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May 2020
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ODELAM: Rapid Sequence-independent Detection of Drug Resistance in Mycobacterium tuberculosis Isolates
ODELAM:结核分枝杆菌耐药性的快速序列独立检测   

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Abstract

Antimicrobial-resistant Mycobacterium tuberculosis (Mtb) causes over 200,000 deaths globally each year. Current assays of antimicrobial resistance require knowledge of the mutations that confer drug resistance or long periods of culture time to test growth under drug pressure. We present ODELAM (One-cell Doubling Evaluation of Living Arrays of Mycobacterium), a time-lapse microscopy-based method that observes individual cells growing into microcolonies. This protocol describes sample and media preparation and contains instructions for assembling the ODELAM sample chamber. The ODELAM sample chamber is designed to provide a controlled environment to safely observe the growth of Mtb by time-lapse microscopy on an inverted wide-field microscope. A brief description of the ODELAM software is also provided here. ODELAM tracks up to 1500 colony forming units per region of interest and can observe up to 96 regions for up to seven days in a single experiment. This technique allows the quantification of population heterogeneity. ODELAM enables rapid quantitative measurements of growth kinetics in as few as 30 h under a wide variety of environmental conditions.


Graphic abstract:



Schematic representation of the ODELAM platform


Keywords: Mycobacterium tuberculosis (结核杆菌), Drug resistance (抗药性), Growth phenotypes (生长表型), Microbiology (微生物学), Microscopy (显微镜检查), Live imaging (Live imaging)

Background

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), which is responsible for 1.4 million deaths globally each year (World Health Organization, 2020). Despite decades of research, TB treatment is still challenging, and current chemotherapies require the combination of four different drugs (Pai et al., 2016). One of the reasons why Mtb remains a remarkably successful pathogen is its ability to produce heterogeneous phenotypes that exhibit variable responses to a given drug (Manina et al., 2015; Dhar et al., 2016; Rego et al., 2017; Logsdon et al., 2018). Thus, characterizing the growth phenotypes of individual Mtb cell populations is critical for understanding how mycobacteria sense and respond to stress conditions such as prolonged drug exposure (Santi et al., 2013; Logsdon et al., 2018).


To date, little is known about the sources and consequences of cell-to-cell differences, mostly due to limitations in the tools and techniques available for precise measurement of growth phenotypes of individual bacterial cell populations. Mtb is especially challenging to study due to its very long doubling time of approximately 24 h. Recent platforms to study single bacterial cells are limited either by the number of cells that can be observed or by the extensive time required (few weeks) to monitor the growth of mycobacterial colonies (Aldridge et al., 2012; Golchin et al., 2012; Wakamoto et al., 2013; Choi et al., 2014; Manina et al., 2015; Barr et al., 2016).


ODELAM (One-cell Doubling Evaluation of Living Arrays of Mycobacterium) was developed to overcome these limitations and is based on ODELAY, developed originally to analyze yeast (Herricks et al., 2017a, 2017b and 2020). ODELAY is a versatile method that has been utilized to investigate the structure-function relationship of the nuclear pore complex and peroxisome biogenesis (Kim et al., 2018; Mast et al., 2018). ODELAM uses time-lapse microscopy to quantify growth phenotypes of populations of individual Mtb colony-forming units (CFU). By direct monitoring of bacterial growth, we can assess the heterogeneity of Mtb strains, arising either naturally or in response to stresses, such as drug treatment. ODELAM can observe up to 1500 CFU per region of interest (ROI) and measure four main growth kinetic parameters: lag time, doubling time, exponential time (the time when colonies stop growing and enter stationary phase), and number of doublings for each identified CFU. ODELAM can track up to 100,000 CFUs in one experiment, which provides statistical power to detect phenotypic variations in the population. Importantly, ODELAM can screen between 80 and 96 samples growing in up to five media conditions within each experiment. In less than 48 h, ODELAM identifies resistant cells in a population of sensitive bacteria down to 1 per 1000 and predicts the minimum inhibitory concentration (MIC) of drugs with high accuracy. Additionally, the ODELAM platform is broadly applicable as a laboratory screening tool, as it can be easily adapted to study most colony-forming microorganisms. ODELAM can quantify heterogeneity and detect heteroresistance in Mtb clinical isolates. This cutting-edge technology will have a meaningful impact on studies on bacterial phenotypic heterogeneity and emergence of drug resistance and help to better understand bacterial adaptation to unfavorable environmental conditions, which will facilitate the development of new powerful therapies.

Materials and Reagents

  1. Mycobacterium tuberculosis culture

    1. 1.8 ml cryotubes (Nunc, catalog number: 375418)

    2. 50 ml conical bottom tubes (Sarstedt, catalog number: 62.547.254)

    3. 10 ml serological pipettes (VWR, catalog number: 89130-898)

    4. 200 μl tips (Genesee Scientific, catalog number: 24-412)

    5. 1,000 μl tips (Genesee Scientific, catalog number: 24-430)

    6. Steriflip-GP Sterile Centrifuge Tube Top Filter Unit (MilliporeSigma, catalog number: SCGP00525)

    7. Mtb strains (storage: -80°C)

      1. H37Rv (background strain available from BEI, catalog number: NR-123)

      2. Other strains of interest

    8. Difco Middlebrook 7H9 Broth (BD, catalog number: 271310, storage: room temperature (RT))

    9. Middlebrook OADC Enrichment (BD, catalog number: 212351, storage: 4°C)

    10. Glycerol (Sigma Aldrich, catalog number: G5516, storage: RT)

    11. Tween 80 (Sigma-Aldrich, catalog number: P1754, storage: RT protected from light)

    12. Disinfectants:

      1. 70% ethanol (Fisher Scientific, catalog number: 04-255-92, storage: RT)

      2. PREempt RTU Disinfectant Solution (Contec, catalog number: 21105, storage: RT)

      3. LoPhene Concentrate Rotational Disinfectant (Decon Labs, catalog number: 8801, storage: RT)


  2. ODELAM agarose gel preparation

    1. 50 ml conical bottom tubes (Falcon, catalog number: 352070)

    2. 15 ml conical bottom tubes (Falcon, catalog number: 352096)

    3. 5 ml syringe (BD, catalog number: 309646)

    4. 18 G × 1½ needle (BD, catalog number: 305196)

    5. Micro Slides 2 in × 3 in 1 mm thick (VWR, catalog number: 48382-180)

    6. UltraPure agarose (Invitrogen, catalog number: 16500, storage: RT)

    7. Difco Middlebrook 7H9 Broth (BD, catalog number: 271310, storage: RT)

    8. Middlebrook OADC Enrichment (BD, catalog number: 212351, storage: 4°C)

    9. Glycerol (Sigma Aldrich, catalog number: G5516, storage: RT)


  3. ODELAM spotting cell cultures

    1. 96-well plate, F-bottom (Greiner bio-one, catalog number: 655180)

    2. Pipette tips RT LTS 20µL 960A/10 (Raining, catalog number: 30389200)

    3. 7H9-GOT media (see Recipes)

    4. 10× 7H9-G media (see Recipes)

Equipment

  1. BSL1 or BSL2

    1. Pipettes

      1. Rainin 2-20 μl, 20-200 μl, 100-1,000 μl

      2. 0.1-10 μl 12-multichannel pipette (Pipet-Lite Multi Pipette L12-10XLS+) (Rainin, catalog number: 17013807)

      Note: The ODELAM chamber is designed for this specific pipette.

    2. 2 × hot plate stirrer (Corning, catalog number: PC-420D)

    3. 2 × 1 L beakers (Sigma-Aldrich, catalog number: CLS10001L-1EA)

    4. 2 × stir bars (VWR, catalog number: 58948-150)

    5. 2 × glass giant Petri dishes to cover the beakers

    6. Vortexer (Scientific Instruments, model: Vortex-Genie 2, catalog number: SI-0256)

    7. Thermometer (VWR, catalog number: 89095-640)

    8. Top-loading Electronic scale with 1 kg capacity and 0.01 g accuracy

    9. Medium (3 per one 5 glass-gasket-glass chambers) and large (2 per one single glass-gasket-glass chamber) binder clips

    10. 3D printer (e.g., Ender 3D, BCN3D Sigma v19, makerbot 2 ×)


  2. BSL3

    1. Pipettes:

      1. Eppendorf 2-20 μl, 20-200 μl, 100-1,000 μl

      2. 0.1-10 μl 12-multichannel pipette (Pipet-Lite Multi Pipette L12-10XLS+) (Rainin, catalog number: 17013807)) with attached binder clips

    2. Motorized inverted microscope with heated incubator (tested on Leica DMI6000 and Nikon TiE)

    3. Personal Protective Equipment (PPE):

      1. Double nitrile gloves

      2. Protective Tyvek coveralls

      3. Shoe covers

      4. Powered air-purifying respirator (PAPR)

    4. Absorbent pad (for working in the biosafety cabinet)

    5. ODELAY microscope chamber (Figure 1)

      Top Plate (machined from 6061-T6 aluminum)



      Figure 1. ODELAM sample chamber. The sample chamber for ODELAM experiments is shown in an exploded view (A) and disassembled with all relevant parts (B).


    6. Top PDMS Gasket (cut from McMaster-Carr Durometer 30A 9010K11)

    7. Indium Tin Oxide (ITO) coated -glass slide 1.1 mm thick (SPI Supplies, catalog number: 06404-AF)

    8. Base Plate (machined from 6061-T6 aluminum)

    9. Glass slide with 3D printed gasket and cast agarose media

    10. Bottom PDMS gasket (cut from McMaster-Carr Durometer 30A 9010K11)

    11. Bottom plate (machined from 6061-T6 aluminum)

    12. Pipette spotting alignment tool (machined from 6061-T6 aluminum)

    13. 1/16 hex screwdriver (McMaster-Carr, catalog number: 5497A23)

    14. 1/16 hex screwdriver (McMaster-Carr, catalog number: 5497A23)

    15. 12 × 4-40 5/8 inch stainless steel button-head screws (McMaster-Carr, catalog number: 98164A433)

    16. 8 × 4-40 1/4 inch stainless steel flat-head screws (McMaster-Carr, catalog number: 90585A200)

    17. 4 × 4-40 1/8 inch nylon socket head screws (McMaster-Carr, catalog number: 95868A254)

    18. Copper electrical contacts (cut from a copper sheet)

    19. 37 °C incubator (VWR 1545 Digital Incubator)

    20. Culture rotator (GEL GRO Tissue culture rotator, LAB-LINE)

Software

  1. Anaconda Python (https://www.anaconda.com/)

  2. Visual Studio Code (https://code.visualstudio.com/)

  3. ODELAY software package (https://github.com/AitchisonLab/)

  4. MicroManager 1.4 or 2.0 gamma (https://micro-manager.org/)

  5. Microsoft Office Excel or another spreadsheet-compatible program (https://www.office.com/)

  6. Cura 3D modeling (https://ultimaker.com/)

Procedure

  1. Mycobacterium tuberculosis culture

    Note: All work with Mtb should be conducted in a Biosafety Level 3 (BSL3) laboratory by trained personnel wearing appropriate PPE and be approved by the Institutional Biosafety Committee (IBC) and Environmental Health and Safety (EHS).

    1. Prepare the pre-culture by inoculating 10 ml of 7H9-GOT (Middlebrook 7H9 media supplemented with 0.2% glycerol, 10% OADC supplement, and 0.05% (v/v) Tween-80) with 0.5 ml of the thawed Mtb glycerol stock (1:20 dilution). Grow cells in 50 ml conical tubes at 37°C for 48-72 h, until cultures reach an OD600 of 0.2-0.4 (early logarithmic phase).

    2. Subculture by diluting 0.5 ml of the pre-culture in 10 ml of 7H9+GOT (1:20 dilution). Grow cells in 50 ml conical tubes at 37°C for 48-72 h, until cultures reach an OD600 of 0.2-0.4 (early logarithmic phase).

    3. Dilute the culture for spotting on the ODELAM plate (see Part E of Procedure for further instructions).


  2. ODELAM agarose aliquot

    1. Gather the 1 L media bottle, high purity agarose, pipettes, and Falcon tubes.

    2. Weigh 2 ± 0.02 g of agarose and place into 1 L media bottle.

    3. Add 150 g of 18 MΩ H2O to the media bottle.

    4. Record the total weight of the bottle, agarose, and water.

    5. Microwave the media bottle, water, and agarose until all agarose is dissolved.

    6. Weigh the media bottle, water, and agarose again to evaluate the amount of water lost during boiling.

    7. Add 18 MΩ H2O until the mass equals the previously recorded total weight.

    8. Aliquot agarose by mass, 15.1 g into 50 ml Falcon conical tubes or 3.0 g into a 15 ml Falcon conical tube.


  3. ODELAM agarose media preparation

    Note: The steps in this section can be performed outside of a biological safety cabinet if the lab is relatively dust free.

    1. Clean the previously printed gasket slide (see 3D printing gaskets of Notes section) using lab labeling tape.

    2. Heat two 1 L beakers with approximately 750 ml of Di H2O, one with boiling water and the other with water warmed to 40°C.

    3. Add the appropriate amounts of 10× media and additional reagents needed to the prepared agarose aliquots (Table 1).

      Note: Do not add reagents that are temperature sensitive.


      Table 1. Volume recipes for the 7H9-GO Media

      Final Media Volume 1.33% w.v. agarose 10× 7H9-G media 18 MΩ H2O OADC

      1,000× Drug
      (optional)

      20 ml 15.1 g 2 ml 1 ml 2 ml 20 μl
      10 ml 7.5 g 1 ml 0.5 ml 1 ml 10 μl
      4 ml 3.05 g 0.4 ml 0.2 ml 0.4 ml 4 μl


    1. Clean a 50 mm × 75 mm × 1 mm glass slide using 70% ethanol. When dry, wipe with lens paper to remove any residual particles and fibers.

    2. Place the clean glass slide on top of the printed gasket slide and secure slides with binder clips (Figure 2A).



      Figure 2. Assembly of agar slide. A. Ensure the gaskets are clamped directly over the gasket; otherwise, the glass will flex, and the agar will be uneven. B. Fill the slides with agarose media by injecting with a syringe needle. C-D. After allowing the media to set, use binder clip loops to pry slides apart gently (D).


    3. Assemble the syringes and 20-gauge needles for each media condition.

    4. Place the tube with the media formulation in boiling water. The 20 ml media tubes should be in the boiling water for 18 min and the 4 ml media tubes for 8 min.

      Note: Make sure the cap does not go below the water surface as this will cause water to leak into the tube. Additionally, ensure the beaker is covered; otherwise, the agarose will not melt completely.

    5. After boiling the appropriate amount of time and the agarose is melted, vortex the agarose media tube and place it in the 40°C water bath.

    6. Wait 3-5 min for the agarose to equilibrate at 40°C and then add the temperature-sensitive components (e.g., drug and OADC additive).

    7. Vortex the tubes thoroughly to mix the media and additives.

    8. Quickly draw the media into the syringe and then push the plunger slightly down and back up to remove air bubbles from the syringe.

    9. Fill the glass slide by injecting the media into the gasket slide assembly (Figure 2B). Note which media is injected into each chamber.

    10. Wait approximately 20-30 min for the agarose to solidify (Figure 2C).

    11. Remove the binder clips.

    12. Using a binder clip loop (or other convenient leverage), slowly and carefully pry the glass slides apart (Figure 2D). Try to keep the agarose from sticking to the blank slide.

      Note: This step takes some practice.

    13. Store the agarose slide in a clean plastic container (e.g., an empty tip box) for transport into the BSL-3 environment. Agarose slides can be stored 3-4 h at RT. Only use agarose slides prepared on the day of the ODELAM experiment.


  4. Preparation for ODELAM experiment

    1. Select the cultures and media conditions of interest.

    2. Prepare directories and data storage space. Create file directories and ensure there is sufficient storage capacity for the experiment.

    3. Create-Spot layout files. Double-check the file to ensure the strain layout is correct. Common mistakes include not correcting for flipping the array and not sorting the array correctly.


  5. ODELAM Spotting Cell Cultures

    1. Clean and sterilize all ODELAM chamber components (except the agarose slide and electrical contacts) with 70% ethanol and dry them with Kimwipes®.

    2. Use a clean plastic container to transfer the ODELAM chamber components to the Biological Safety Cabinet.

    3. Measure the OD600 of all Mtb cultures.

    4. Dilute cultures to an optimal OD600. The goal is to have 500-1,000 cells per ROI. The ratio of OD600 to CFU varies from strain to strain; therefore, the OD600 value may need to be adjusted slightly for different organisms and strains. An OD600 value of 0.03-0.05 is optimal for many mycobacterium strains.

    5. Array 150 μl of the diluted cultures into a 96-well plate. The locations in the 96-well plate should be determined by the spot layout pattern (see examples in Figures 5 and 6).

    6. Organize and layout the tips in tip-boxes for spotting (see Notes for suggestions).

    7. Place the base plate on the pipette alignment tool. Ensure the screws that level the alignment tool fit into the three recessed holes in the base plate (Figure 3A).

      Note: The following steps (E7-E10) should be performed in a Biological Safety Cabinet over an absorbent pad soaked in a tuberculocidal agent.



      Figure 3. Assembly of ODELAM chamber. A. The base plate has recessed holes for mounting the pipette alignment tool, as indicated by the red arrows. B. Place the agar slide in the recession. C-D. Place the clean silicon gasket on the bottom plate (C) and secure it with the appropriate button head and countersunk screws (D). E. Spot the cultures onto the agar pads using the correct tip sequence. F. Complete assembly of the chamber by adding the ITO slide, top plate, top gasket, and electrical contacts.


    8. Place the previously prepared agar slide in the recessed section of the ODELAM base plate (Figure 3B).

    9. Place the bottom silicon gasket on the ODELAM bottom plate. Smooth out the gasket and ensure the screw holes are correctly aligned and the gasket has a minimal overhang around the edges of the bottom plate (Figure 3C).

    10. Place the bottom plate onto the base plate and secure the two with the appropriate screws (Figure 3D).

    11. Flip the assembly over and place the pipette alignment tool onto the base plate. Ensure the screws protruding from each leg are set into the three corresponding holes in the base plate.

      Note: Be careful that the forward screw does not fall into the vent hole, as this sometimes happens and will cause the pipette tips to mark the agar.

    12. Check the agar surface to see if the pipette tips mark the agar (Figure 3E). If they do, adjust the height of the alignment tool using the screws on the legs until the tips are within 1 mm of the surface. They should be close but not mark the media.

    13. Use diagrams to guide spotting patterns for transferring from 96-well plate to ODELAM (see Notes)

    14. Spot 0.8-1.1 μl of culture. If needed, increase or decrease the volume of culture spotted to adjust its diameter slightly.

    15. Place the ITO glass slide into the recession in the base plate. Make sure the ITO conductive side of the slide is facing up.

      Note: Mark the conductive side of the ITO slide with an alcohol-resistant marker to identify the conductive side.

    16. Place the upper silicon gasket onto the upper plate (similar to Step E6, Figure 3C). Ensure the gasket does not overhang the center hole in the plate as this could interfere with the electrical contacts to the ITO slide.

    17. Secure the top plate to the base plate with screws.

    18. Screw in the vent plugs on the bottom plate.

    19. Disinfect the ODELAM chamber by wiping with a disinfectant-soaked towel but avoid touching or getting disinfectant on the ITO slide as this will cause condensation that will interfere with imaging.

    20. Attach the electrodes and silicon gasket electrode spacers with the plastic screws to the top plate and flip the chamber over (Figure 3F).

    21. Keeping track of plate orientation, place the assembled chamber onto the microscope stage.

    22. Connect electrodes to the power supply, turn on the heater power supply, and ensure that the current flows through the ITO slide. If the electrodes are in contact with aluminum, the power supply may indicate a short or have a high current. Look for a current from 0.15 A to 0.2 A at 10 V.

    23. Proceed to the next section on ODELAM microscope control.


  6. ODELAM microscope control

    1. Open the Visual Studio Code.

    2. Create a python terminal and activate the odelay virtual environment.

    3. Run the python file ODELAY microscopecontrol.py (e.g., python ODELAYmicroscopecontrol.py).

    4. Select the appropriate directories, files, and ODELAY experiment type for the experiment.

    5. Once the new graphical user interface has loaded, check to ensure the ROI layout on the microscope control panel matches that of the desired experiment (Figure 4).



      Figure 4. ODELAM microscope control interface and image display. The ODELAM microscope interface consists of a microscope control panel and an image display panel. An image of mycobacterium is displayed. The red circles in the control panel center indicate the ROIs that will be recorded, and the grey circles indicate the ROIs that will be ignored. Autofocus parameters for phase 1 and phase 2 are indicated and editable on the right. Camera exposure times are listed above the fluorescent cube selected.


    6. Start the camera by pressing the focus button.

    7. Move the stage to a spot in the upper left corner of the array, where cells are present.

    8. Focus the image and then move the upper left most ROI on the array (Spot E06 is the origin).

    9. Press the set origin button.

    10. Move to a ROI close to a leveling screw that has cells to focus on and use the leveling screw to focus the image.

    11. Repeat this step for all three leveling screws until the image is in focus at all three ROIs next to the leveling screws.

    12. Evaluate other ROIs and use the Autofocus 1 button to evaluate if those can be brought into focus by the default Autofocus parameters. Increase or decrease the Autofocus as needed to ensure all ROI can be found by the Autofocus settings (see Notes for additional Instructions).

    13. Press the ODELAY button to begin recording data.


  7. ODELAM Chamber Sterilization

    1. Fill two small plastic containers (e.g., tip box lids) and one the size of the ODELAM chamber with LoPhene. Prepare the biological safety cabinet with LoPhene-soaked absorbent pads as a working surface.

    2. After the experiment, remove the chamber from the microscope. Check for leaks. If necessary, apply disinfectant.

    3. Remove the copper contacts using the 3/32 hex driver and place the contact and screws in an appropriate storage container.

    4. Move the chamber to the biological safety cabinet.

    5. Use the 1/16-inch driver to remove the 10 flat head and button head screws from the bottom plate.

    6. After removing each screw, drop them into one of the two tip box lids with LoPhene.

    7. Gently but firmly, pry the bottom plate and gasket from the base plate. Do not use a tool for this as it could damage the gasket or either plate. Take your time as the silicon will stick to two plates together but will slowly release with firm but constant pressure.

    8. Gently pry the agarose slide from the gasket and place the slide in the second tip box filled with LoPhene. Make sure LoPhene completely covers the agar. Usually, some condensation forms on the edges of the slide at this point. Avoid touching it and clean the gloves thoroughly as they can become contaminated with Mtb at this point.

    9. Completely separate the bottom gasket from the bottom plate and place both in the plastic container filled with LoPhene. Make sure each part is covered.

    10. Repeat Steps G5-G7 for the top plate. Gently but with constant and firm pressure, pry the top plate and top gasket from the base plate.

    11. Remove the ITO coated slide and place it in the tip box lid with the screws. Ensure the ITO slide is immersed in LoPhene.

    12. Separate the top gasket from the top plate and the top plate, top gasket, and base plate into the plastic container with LoPhene. Add extra LoPhene if needed to cover all parts completely.

    13. After approximately 15-20 min, remove the agarose slide from the LoPhene. Place the agarose into a solid waste disposal within the hood and glass slide in the sharp container.

    14. Dump the LoPhene that the agarose slide was in into a liquid waste container.

    15. Spray LoPhene on the other containers and remove them from the biological safety cabinet.

    16. Wash all parts and screws with generous amounts of deionized water to remove the LoPhene. Finally, spray all parts with 70% ethanol and dry with a paper towel.

    17. Store the disassembled chamber for future use.

Data analysis

Note: This section assumes that all data analysis is performed with the ODELAYTools python package executed in the command line and utilizing High Performance Compute cluster as described in the ODELAY ReadMe.md file. Please follow the instructions there to install the software and package commands. Additional features to perform analysis on standalone desktops are in development.


  1. Activate the ODELAY python environment (e.g., >conda activate “name of odelay environment”).

  2. Create a data directory where the processed data of the experiment will be written.

  3. Set the image directory where the microscope images were written using the command “odelay set-image-dir”, and enter the path to the image directory at the command prompt.

  4. Set the data directory using the command “odelay set-data-dir”.

  5. Initialize the data processing with “odelay initialize”. Wait for the response that the experiment is initialized.

  6. Finally, enter the command prompt >odelay process all.

  7. Make sure that a *Spot-Layout.xlsx file is correctly filled out and in the data directory.

  8. After the data has finished processing, enter the command >odelay summarize-experiment. This command will reduce the dataset to a single hdf5 file.

  9. Plot summary histograms of all regions of interest using the command >odelay plot-summary Mtb (Figure 5A).

  10. Plot growth curves for histograms with the command >odelay plot-gc all Mtb. This will plot all regions of interest successfully processed in the experiment (Figure 5B).



    Figure 5. Example data from ODELAM experiments. Summary histograms for each region of interest in an ODELAM experiment. A. From left to right are histograms for the kinetic parameters of doubling time (Td), lag time (Tlag), time in exponential phase (Texp), and number of doublings (Num Dbl). The total population given on a Log10 scale is in the rightmost column. B. Plot of CFU growth curves observed in ROI E09. Histograms for doubling time, lag time, exponential time, and number of doublings are shown and can be inspected.

Notes

  1. Layout Patterns for 96-Well plates

    ODELAM spotting requires practice and organization to transfer samples from a 96-well plate to the 384-well pitch of the ODELAM plate. The following examples describe two strategies for arranging samples in a 96-well source plate. The first example shows how to spot a 96-well plate.

    1. Arrange the 96-well plate according to how many samples are required. Usually, multiples of four are best.

    2. Arrange the tips in the tip boxes according to the number of conditions required for the configuration of the agar pads (Figure 6A).

    3. Alternate from left to right every row when spotting to generate a checkerboard pattern on the agarose slide (Figure 6B).

    4. Repeat step 3 for the rows that were skipped in the previous step (Figure 6C).



      Figure 6. 96-well layout patterns for ODELAM experiments. A. Samples from a 96-well source plates (left) are transferred to the ODELAM agarose slide using the patterns shown in panels B and C to arrive at the final patterning on the top right. B. First step in patterning, selecting the samples shown on the left using the tip box patterns shown in the middle (grey) to produce the pattern on the agarose slide shown on the right. It is important to alternate every other row to prevent cultures from mixing while spotting. C. After the initial round of spots adsorb,spot the remaining wells as shown. Following these steps produces the interleaved pattern (top right) from the 96-well source plate (top left).


    5. Alternate patterns can be generated for spotting fewer colonies. A 12 or 24 sample pattern can be generated (Figure 7A) or for spotting on multiple conditions (Figure 7B).



      Figure 7. An example of 12 samples replicated eight times on a single condition. Each color represents a sample type. A. As described above, each row is sampled four times. B. Example with a five-condition four-sample layout. Four samples are arrayed across rows A, C, E, and G. Each is replicated four times per condition in the final layout. Pipette tips are marked using gray circles. Empty wells or tip positions are shown in white.

      Note: Columns 8,11,14, and 17 on the agarose slide are unused due to the presence of the gasket dividing the agar pads (refer to Figures 2 and 3 for gasket geometry).


  2. Autofocus Settings

    The autofocus settings are flexible to ensure that all spots will stay in focus throughout the experiment. There are two autofocus settings, Autofocus 1 and Autofocus 2.

    1. Autofocus 1 is utilized to find focus for the first timepoint of an ROI.

    2. Thereafter, Autofocus 2 uses settings that increase the speed of image acquisition.

    3. When setting Autofocus 1, ensure all ROIs are within ± range setting.

    4. With larger ranges, it is important to increase the number of images collected so that the focus algorithm will converge on a good focus. Ideally, a good print of the gasket should enable ROI focus to stay within 60 μm of each other across the spotted array.


  3. 3D Printing Gaskets

    Successfully printing 3D gaskets onto a microscope slide requires editing the G-code file that defines the printer movements. Most 3D printing software does not allow printing an object above the build plate that, in this case, is required to print on a 1 mm thick glass slide. TPU gasket 3D printing can be tricky and inconsistent. The printing speed, feed rates, extrusion temperature, and print cooling parameters that work will vary depending on the filament diameter and printer utilized. In general, a print head speed of 20 mm/s, removing filament retraction, an extruding temperature of 230°C, and a build plate temperature of 65°C are good starting points. We have found that drying the filament overnight at 70°C in an oven can dramatically increase print quality. This is due to water being absorbed into the polymer filament and then boiling out when the filament melts. The voids created from the water vaporizing make the extruded filament uneven, leading to flaws in the print. The following instructions give a general description of how to generate G-code files and edit the G-code so that the printer will print a gasket directly onto a glass slide. G-code files are text files with commands that tell the printer where and how to move the printing head. Correctly following these steps helps generate a gasket that allows molding of the agarose media for reliable time-lapse imaging.

    Printing Slide Build Plate mount:

    1. Generate a *stl file of a rectangular frame with an inner opening of approximately 50.75 mm × 75.75 mm and an outer measurement of 70 mm × 95 mm and thickness of 0.5 mm to 0.75 mm.

    2. Center the frame on the build plate and generate the G-code file using standard PLA filament parameters. Do not use a raft or any support material.

    3. Transfer the G-code file to the printer.

    4. Print the frame on a glass plate and use Kapton tape around the outer edges of the frame to hold it down afterward. This frame will center the glass slide for printing the gasket and can be used several times. It is recommended to have a dedicated glass plate for printing slide gaskets that can be reliably removed and relocated on the build plate.


    Generating G-Code for printing a gasket:
    1. Import the gasket design (usually a *.stl file) into the 3D printer software (e.g., Cura3D).

    2. Place the gasket on the center of the build plate.

    3. Enter the appropriate printing parameters for TPU and generate the G-code file.

    4. Open the ZchangerGcode.py file and change fileName variable to the G-code file generated in the previous step.

      Note: Line 19 of the ZchangerGcode.py';TYPE:WALL-OUTER' may need to be changed to a different value depending on the printer software generating the G-code. Generally, there are notes added to the G-code. It may take some inspection of the file and trial and error to get this section correct.

    5. Additionally, change the writeFileName to a name that is easy to recognize.

    6. Run the file using >python ZchangerGcode.py. This will create a file where the z-height of the gasket is offset by 1.1 mm from the print bed, allowing the gasket to be printed on a 1 mm thick slide.

    7. Transfer the file (named from writeFileName) the printer.

    8. Place a 50 × 75 × 1 mm glass slide into the rectangular frame previously printed.

    9. Tape the corners of the slide to the rectangular frame using Kapton tape.

    10. Print the slide gasket.

    11. Repeat steps 8-10 as necessary.

Recipes

  1. 7H9-GOT media (1,000 ml)

    1. Weigh 4.7 g of 7H9 powder into a 1,000 ml glass media bottle

    2. Add 900 ml of 18 MΩ H2O and dissolve the 7H9 powder

    3. Add 4 ml of sterile 50% glycerol

    4. Autoclave at 120°C for 30 min

    5. Allow the liquid to cool to 55°C.

    6. Add the following ingredients aseptically:

      2.5 ml of sterile 20% Tween 80

      100 ml of sterile OADC enrichment

  2. 10× 7H9-G media (41.6 ml)

    1. Weigh 1.88 g of 7H9 powder into 50 ml Falcon tube

    2. Add 40 ml of 18 MΩ H2O and dissolve the 7H9 powder by vortexing and slight heating

    3. Add 1.6 ml of 50% glycerol

    4. Filter sterilize using a 50 ml steriflip filter and conical tube

Acknowledgments

This project was funded by the National Institutes of Health (grant number, U19 AI106761 and NIH P41 GM109824).

Competing interests

The authors declare no competing interests.

References

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  7. Herricks, T., Dilworth, D. J., Mast, F. D., Li, S., Smith, J. J., Ratushny, A. V. and Aitchison, J. D. (2017a). One-Cell Doubling Evaluation by Living Arrays of Yeast, ODELAY! G3 (Bethesda) 7(1): 279-288.
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简介


[摘要]抗菌耐药结核分枝杆菌(结核杆菌)造成超过20万人死亡全球每一年。当前的抗药性分析需要了解赋予抗药性的突变或较长的培养时间以测试药物压力下的生长。我们提出ODELAM(一种分枝杆菌的活细胞阵列的单细胞加倍评价),一种基于延时显微镜的方法,观察单个细胞长成小菌落。该协议描述了样品和介质的制备,并包含有关组装ODELAM样品室的说明。所述ODELAM样品室被设计成提供受控环境以安全地观察的生长的Mtb在倒置宽延时显微镜-场显微镜。这里还提供了ODELAM软件的简要说明。ø DELAM跟踪多达1500个菌落形成的每单位区域的兴趣,并且可以观察到96个区域长达七天单次实验。这种技术允许的群体异质性的定量。ODELAM能够快速定量量度换货小号生长在作为动力学几个作为在各种各样的环境条件下30小时。


图形摘要:

ODELAM平台的示意图



[背景]结核分枝杆菌(Mtb )是结核病(TB)的病原体,每年导致全球140万人死亡(世界卫生组织,2020年)。尽管d的研究ecades,结核病治疗仍challeng ING ,和目前的化疗需要四个不同的药物(联合排等。,2016)。ø NE的为什么原因的Mtb仍然是一个非常成功的病原体是它能够产生表现出可变响应异构表型能力小号到给定的药物(Manina等人。,2015;达尔等人,2016;雷哥等人。,2017; Logsdon et al 。,2018)。因此,表征的个体的生长表型的Mtb细胞群小号是理解如何临界分枝杆菌感和响应应激条件如长时间暴露于药物(桑蒂等人。,2013;劳格斯登。等人,2018)。

迄今为止,关于细胞间差异的来源和后果知之甚少,这主要是由于可用于精确测量个体细菌细胞群生长表型的工具和技术的局限性所致。由于Mtb的加倍时间非常长(大约24小时),因此其研究尤其具有挑战性。最近平台到研究单个细菌细胞是由可观察或通过大量的时间单元的数量的限定需要(几周)来监视所述分枝杆菌集落的生长(阿尔德里奇等人,2012; Golchin等人,2012 ;若本等人。,2013;彩。等人,2014; Manina等人。,2015;巴尔等人。,2016)。

ODELAM(单细胞倍增分枝杆菌的生活阵列的评价)的开发是为了克服这些限制,并且基于ODELAY ,开发原本以分析酵母(Herricks等人。,2017年一,2017b和2020年)。ODELAY是已被利用来调查核孔复合体和过氧化物酶体生物的结构-功能关系的通用方法(金等人。,2018;肥大等人。,2018)。ODELAM使用时间推移显微镜定量个体的群体的生长表型的Mtb菌落-形成单位(CFU)。通过直接监测细菌的生长,我们可以评估Mtb菌株的异质性,它既可以自然产生,也可以响应压力(例如药物治疗)而产生。ODELAM可以观察高达1500 CFU每感兴趣区域(ROI),并测量四个主要生长动力学参数:滞后时间,倍增时间,指数时间(该时间时菌落停止生长并进入固定相),并确定倍增为每个数CFU。在一项实验中,ODELAM可以追踪多达100,000个CFU,这提供了检测种群中表型变异的统计能力。重要的是,ODELAM可以筛选每个实验中在多达5种培养基条件下生长的80至96个样品。在不到48小时,ODELAM识别抗性细胞群体中的敏感的细菌下降到1每1000和预测的药物以高准确度的最小抑制浓度(MIC)。此外,ODELAM平台可广泛地应用于作为实验室筛选工具,因为它可以很容易的广告一个PTED来研究最菌落-形成微生物。ODELAM可以量化的异质性和检测heteroresistance在结核分枝杆菌临床分离株。这些尖端技术将会对细菌的表型异质性研究产生有意义的影响和耐药性和帮助的出现,以更好地了解细菌适应不利的环境条件,这将有利于在新的强大的疗法的发展。

关键字:结核杆菌, 抗药性, 生长表型, 微生物学, 显微镜检查, Live imaging

材料和试剂

结核分枝杆菌培养
1.8 m l冷冻管(Nunc,目录号:375418)
50 m l锥形底管(Sarstedt ,目录号:62.547.254)
10米升血清移液管(VWR,目录号:89130-898)
200个μ升提示(杰纳西科学,目录号:24-412 )
1 ,000 μ升提示(杰纳西科学,目录号:24-430)
Steriflip -GP无菌离心管顶部过滤器单元(MilliporeSigma ,目录号:SCGP00525)
Mtb菌株(存储:-80°C)
H37Rv(可从BEI获得的背景菌株,目录号:NR-123)
其他感兴趣的菌株
Difco Middlebrook 7H9汤(BD,目录号:271310,储存:室温(RT))
Middlebrook OADC Enrichment(BD,目录号:212351,存储:4°C)
甘油(Sigma Aldrich,目录号:G5516,储存:RT)
Tween 80(Sigma-Aldrich,目录号:P1754,存储:RT避光)
消毒剂:
70%乙醇(Fisher Scientific,目录号:04-255-92,储存:RT)
PREempt RTU消毒剂解决方案(Contec ,目录号:21105,存储:RT)
LoPhene浓缩旋转消毒剂(Decon Labs,目录号:8801,存储:RT)


ODELAM琼脂糖凝胶制剂
50 m l锥形底管(Falcon,目录号:352070)
15 m l锥形底管(Falcon,目录号:352096)
5 ml注射器(BD,货号:309646)
18 G × 1½针(BD,目录号305196)
微型幻灯片2英寸× 3英寸1毫米厚(VWR,目录号:48382-180)
UltraPure琼脂糖(Invitrogen,目录号:16500,存储:RT)
Difco Middlebrook 7H9汤(BD,货号:271310,存储:RT)
Middlebrook OADC Enrichment(BD,目录号:212351,存储:4°C)
甘油(Sigma Aldrich,目录号:G5516,储存:RT)


ODELAM点样细胞培养
96孔板,F底(Greiner bio-one,目录号:655180)
移液器吸头RT LTS 20µL 960A / 10(雨淋,目录号:30389200)
7H9-GOT介质(请参阅食谱)
10 × 7H9-G介质(请参阅食谱)


设备


BSL1或BSL2
移液器
RAININ 2-20 μ升,20-200 μ升,100-1 ,000微升
0.1-10微升12多道移液器(吸取-精简版多移液器L12-10XLS + )(的Rainin ,目录号:17013807)
注:该ODELAM室是专为这一特定吸管。


2 ×热板搅拌器(Corning,目录号:PC-420D)
2 × 1 L烧杯(Sigma-Aldrich,目录号:CLS10001L-1EA)
2 ×搅拌棒(VWR,目录号:58948-1 50)
2 ×玻璃巨培养皿覆盖烧杯
Vortexer (科学仪器,型号:Vortex-Genie 2 ,目录号:SI-0256)
温度计(VWR,目录号:89095-640)
顶部装载电子秤,容量为1 kg,精度为0.01 g
中号(每5个玻璃垫片玻璃室3个)和大号(每1个玻璃垫片玻璃室2个)活页夹
三维打印机(例如,安德3D,BCN3D西格玛V19,makerbot 2 × )


BSL3
移液器:
的Eppendorf 2-20 μ升,20-200 μ升,100-1 ,000 μ升
0.1-10 μ升12多道移液器(吸取-精简版多移液器L12-10XLS + )(的Rainin ,目录号:17013 807)),与附接长尾夹
带加热培养箱的电动倒置显微镜(在Leica DMI6000和Nikon TiE上测试)
个人防护装备(PPE):
d ouble丁腈手套
P rotective特卫强工作服
小号锄头盖
P owered空气-净化呼吸器(PAPR)
吸水垫(用于在生物安全柜中工作)
ODELAY显微镜室(图1)
顶板(由6061-T6铝加工)




图1. ODELAM样品室。用于ODELAM实验的样品室以分解图(A )示出,并与所有相关部件一起分解(B )。


顶级PDMS垫圈(从McMaster-Carr Durometer 30A 9010K11切下)
氧化铟锡(ITO)涂层玻璃载玻片1.1毫米厚(SPI S提供,目录号:06404 -AF)
底板(由6061-T6铝加工)
玻璃与3D滑动印刷衬垫,并投琼脂糖介质
底部PDMS垫圈(从McMaster-Carr Durometer 30A 9010K11切下)
底板(由6061-T6铝加工)
移液器点对中工具(由6061-T6铝加工)
1/16六角螺丝刀(McMaster-Carr ,目录号:5497A23)
1/16六角螺丝刀(McMaster-Carr ,目录号:5497A23)
12 × 4-40 5/8英寸不锈钢纽扣式螺钉(McMaster-Carr ,目录号:98164A433)
8 × 4-40 1/4英寸不锈钢平头螺钉(McMaster-Carr ,目录号:90585A200)
4 × 4-40 1/8英寸尼龙内六角螺钉(McMaster-Carr ,目录号:95868A254)
铜电触点(从切割一铜薄片)
37 °C培养箱(VWR 1545数字培养箱)
培养旋转仪(GEL GRO组织培养旋转仪,LAB-LINE)


软件


Anaconda Pytho n(https://www.anaconda.com/)
Visual Studio代码(https://code.visualstudio.com/)
ODELAY软件包(https://github.com/AitchisonLab/)
MicroManager 1.4或2.0伽玛(https://micro-manager.org/)
Microsoft Office Excel中或在其他电子表格-兼容的程序(https://www.office.com/)
Cura 3D建模(https://ultimaker.com/)


PROC edure


结核分枝杆菌培养
注:与所有的工作结核杆菌应该进行一个3级生物安全(BSL3)实验室穿着受过培训的人员适当的PPE和可以由生物安全委员会(IBC)和环境健康与安全(EHS)的批准。


制备的接种10米预培养升7H9-GOT的(的Middlebrook 7H9介质补充有0.2%甘油,10%OADC补充剂,和0.05%(V / V)吐温80)用0.5M升的解冻的Mtb甘油原液(1:20稀释)。在37°C的50 ml锥形管中培养细胞48-72 h,直到培养物的OD 600为0.2-0.4(对数早期)。
通过稀释0.5米传代培养升10毫升预培养的升7H9 + GOT(1:20稀释)的。在37°C的50 ml锥形管中培养细胞48-72 h,直到培养物的OD 600为0.2-0.4(对数早期)。
稀释的培养物用于点滴的ODELAM板(小号程序EE部分E的进一步说明)。


ODELAM琼脂糖等分试样
收集的1升瓶媒体,高纯度的琼脂糖,移液管,和˚F爱尔康管。
称取2±0.02 g琼脂糖,放入1 L培养基瓶中。
添加150克18MΩH的2 O到媒体瓶。 
记录瓶,琼脂糖的总重量,和水。 
微波的媒体瓶,水和琼直到所有琼脂糖溶解。 
称该媒体瓶,水,和琼再次评估的水沸腾过程中损失的金额。
添加18MΩħ 2 O键,直到质量等于š先前记录的总重量。
等分试样的琼脂糖质量,15.1克成50米升猎鹰锥形管或3.0克到15微米升˚F爱尔康锥形管中。


ODELAM琼脂糖培养基的制备
注意:如果实验室相对无尘,则可以在生物安全柜外部执行本节中的步骤。


清洁的预先印刷的垫圈滑动(见使用实验室标签带注释部分的3D印刷垫圈)。
热用两个1升烧杯pproximately 750米升狄的ħ 2 O,一个用沸水和其他与水加温至40℃。
添加了10个的合适的量×媒体和需要制备的琼脂糖等分试样的另外的试剂(表1) 。
注意:不要不不要再增加对温度敏感的试剂。


表1 。量食谱的7H9-GO媒体


使用70%乙醇清洁50 mm × 75 mm × 1 mm的载玻片。干燥后,用镜纸擦拭以除去所有残留的颗粒和纤维。
将干净的玻璃载玻片放在印刷的垫片载玻片的顶部,并用活页夹固定载玻片(图2 A)。




图2 。琼脂载玻片的组装。一。确保垫圈直接夹在垫圈上; 否则,玻璃会弯曲,琼脂将不均匀。B.用注射器针头将琼脂糖培养基装满玻片。光盘。允许后的媒体集合,使用装订夹环到撬载玻片分开轻轻(d)。


组装注射器和20 -针对每个媒体条件表针。
放置管的在沸水中的培养基配方。所述20米升介质管应该在沸水中18分钟,并在4米升8分钟介质管。
注意:确保盖子不低于水面,因为这会导致水泄漏到管子中。此外,Ë ñ确保烧杯覆盖; 否则,将琼脂糖不会熔化完全。


后煮沸的时间合适的量和琼脂糖熔化,涡流的琼脂糖介质管和地点它在第E-40 ℃水浴中。
等待3-5分钟,让琼脂糖在40 °C达到平衡,然后添加对温度敏感的成分(例如,药物和OADC添加剂)。
彻底涡旋试管,以混合介质和添加剂。
快速绘制介质插入注射器中,然后推动柱塞稍微向下和备份以除去气泡从注射器。
通过将介质注入到密封垫玻片组件中来填充玻片(图2 B)。注W¯¯ HICH媒体被注入到每个腔室。
等待约20-30分钟,以使琼脂糖固化(图2 C)。
卸下活页夹。
使用活页夹夹环(或其他方便的杠杆作用),缓慢小心地将载玻片撬开(图2 D)。尝试防止琼脂糖粘在空白载玻片上。
注意:此步骤需要一些练习。


将琼脂糖载玻片存储在干净的塑料容器(例如,空的笔尖盒)中,以运输到BSL-3环境中。琼脂糖玻片可以在室温下保存3-4小时。Ø NLY ü本身琼脂糖幻灯片上ODELAM实验当天准备。


准备ODELAM实验
选择感兴趣的文化和媒体条件。
准备目录和数据存储空间。创建文件目录,并确保实验有足够的存储容量。
创建点布局文件。双重-检查的文件,以确保应变布局是正确的。常见的错误包括不正确地翻转数组和不正确地对数组进行排序。


ODELAM点样细胞培养
清洁和消毒所有ODELAM腔室部件(除了琼脂糖滑动和电触点)用70%乙醇和干燥它们与的Kimwipes ® 。
使用干净的塑料容器将ODELAM腔室组件转移到生物安全柜中。
测量的OD 600的所有结核杆菌培养秒。
将培养物稀释至最佳OD 600 。我们的目标是有500-1 ,每000个细胞的投资回报率。OD 600与CFU的比例因菌株而异; 因此,该OD 600值可能需要针对不同的生物体和菌株进行略微的调整。对于许多分枝杆菌菌株而言,OD 600值为0.03-0.05是最佳的。
阵列150 μ升稀释的培养物在96 -孔平板中。在96的位置-孔板应该由点布局图案(确定小号EE例子在图5和6 )。
组织和布局的叶顶盒技巧为察觉(小号EE须知建议)。
将基板放在移液器对齐工具上。恩确保平对准工具配合到在基板(图三个凹孔的螺钉3的A)。
注意:下面的步骤(Ë 7- Ë 10)应在生物安全柜中超过浸泡吸收垫来执行一个杀结核菌剂。




图3 。组装ODELAM腔室。A.基板具有凹入孔,用于安装在移液管对准工具,由所指示的红色箭头。B.将琼脂载玻片放入凹槽中。C- D 将干净的硅垫圈放在底板(C)上,并用适当的按钮头和沉头螺钉(D)固定。E.使用正确的吸头顺序将培养物点到琼脂垫上。F.通过添加ITO滑动,顶板,顶部垫圈腔室的完整的装配,以及电触点。


将之前准备好的琼脂玻片放在ODELAM基板的凹入部分中(图3 B)。
将底部硅垫片放在ODELAM底板上。弄平垫圈,并确保螺丝孔正确对准,并且垫圈在底板边缘周围的悬垂最小(图3 C)。
将底板放在底板上,并用适当的螺钉固定两者(图3 D)。
将组件翻转过来,然后将移液器对准工具放在底板上。恩确保从每个腿部突出的螺钉被设置成在基板上的三个相应的孔中。
注意:请注意,向前的螺钉不要掉入通风孔,因为这种情况有时会发生,并会导致移液器尖端在琼脂上留下痕迹。


检查琼脂表面,看移液器尖端是否标记了琼脂(图3 E)。如果是这样,请使用支腿上的螺钉调整对中工具的高度,直到尖端距离表面1毫米以内。它们应该靠近但不能标记媒体。 
利用图来引导斑点图案从转印96 -孔板到ODELAM(见注释)
现货0.8-1.1 μ升培养物。如果需要,可增加或减少点样的培养物的体积,以稍微调整其直径。
将ITO玻璃载玻片放入底板的凹槽中。确保滑块的ITO导电面朝上。
注意:用耐酒精的标记物标记ITO载玻片的导电面,以识别导电面。


将上硅垫圈到所述上板(类似于小号TEP È 6 ,图3 C)。确保垫圈不会悬在板上的中心孔上,因为这可能会干扰与ITO载玻片的电接触。
用螺钉将顶板固定到底板上。
拧紧底板上的排气塞。
通过用消毒剂擦拭消毒ODELAM室-浸泡过的毛巾但避免触及或获取消毒剂在ITO上滑动,因为这会导致冷凝,这将干扰成像。
用塑料螺钉将电极和硅垫片电极的垫片固定在顶板上,然后将反应室翻转过来(图3 F)。
跟踪板的方向,将组装好的腔室放置在显微镜载物台上。
连接电极到电源,接通加热器电源,并确保该电流流过ITO滑动。如果电极与铝接触,则电源可能表示短路或电流过大。寻找一个电流从0.15 A至0.2 A 10 V.
继续进行ODELAM显微镜控制的下一部分。


ODELAM显微镜控制
打开的Visual Studio代码。
创建一个python终端并激活odelay虚拟环境。
运行Python文件ODELAY microscopecontrol.py(É 。克。,蟒ODELAYmicroscopecontrol.py)。
选择适当的目录,文件,以及ODELAY实验的实验类型。
一旦新的图形用户界面已加载,检查连接确保ROI上所期望的实验中(图的显微镜控制面板布局的匹配4 )。




图4. ODELAM米icroscope Ç ONTROL接口和我法师d isplay。ODELAM显微镜界面由显微镜控制面板和图像显示面板组成。显示分枝杆菌的图像。红色圆圈在控制面板中心指示ROI小号将被记录,并且所述灰色圆圈表示的ROI小号将被忽略。第1阶段和第2阶段的自动对焦参数在右侧指示并可以编辑。相机的曝光时间在所选的荧光立方体上方列出。


按下对焦按钮启动相机。
移动载物台,以在所述阵列的左上角的点,其中细胞存在。
聚焦图像,然后将最左上角的ROI移动到阵列上(点E06为原点)。
按下原点设置按钮。
移至靠近要使单元聚焦的水平调节螺钉的ROI,然后使用水平调节螺钉聚焦图像。
重复此步骤,为所有三个脚螺丝,直到图像对焦在所有3个ROI小号旁边平螺丝。 
评估其他ROI小号,并使用自动对焦1按钮来评估,如果这些可以通过默认的自动对焦参数成为关注的焦点。增加或根据需要,以确保所有的投资回报率可以通过自动对焦设置(可以找到降低自动对焦小号的附加说明EE注解)。
按ODELAY按钮来开始记录数据。


ODELAM灭菌箱
填2个小的塑料容器(É 。克。,尖端盒盖)和一个所述与ODELAM室的大小LoPhene 。准备与生物安全柜LoPhene -浸泡吸水垫作为工作表面。
实验结束后,从显微镜中移出培养箱。检查是否泄漏。如有必要,请使用消毒剂。
使用3/32六角螺丝刀卸下铜触点,然后将触点和螺钉放在适当的存储容器中。
将腔室移至生物安全柜。 
我们ë 1/16 -英寸驱动器,以从底板卸下10个平头和按钮头螺钉。 
卸下每个螺丝后,用LoPhene将它们放入两个烙铁头盒盖之一。
温和而坚定地,撬从底板底板和垫片。请勿使用任何工具,因为这可能会损坏垫圈或任一板。慢慢来,硅会粘在两个板上,但会在牢固但恒定的压力下缓慢释放。
从垫圈上轻轻撬开琼脂糖玻片,然后将其放入装有LoPhene的第二个吸头盒中。确保LoPhene完全覆盖琼脂。通常,此时在滑块的边缘上会形成一些凝结。避免接触和清洁的彻底手套等,他们可以用被污染结核杆菌在这一点上。
Ç ompletely š从底板eparate底部垫圈,并放置在两个填充有塑料容器LoPhene 。确保每个部分都被覆盖。
重复小号TEPS ģ 5- ģ 7为顶板。轻轻但以恒定且牢固的压力,从底板撬起顶板和顶垫。
取下涂有ITO的载玻片,并用螺钉将其放在吸头盒盖中。恩确保ITO滑动浸入LoPhene 。
分离从顶板和顶板,上垫片顶部垫圈,和基底板与塑料容器LoPhene 。如果需要完全覆盖所有部件,请添加额外的LoPhene 。 
后约15-20分钟,除去从琼脂糖滑动LoPhene 。将琼脂糖放入通风橱中的固体废物处理中,并将玻璃片放入锋利的容器中。
将琼脂糖玻片所在的LoPhene倒入废液容器中。
喷雾LoPhene在其他容器,并删除它们从该生物安全柜。
用大量的去离子水清洗所有零件和螺丝,以除去LoPhene 。最后,用70%乙醇喷洒所有零件,并用纸巾擦干。
存放拆卸后的腔室,以备将来使用。 


数据分析


注意:本节假定所有数据分析都是通过在命令行中执行的ODELAYTools python包并按照ODELAY ReadMe.md文件中所述的高性能计算群集执行的。请按照那里的说明安装软件和软件包命令。正在开发在独立台式机上执行分析的其他功能。


激活ODELAY蟒环境(É 。克。,>康达激活“的名称ODELAY环境”)。
创建在处理数据的数据目录中的实验将被写入。
组,其中所述显微镜图像使用命令“写入的图像目录ODELAY设置图像- DIR ” ,并进入到在命令提示的图像目录的路径。
使用命令“ odelay set-data- dir ”设置数据目录。
使用“ odelay初始化”初始化数据处理。等待实验已初始化的响应。
最后,进入命令提示符> odelay process all。
确保正确填写* Spot-Layout.xlsx文件,并将其放在数据目录中。
数据处理完毕后,输入命令> odelay summary -experiment。此命令会将数据集缩减为单个hdf5文件。
使用命令> odelay plot-summary Mtb绘制所有关注区域的摘要直方图(图5 A)。
用于与所述命令>直方图情节生长曲线ODELAY plot- GC所有的Mtb 。这将绘制出在实验中成功处理的所有感兴趣区域(图5 B)。




图5 。来自ODELAM实验的示例数据。ODELAM实验中每个感兴趣区域的汇总直方图。答:从左到右是直方图,表示倍增时间(Td),滞后时间(Tlag ),指数相时间(Texp )和倍增次数(Num Dbl )的动力学参数。Log10量表给出的总人口在最右边的列中。B. P很多的CFU生长曲线在ROI E09观察。显示并可以检查倍增时间,滞后时间,指数时间和倍增次数的直方图。 



笔记


布局模式为96 -井板
ODELAM斑点需要从96实践和组织转移样品-孔板到384 -的ODELAM板的孔间距。以下实施例描述2个为在96布置样品策略-井源板。第一个例子示出了如何发现96 -孔平板中。


安排96 -根据多少样本需要孔板中。通常,最好是四的倍数。
根据琼脂垫配置所需的条件数,将尖端排列在尖端盒中(图6 A)。
当在琼脂糖载玻片上点按以在棋盘图案上产生时,从左到右每行交替(图6 B)。
对上一步中跳过的行重复步骤3(图6 C)。




图6 。用于ODELAM实验的96孔布局图。A.使用B板和C板中所示的图案,将来自96孔源板(左)的样品转移到ODELAM琼脂糖玻片上,以得到右上角的最终图案。B.图案化的第一步,使用中间(灰色)所示的笔尖盒图案选择左侧所示的样品,以在右侧所示的琼脂糖玻片上产生图案。我t是重要交替每ö疗法行吨ö防止培养物而斑点混合。C.初始圆斑后吸附,发现剩余的孔中作为示出。在这些步骤之后产生交织模式(右上)从所述96孔源板(左上)。


可以生成替代模式以发现较少的菌落。A 12或24的样本图形可以产生(图7 A)或用于点滴多个条件(图7的B)。




FIGUR ê 7 。一个12个样本在一个条件下重复八次的示例。每种颜色代表一个样本类型。A.如描述以上,每行被采样的4倍。B. Ë xample与五-状态4 -样品布局。四个样品跨越行A,C,E排列,和G.每一个被复制的4每种条件倍的最终布局。移液器吸头用灰色圆圈标记。空孔或尖端位置以白色显示。


注:Ç olumns 8 ,11,14 ,和17上的琼脂糖滑动是与垫圈将所述琼脂垫的存在未使用的,由于(参照˚F IG URE小号2和3为垫圈几何形状)。


自动对焦设置
自动对焦设置非常灵活,可以确保所有斑点在整个实验过程中始终保持焦点对准。有两种自动对焦设置,自动对焦1和自动对焦2。


自动对焦1用于在ROI的第一个时间点找到焦点。
此后,自动对焦2使用可提高图像获取速度的设置。
当设置自动对焦1,EN确保所有ROI小号是±范围设置内。
对于较大的范围,重要的是增加收集的图像数量,以便聚焦算法将聚焦在良好的聚焦上。理想地,所述垫圈的良好的打印应使ROI焦点留60内微米横跨斑点阵列彼此的。


3D打印垫片
要在显微镜载玻片上成功打印3D垫圈,需要编辑定义打印机移动的G代码文件。大多数3D打印软件都不允许在模板上打印对象,在这种情况下,必须在1毫米厚的载玻片上进行打印。TPU垫圈3D打印可能很棘手且不一致。可用的打印速度,进给速度,挤出温度和打印冷却参数将根据细丝直径和所用打印机的不同而有所不同。通常,打印头速度为20 mm / s,消除细丝缩回,230°C的挤出温度和65°C的模板温度是不错的起点。我们发现,在烤箱中将细丝在70°C下干燥过夜可以显着提高打印质量。这是由于水被吸收到聚合物长丝,然后沸腾出当灯丝熔体小号。水汽化产生的空隙使挤出的长丝不均匀,从而导致印刷缺陷。下面说明得到一般性描述的如何生成的G代码文件和编辑G代码,使得打印机将直接打印的垫圈在载玻片上。G代码文件是带有命令的文本文件,这些命令告诉打印机将打印头移动到何处以及如何移动。正确地执行以下步骤有助于产生,其允许可靠的时间模制的琼脂糖介质的垫片-移成像。


打印幻灯片构建板安装座:


生成* STL的矩形框的文件用的内开口大致50.75毫米× 75.75毫米和70毫米的外测量× 95毫米和厚岬的0.5毫米至0.75毫米。
将框架居中放在模板上,并使用标准的PLA灯丝参数生成G代码文件。请勿使用木筏或任何支撑材料。
将G代码文件传输到打印机。
打印在玻璃板上的帧,并使用卡普顿胶带围绕框架的外边缘以将其保持下来之后。该框架将玻璃载玻片居中以打印垫片,并且可以使用多次。建议使用专用的玻璃板来印刷滑动垫片,可以将其可靠地卸下并重新放置在构建板上。


摹enerat荷兰国际集团G代码用于打印垫片:


导入的垫片设计(通常是*。STL文件)到3D打印机软件(é 。克,Cura3D) 。
将垫圈放在构建板的中央。
输入TPU的适当打印参数并生成G代码文件。
打开ZchangerGcode.py文件,并将fileName变量更改为上一步中生成的G代码文件。
注:大号的INE 19的ZchangerGcode。py ' ; TYPE:WALL -OUTER'可能需要更改为其他值,具体取决于生成G代码的打印机软件。通常,在G代码中添加了注释。为了使本节正确无误,可能需要检查文件和反复试验。


此外,将writeFileName更改为易于识别的名称。
使用> python ZchangerGcode.py运行文件。这将创建其中所述垫圈的所述z高度由1.1偏移的文件从打印床毫米,允许荷兰国际集团的垫圈上的1mm厚的幻灯片将被打印。
将文件(从writeFileName命名)传输到打印机。
将50 × 75 × 1毫米的载玻片放入先前打印的矩形框架中。 
使用Kapton胶带将幻灯片的边角粘贴到矩形框架上。 
打印滑动垫片。 
根据需要重复步骤8-10。 


菜谱


7H9-GOT介质(1 ,000毫升)
一种。称取4.7克的7H9粉末成一个1 ,000米升玻璃介质瓶     

b。添加900米升的18MΩħ 2 O和溶解7H9粉末     

C。添加4米升无菌的50%甘油的     

d。在120°C高压灭菌30分钟     

e。允许所述液体冷却到55℃ 。     

F。添加了以下成分在无菌条件下:       

2.5米升的无菌20%的吐温80


百米升无菌OADC富集的


10 × 7H9-G介质(41.6 m l )
称取1.88克的7H9粉末成50米升Falcon管
添加40米升的18MΩħ 2 O和溶解7H9粉末通过涡旋和轻微加热
添加1.6米升50%的甘油
使用过滤消毒一个50米升steriflip滤波器和锥形管


致谢


该项目被资助通过了美国国立卫生研究院(授权号,U19 AI106761和NIH P41 GM109824 )。


利益争夺


在一个uthors声明没有竞争利益。


参考


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Copyright Herricks et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Herricks, T., Donczew, M., Sherman, D. R. and Aitchison, J. D. (2021). ODELAM: Rapid Sequence-independent Detection of Drug Resistance in Mycobacterium tuberculosis Isolates. Bio-protocol 11(10): e4027. DOI: 10.21769/BioProtoc.4027.
  2. Herricks, T., Donczew, M., Mast, F. D., Rustad, T., Morrison, R., Sterling, T. R., Sherman, D. R. and Aitchison, J. D. (2020). ODELAM, rapid sequence-independent detection of drug resistance in isolates of Mycobacterium tuberculosis. Elife 9: e56613.
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