与适当的替代品互换。
所有这些成分都可以在室温下干燥的条件下存储。
 


不锈钢安装晶片
铜线
封口膜
22 x 22毫米玻片
显微镜盖玻片
细菌样本
钛金合金
Difco TM 琼脂,颗粒状(VWR,BD Biosciences,目录号:90000-786 )
L-谷氨酸水合钠(Sigma-Aldrich,目录号:G1626)
甘油99%纯度(Fisher Chemical,目录号:G / 0650/17)
3-(N-吗啉代)丙烷磺酸(MOPS)(Sigma-Aldrich,目录号:M9381)
二水合磷酸氢钾,K 2 HPO 4 ·7H 2 O (Sigma-Aldrich,目录号:431478)
正磷酸二氢钾,KH 2 PO 4 (Fisher Chemical,目录号:7778-77-0)
D-葡萄糖(Sigma-Aldrich,目录号:D9434)
氯化铵,NH 4 Cl(Fisher Chemical,目录号:12125-02-9)
氯化钙,CaCl 2 ·2H 2 O(Fisher Chemical,目录号:10035-04-8)
氯化镁,MgCl 2 (Fisher Chemical,目录号:7791-18-6)
氯化亚铁(FeCl 3 ·6H 2 O)(Fisher Chemical,目录号:10025-77-1)
氯化锰MnCl 2 (Sigma-Aldrich,目录号:244589)
氯化锌ZnCl 2 (Fisher Chemical,目录号:7646-85-7)
盐酸硫胺素(Sigma-Aldrich,目录号:T4625)
硫黄素T(Sigma Aldrich,目录号:T3516)
氢氧化钠
LB汤(Fisher Scientific,目录号BP1426-2 )
0. 5 M磷酸钾缓冲液(pH 7)(请参阅食谱)
MOPS缓冲区(请参阅食谱)
修改后的MSgg (请参阅食谱)
 


设备


 


注:牛逼,他列出斯特拉特福德等人所使用的特定设备。,2019 可在表中找到1 - 2 。


 


基本设备


倒置荧光显微镜
显微镜培养箱
温度控制器
荧光灯光源
任意函数发生器
定制电极盘(材料清单在表3中)。
继电器电路(该图在图4中)。
电子束气相沉积系统或等效合金沉积方法
注意:使用CytePulse 方法不需要#5- 8 。


 


辅助设备


pH计或试纸
体重秤
摇摇培养箱
音量可调的移液管,从0.1 微升至1000 微升
量筒或血清移液管从5 - 100毫升
解剖刀
镊子
本生燃烧器




表1 。基本设备示例设备清单


基本设备


 


正文参考


基础学习设备


多级荧光显微镜


 


配备Leica CTR高级电子盒的Leica DMi8显微镜(Leica)


孵化室


佩康有限公司(PeCon )


温度控制器


TempController 2000-2加热器单元(Pecon )


荧光灯光源


Lumencor SOLA SE光引擎光源(Lumencor )


任意函数发生器


泰克AFG1062函数发生器(RS,目录号:898-6858)


定制电极盘


施工过程:s 程序A


物料清单:表3


CytePulse 易损件。


CytePulse 耗材(Cytecom )


继电器电路


物料清单:表3 。


电路图:图4


电子气相沉积系统


定制模型II2000EB(科学真空系统)


 


表2 。辅助设备示例设备清单


辅助设备


 


正文参考


基础学习设备


pH计


pH计(Fisher-Scientific,目录号:AE-150)


体重秤


分析天平(Fisherbrand ,目录号:PS100)


摇摇培养箱


摇动培养箱(Labnet ,目录号:311DS)


可调量移液器


可调量移液器(Eppendorf,Research Plus)


分光光度计


Jenway 7305分光光度计


 






表3 。定制电极盘物料清单


零件


制造商


制造商零件编号


数量


玻璃底显微镜皿


威尔科·威尔斯


HBST-5040


4


爱牢达两部分环氧树脂


一个红宝石


2457-H


1个


钕磁铁


中号agnets4U


SP515-50


50


不锈钢口罩


激光微加工


不适用


1个


M 6平板维修洗衣机


奇异用品


不适用


4


Arduino Uno(版本3)


Arduino的


0000


1个


中继


RS PRO


291-9704


7


凤凰联络人


凤凰联络人


1725656


4


BNC母连接器


RS PRO


526-5864


4


电阻1MΩ


TE Connectivity


LR1F1M0


7


原型面包板


维罗技术


01-0021


1个


10路IDC连接器(公)


RS PRO


625-7252


7


10路IDC连接器(母)


RS PRO


625-7369


4


10向带状电缆卷盘


RS PRO


360-093


1个


2.54 mm间距排针


RS PRO


251-8121


3


面包板跳线


RS PRO


791-6463


1个


 


软件


 


显微镜控制软件(例如,的MetaMorph ,微经理[ micro-manager.org/ ] )
Arduino IDE(arduino.cc)(Cytepulse 方法不需要)
图像处理软件(例如,Fiji / I mageJ(fiji.sc)(Schindelin 等人,2012 ))
数据处理软件(例如,Matlab的,水蟒的Python / R分布[ anaconda.com ] )。
 


程序


 


对于那些使用定制方法的人员,如Stratford 等人所述。(2019 )必须遵循所有步骤。对于使用CytePulse 系统(Cytecom Ltd)的用户,请跳至步骤C “ 样品制备”部分。另外,值得注意的是,该过程中标有星号(*)的步骤仅适用于采用定制方法的步骤。如果使用CytePulse 产品和耗材,请忽略这些步骤。


 


定制电极盘结构
在下面的方法是基于使用电子束汽相淀积系统。不同的方法可以不同的结构之间变化小号。图2提供了说明性指令。


CON STRUCT负不锈钢掩模(图1)。这是由一家激光制造公司完成的。
 


 


图1.负极掩模。用于定制电极皿构造的负电极掩模示意图。


 


构造安装晶片(图2)。
 


 


图2.安装晶片的过程。一世。安装晶片结构所需的材料(1 个不锈钢安装晶片; 4个不锈钢垫圈;双面胶带)。ii。在每个洗衣机的底部粘贴双面胶带。iii。将每个垫圈固定到安装晶片上。


 


将面罩放入玻璃底显微镜皿中(图3)。
 


 


图3. Bespo ke电极盘结构;步骤A3-A6。所有数字对应于定制电极构建过程中的它们各自的步骤。


 


使用非磁性镊子在与每个电极平行的位置放置12个镍磁体。这样可以确保面罩与玻璃接触。
将带有面罩,磁铁和电线的碟子安装到安装晶片上的不锈钢平台上。放入电子气相沉积系统。
使用电子气相沉积法(20 nm Ti ; 80 nm Au)将钛金合金沉积到培养皿上。
从蒸镀机上取出碟子。使用光学显微镜检查每个位置的电极间隙是否连续。
将大约5厘米的7根铜线焊接到公IDC连接器的相应引脚上。
使用烙铁,在现在已图案化的玻璃底电极盘的塑料边缘上创建7个小孔。将每个铜线穿过一个孔。用Araldite两部分环氧树脂将公IDC连接器固定到培养皿的边缘。
将铜线修剪成适当的长度,以接触每个电极端子(包括接地),如图3 所示。用导电石墨环氧树脂将铜线粘合在一起。石墨环氧树脂固化后,进一步用Araldite两部分环氧树脂涂覆石墨结合剂以防水。
 


继电器电路构造
以下电路用于控制跨电极的电刺激。门控各个电极,直到手动触发或在预定数量的显微镜图像后触发为止,具体取决于用户显微镜的配置。物料清单可在表2中找到。运行该电路的代码位于github (github.com/ ConorEd / ElectrodeStimulation )。


焊接两个BNC母连接器;6个继电器 原型板有6个1MΩ电阻器和2个10通IDC公头连接器,如图4所示。
沫ü NT中的Arduino UNO微控制器的原型板并连接Arduino的GPIO引脚到所述电路如图4这可以通过从电路或者挤压试验板跨接线来实现,并将它们插入到销,或创建“屏蔽通过将2.54 mm间距的排针焊接到电路板上。
将带状电缆切成适当的长度,以到达显微镜和信号记录设备。
将两个10通IDC母头连接器连接到显微镜电缆的任一端。
使用USB B电缆将Arduino UNO微控制器连接至计算机,然后上传位于www.github.com/ConorEd/ElectrodeStimulation的代码。
 


 


图4.继电器电路图。继电器电路构造的详细示意图。


 


样品preparatio Ñ (图小号5 和6 )
该方法可适用于许多细菌菌株或物种,需适当选择液体和固体生长培养基。威尔D型枯草芽孢杆菌(NCIB3610),或大肠杆菌(K12),在OD的密度600 = 1.5,作为粘性物的方法d模式生物和斯特拉特福使用等。,2019。


  以下协议细节的野生型培养物的程序枯草芽孢杆菌(NCIB3610),OD 600 〜1.5。使用经过或未经过抗生素万古霉素处理的培养物,该程序可确定对抗生素敏感的细胞和对耐药的细胞。在斯特拉特福等。(2019 ),我们显示了用UV,乙醇或CCCP处理过的细胞显示出与未经处理的增生细胞不同的反应。可以测试用不同的抗菌剂或程序处理过的细胞的快速AST的电响应。


 


注意:虽然此方法应适用于广泛的微生物,但可能需要适应于具有不同生长要求的物种和/或菌株。






LB文化


将枯草芽孢杆菌的单个菌落放入LB液体培养基的5 ml等分试样中。使用微生物学的标准无菌技术。
放置在振荡培养箱(37 °C; 200 rpm)中过夜。监测使用分光光度计细胞培养物的光密度,基准OD 600 在此过程中在所述时间实验的是大约1.5。
 


MSgg 液体培养


将枯草芽孢杆菌LB培养物在3,220 xg (4,000 rpm)下离心5分钟; 保留沉淀并丢弃液体上清液。
在等体积的补充或不加万古霉素(或其他抗菌剂)的MSgg 液体培养基中重悬沉淀。
将培养物置于振荡培养箱(30 °C; 200 rpm)中1 h 。
 


创建琼脂垫*


提取培养物之前〜30分钟,创建琼脂垫
放置在显微镜盖玻璃上的P arafilm,这确保了液体MSgg 琼脂溶液保持小号上Ç 超过玻璃。加入1 米升液体的MSgg 琼脂到单个22×22毫米的玻璃滑,小心覆盖整个滑移。


立即在琼脂上放置另一个盖玻片,然后等待琼脂凝固。避免形成气泡。
用镊子和手术刀将顶部的玻璃片从固体琼脂上倒出,然后将琼脂切成约50 mm 2的方形垫。
 


装载样品放入电极盘或Cytecom 样本保持器(图小号5 和6 )
将1.5μl 细胞悬液添加到每个琼脂垫上,确保液滴在中心且不接触垫的边缘,以防止由于细菌生长而导致垫侧向漂移。
等待10分钟,使滴液在室温下干燥至琼脂垫中。
用手术刀和镊子将琼脂垫从底部缓慢提起并翻转到电极上,使样品的一面朝下,以使样品直接与电极接触。*             
注意:琼脂垫可能非常脆弱,请注意不要破坏或划伤琼脂垫。确保将接种面朝向电极。


关闭培养皿或Cytecom样品架上的盖子,以防止样品干燥。
注意:如果使用所述定制的电极盘,确保P arafilm密封盘关闭,以防止从样本过多水分流失。


 


准备样品进行显微镜检查
将培养皿固定在显微镜载物台插入物中。如果使用油浸透镜,则在每个电极上方,将浸油浸入培养皿或Cytecom样品架的底部。
将载物台插入显微镜载物台。
打开MetaMorph (或等效的显微镜操作软件)。
找到电极的位置,并在相衬,DIC或明场照明下找到正确的焦点。使用显微镜软件保存电极的位置。对所有6个电极重复此操作。
注意:在远离电极的位置添加一个“静止”位置,这应该是物镜在成像序列之间保持远离电极的位置。这有助于防止垫变干。


将样品在30 °C下在显微镜室中静置1 h,以防止由于热移动而引起的焦点漂移。
 


延时显微镜设置
按照表4 设置显微镜参数。时差分别用于每个位置。
 


表4 。蒂姆· E-推移显微镜参数


参数


设置


总时长


1分钟


影像间隔


500 毫秒


波长


434 nm(CFP滤光片)


曝光时间


150 毫秒


荧光强度


X


 


将代码(https://github.com/ConorEd/ElectrodeStimulation)上传到Arduino UNO。* 微控制器,确保所有继电器均设置为闭合。在Arduino IDE上载之前,可以设置手动或显微镜触发的继电器控制。
              验证电刺激设置正确(如表5所示)。
打开信号发生器。*
通过IDC连接器将电极盘连接到设备(图7 )。
 


表5 。电刺激参数


参数


设置


持续时间


2500 毫秒


波形图


正弦波


频率


100赫兹


电压


±1.5伏


偏移量


0伏


 


 


图5. 从细胞培养到显微镜的定制方案概述。用于创建细胞培养物,MSgg 液体培养物,创建琼脂垫并将样品加载到Cytecom 样品架中的示意图。


 


图6. cytecom 协议概述;从细胞培养到显微镜。硅。创建细胞培养物(方法见补充信息)。慈 MSgg 液体培养。D.将样品装入Cytecom 样品架。


 


 


图7.将碟子连接到设备。将信号输出IDC连接器分别连接到i)定制碟或ii)CytePulse载物台插件。


 


通过显微镜软件开始图像采集。
 


数据分析


 


将延时图像序列加载到Fiji / ImageJ中(Schindelin 等,2012)(或平衡图像分析软件)
注意:建议将数据集作为电影播放,以确保没有因电解而引起的焦点漂移,图像漂移或气泡形成。


使用ImageJ中的ROI管理器在单个单元格周围添加感兴趣的区域(ROI)。
使用ROI Manager中的多项测量功能来测量随时间变化的强度值。在基础研究中,我们观察到增殖细胞中的荧光强度增加,而抑制细胞中的荧光强度降低。
 


菜谱


 


ñ OT E:所有的媒体需要进行高压灭菌,用标准的微生物无菌技术处理。



按照瓶子上的指示混合25 g / L


0. 5 M磷酸钾缓冲液(pH 7)
混合68.5 ml 1 M K 2 HPO 4 和31.5 ml 1 M KH 2 PO 4
加100 毫升水稀释至0. 5 M
1 M MOPS缓冲区
将1 摩尔MOPS盐倒入900毫升水中
用NaOH滴定pH至7.0
质量高达1 L
修改后的MSgg
1)20毫升小号terile 中号illQ 瓦特亚特      


2)200 微升的1M MOPS b uffer      


3)200 微升的0.5M钾,对hosphate b uffer      


4)仅适用于MS gg 琼脂:0.3克琼脂      


在固体MSgg 琼脂的情况下,加入成分1)-3)[1)-4)后高压灭菌]。对于固体MSgg 琼脂,在过程中使用培养基之前,先添加成分5)-14)[5)-15)。


5)200 微升50%的甘油(V / V)      


6)200 微升25%葡萄糖的(W / V)      


7)400 微升的25%L-谷氨酸(W / V)      


8)200 微升的0.1氯化物M铵      


9)20 微升的0.7M的CaCl 2      


10)20 微升2 M MgCl 2   


11)20 微升的0.1M的的FeCl 3   


12)20 微升的0.05M的的MnCl 2   


13)0.4 微升0.1M的硫胺的   


14)0.2 微升的0.1M氯化锌的2   


15)对于MSgg 琼脂只:20 微升的10mM的ThT的   


16)对于AST,根据需要补充抗菌剂   


 


致谢


 


该协议改编自St r atford 等。,2019年。这项工作得到了生物技术和生物科学研究委员会(BBSRC)/工程和物理科学研究委员会(EPSRC)对沃里克综合合成生物学中心(Grant BB / M017982 / 1)的资助。


 


利益争夺


 


JPS和MA是Cytecom Ltd(Cytecom.co.uk)的创始人和股东。JPS和CLAE是Cytecom Ltd.的雇员。JPS和MA已为该报告的技术申请了专利。


 


参考文献


 


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引用:LA Edwards, C., Malyshev, D., Stratford, J. P. and Asally, M. (2020). Rapid Detection of Proliferative Bacteria by Electrical Stimulation. Bio-protocol 10(3): e3508. DOI: 10.21769/BioProtoc.3508.
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