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Jul 2021
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An Inexpensive Imaging Platform to Record and Quantitate Bacterial Swarming
记录和定量细菌群集的廉价成像平台   

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Abstract

Bacterial swarming refers to a rapid spread, with coordinated motion, of flagellated bacteria on a semi-solid surface (Harshey, 2003). There has been extensive study on this particular mode of motility because of its interesting biological and physical relevance, e.g., enhanced antibiotic resistance (Kearns, 2010) and turbulent collective motion (Steager et al., 2008). Commercial equipment for the live recording of swarm expansion can easily cost tens of thousands of dollars (Morales-Soto et al., 2015); yet, often the conditions are not accurately controlled, resulting in poor robustness and a lack of reproducibility. Here, we describe a reliable design and operations protocol to perform reproducible bacterial swarming assays using time-lapse photography. This protocol consists of three main steps: 1) building a “homemade,” environment-controlled photographing incubator; 2) performing a bacterial swarming assay; and 3) calculating the swarming rate from serial photos taken over time. An efficient way of calculating the bacterial swarming rate is crucial in performing swarming phenotype-related studies, e.g., screening swarming-deficient isogenic mutant strains. The incubator is economical, easy to operate, and has a wide range of applications. In fact, this system can be applied to many slowly evolving processes, such as biofilm formation and fungal growth, which need to be monitored by camera under a controlled temperature and ambient humidity.

Keywords: Bacterial swarming (细菌群集), Bacterial motility (细菌运动性), Colony growth (菌落生长), Incubation (孵化), Time-lapse imaging (延时成像)

Background

Bacteria show different motility phenotypes, such as swimming, swarming, gliding, sliding, and twitching (Kearns, 2010). Coordinated multicellular migration across a moist surface, known as bacterial swarming, is an important motility phenotype that has been studied for decades due to its relevance to pathogenicity, virulence, and antibiotic resistance (Kearns, 2010). A systematic study of colony expansion speed and morphological patterns coupled to swarming may provide insight into the correlation between bacterial motility and host health. Here, we build an environment-controlled incubator to perform a swarming assay. Inside the incubator, a digital camera is mounted on the top to take time-lapse images of the swarming activity. After the recording, images are transferred to a laptop for quantitation of the swarm expansion using the ImageJ software. The system was tested by recording over 1,000 swarming events to assure stability and reproducibility.


At first glance, one may consider a swarming assay rather simple: just inoculate bacteria on agar filled in a Petri dish, and then take an image of the plate after incubating for a certain time. However, it can be challenging to perform swarming assays with reproducible results and record clear images for further quantitative analysis. Here, we list a few technical challenges that one may encounter and provide corresponding solutions in our protocol:

  1. A typical swarming event may take 10-20 h for the bacteria to cover a standard 9-cm Petri dish. Besides the coverage time, sometimes we need to know how long the lag phase lasts, at what time branches form at the colony edge; and at the microscopic level, when cell elongation occurs. Thus, the researcher needs to check the plate regularly, aided by microscopy. Suppose one starts the assay during the daytime; one may need to scan the plates every 10-30 min overnight. In our protocol, time-lapse photography helps to capture images so that the researcher does not have to stay up late or check every 10-30 min in order not to miss key events.

  2. Bacterial swarming is highly sensitive to the environment. Fluctuations in ambient temperature and humidity often cause large differences in the swarming rate and colony pattern; therefore, a stable humidity- and temperature-controlled environment is critical for the assay. In our system, we utilize a thermo-insulating tent, a humidity control unit, and a temperature control unit to minimize the environmental fluctuation during the assay. One can readily set different humidities and temperatures for a swarming strain screen.

  3. Since an agar gel typically contains over 97% water by mass, condensation readily forms on the plate lid, which obscures image taking. We designed the incubator in such a way that, when the plates are placed inverted on the platform, the temperature of the lid is slightly higher than that of the agar, which prevents condensation. Note: the agar surface architecture can vary depending on pouring methods, thickness, and percentage of agar used; these need to be individually optimized for the particular bacteria.

  4. Taking a clear photo of the swarming plates is tricky. A swarming plate has three surfaces that affect the optical quality while imaging: the plate lid, the agar surface, and the plate bottom. When the camera flashlight or auxiliary front light is used, some light is reflected by the lid and the bottom of the plate to the camera, forming undesirable light spots on the photos. In our design, for one-plate photo shooting, we use a circular fluorescent tube as the backlight under an adjustable light shield. For multiple-plate (up to 9) assays, an LED light strip is used for sidelight illumination. Image quality is better when using the light shield because of the light field setting; however, the efficiency is higher when using a LED light since it illuminates a larger area that can fit 9 Petri dishes at a time.

  5. Quantitation of the swarming rate takes a lot of effort; researchers generally scan the plates and measure the radius of the swarming colony using a ruler. For an irregular-shaped colony, they usually make a rough estimation of the “effective radius.” When multiple plates are involved in the assay, one should plot the swarming area vs. time instead of just calculating the average swarming rate. A lot of work is required for numerous manual measurements. In our case, we import the digital swarming images to the ImageJ software and calculate the swarming area by outlining the swarm periphery. This computerized process improves the efficiency and reduces the error.

    Compared with conventional practice in this field, our protocol has distinct advantages:

    (1) Affordability. In 2015, a research team headed by Dr. Joshua Shrout at the University of Notre Dame described a protocol for the preparation, imaging, and quantitation of swarming (Morales-Soto et al., 2015). In their protocol, they used the commercial equipment “Bruker in vivo imaging station.” This product is no longer available from the company, and a used one costs over $58,000. Most microbiology labs studying bacterial swarming may not need many of the complex functions that this particular equipment provides, such as x-ray imaging and in vivo fluorescence imaging. Those labs may not be able to afford or be willing to invest so much in an overqualified bacterial swarming chamber. In contrast, the total cost of our system is around $1,000, with everything included.

    (2) Accuracy. For the Bruker imaging station, placing a plate of water next to each swarming plate is suggested in order to maintain humidity. In this way, however, it is hard to control the humidity within a specific range. In our design, the chamber humidity is well controlled; one can set the parameters before the assay starts, and the environment is controlled dynamically using a digital sensor and feedback controllers.

    (3) Efficiency. In 2018, an independent researcher, Peñil Cobo, developed a time-lapse imaging chamber for bacterial colony morphology observation (Peñil Cobo et al., 2018), which allows for photography of one Petri dish at a time. Since the temperature is not controlled in that design, the whole system must be placed in a warm room. In our case, multiple 9-cm Petri dishes can fit inside the incubator, providing convenience and efficient use of space and energy. Having a larger box in our design, necessary to hold multiple plates, alters the optical path in a subtle way, but the geometry of our incubator and light field for photography are well calibrated to ensure the best lighting.


  This protocol is divided into three steps: (1) assembly of an incubator; (2) the swarming assay; and (3) data processing. By following the procedure as detailed below, we guarantee that one can obtain a homemade bacterial plate incubator with stable swarming results and high-quality images. The overall cost depends on which camera is used in the system, but it should be within $1,000 dollars. In practice, any digital camera that has a “Manual Mode” is adequate for the purpose. A swarming assay requires researchers to be meticulous with certain details to ensure reproducibility. For instance, bacterial swarming is very sensitive to small changes in conditions such as the roughness and moisture level of the agar surface. Here, we attempt to elaborate on procedures and notes in as much detail as possible for users to follow. In the photo shooting part, we show in detail how to tune the camera settings. Finally, we explain how to process the data using ImageJ to measure the swarming area.

Materials and Reagents

  1. To build the photographing incubator

    1. Hydroponic Indoor Garden Grow Tent (24 in. × 24 in. × 48 in., Yaheetech, model: YT-2801)

    2. Photography unit

      1. Digital camera (Panasonic, model: DMC-FZ50)

      2. LCD Timer Remote Control (JJC, model: TMD)

      3. AAA battery (2 pcs, Duracell)

      4. Zinc-plated slotted angle (4 pcs, 1.5 in. × 14 Gauge × 36 in., Crown Bolt)

      5. Zinc-plated slotted angle (10 pcs, 1.5 in. × 14 Gauge × 18 in., Crown Bolt)

      6. Aluminum flat bar (0.75 in. × 36 in. × 0.125 in., Everbilt)

      7. Black polyester cloth (20 in. × 20 in., Dazian)

      8. Bolts and nuts (40 pairs, M5, Crown Bolt)

      9. Black acrylic sheet (2 pcs, 18 in. × 18 in. × 0.125 in., National Security Mirror)

      10. LED light strip (3 meters, White, GuoTonG)

      11. Power strip (6-outlet, Belkin)

    3. Backlight shield

      1. Black acrylic sheet (3 pcs, 12 in. × 12 in. × 0.118 in., National Security Mirror)

      2. Fluorescent circline ceiling light (Sunlite, model: FC12T9/CW) with starter ballast

      3. Zinc-threaded rod (4 pcs, 0.25 in. × 12 in., Everbilt)

      4. Hex-plated nuts (24 pcs, 0.25 in., Everbilt)

    4. Temperature and humidity control unit

      1. Heated control module (Coy Lab, serial: DC1807)

      2. Fan (AC Infinity, model: LS1225A-X)

      3. Digital humidity controller outlet (Inkbird, model: IHC200S)

      4. Reptile humidifier (2 L, Evergreen)

      5. Beaker (500 ml)

        Note: Other brands of humidifiers or controllers can be alternatives if their combination can well control the humidity inside the chamber.


  2. For swarming plate preparation

    1. Enterobacter sp. SM1, SM3, SM3_18, SM3_24 (species of bacteria for the swarming assay)

    2. LB broth (see Recipes)

    3. 0.5% LB agar plates (see Recipes)

Equipment

  1. Labconco Purifier Class II Biosafety Cabinet (Delta Series)

  2. Falcon 14-ml Polystyrene Round-Bottom Tubes (17 mm × 100 mm, Corning)

  3. Sterilized wooden sticks (2.5 in.)

  4. Pipet-aid (Drummond, 1-100 ml) with appropriate pipette

  5. New Brunswick Innova 4300 Incubator Shaker

  6. Weighing paper (4 in. × 4 in., Fisher Scientific, catalog number: 09-898-12B)

  7. Laboratory spatula (4 pcs, 6.5 in, stainless steel, Home Science Tools)

  8. Pyrex glass bottles (250 ml, Corning, model: 1395)

  9. Pyrex graduated cylinder (500 ml, Corning, model: 3022)

  10. Hot plate magnetic stirrer (Barnstead International, model: SP46925)

  11. Thermal insulating gloves

  12. Autoclave (AMSCO Scientific, model: SV-120) with autoclavable tray

  13. Petri dishes (100 mm × 15 mm, sterile, polystyrene, Fisher Scientific, catalog number: FB0875712)

  14. Micropipette (0.5-10 μl, Eppendorf, catalog number: 3123000020)

  15. Acrylic cutter (Fletcher)

  16. Circle cutter (Bott)

  17. Hand drill/table drill with appropriate drill bit set

  18. Hacksaw

Software

  1. ImageJ (ver1.59g) (https://imagej.nih.gov/ij/)

Procedure

Part I: Set up the photographing incubator


  1. Assemble the camera stand

    1. Set up the Yaheetech hydroponic tent according to the instruction manual. The manual comes with the product package. Assemble the skeleton first and then cover it with the polyester material.

    2. Use M5 bolts and nuts to assemble the camera frame, as illustrated in Figure 1. Connect the Zinc-plated slotted angles first and then the aluminum bars. The slotted angles have holes by the side, but the aluminum bars do not. Drill holes by the ends of the aluminum bars using a hand or table drill. Since the aluminum bars are used to stabilize the structure, their positions do not have to be precise.



      Figure 1. Schematic showing the structure of the camera frame. Four pieces of 36-in. zinc-plated slotted angle stand perpendicular to the ground and are connected by 10 pieces of 18-in. zinc-plated slotted angle using M5 bolts and nuts. The aluminum bars are to stabilize the whole structure with no specific position requirements. When using the light shield, the distance between the ground and the sample platform is 18 in. When using LED light illumination, the sample platform can be elevated by several inches for photography.


    3. Use an acrylic cutter to cut the sides of 18 in. × 18 in. acrylic sheets slightly to fit in the sample platform.

    4. Use a circular cutter to cut a circle of 9 inches in diameter out of one of the 18 in. × 18 in. acrylic sheets “A” from the center; leave the other sheet “B” uncut.

    5. Fix sheet “A” on the sample platform. Drill the appropriate holes on the side to allow the bolts to go through all the way down to the holes of the slotted angles.

    6. Fix the camera on the camera fixer, with its lens facing downward. Adjust the fixer back and forth to align the camera with the circle on the acrylic sheet.

    7. Tape the LED strip around the sample platform 1 inch above the acrylic sheet on the slotted angle. The position of the light cannot be too high because we want to avoid reflections of the light from the plates.

    8. Cut two pieces of black cloth, around 20 in. × 20 in. Place one of them on the bottom of the tent as a black photography background. For the other piece, cut a hole in the center to allow the camera lens through, and hang the cloth onto the camera platform to shield the reflection from the top.

    9. Place the camera stand inside the tent.

    10. Load the batteries into the LCD timer remote control and connect it to the camera through the hole on the top of the tent. Tighten the hole using the elastic cords around it.


  2. Make the light shield

    1. Cut circles of 3.62 in., 5.75 in., and 9.50 in. diameter out of three pieces of acrylic sheets. Drill 0.25-in. holes in each of the corners, 1.2 in. from both edges. Assemble the light shield according to Figure 2. Place the circular fluorescent light bulb between level I and the sample platform. Stick the starter ballast under the sample platform using tape.

      Pause point: There are two modes of illumination: one uses a backlight, and the other uses a side light (Figure 3). When taking close-up images of a one-plate event, it is preferable to use the light shield with its own light source below a light sheet. To take photos, place the light shield on the sample platform and align it with the camera. Turn off the LED light when using the light shield. On the other hand, when taking photos of more than one plate, the light shield is removed, and the uncut 18 in. × 18 in. acrylic sheet is placed on top of the circularly cut sample platform. As many as 9 agar plates can be placed on the uncut sheet for imaging. To take photos, turn on the LED light as the side light source.



      Figure 2. Image showing a light shield assembly for single-plate imaging. Three pieces of 12 in. × 12 in. a hollow acrylic sheet are connected by four pieces of 12 in. zinc-threaded rod and fixed using hex-plated nuts. The distance between the Level I sheet and the ground is about 1.5 in., leaving just enough space to place the circular fluorescent bulb to fit in. The heights of Level II and Level III can be adjusted to optimize illumination.



      Figure 3. Taking photos using the light shield or LED light strip. (A) Taking photos using the light shield assembly. The circular fluorescent bulb is placed between Level I and the sample platform (noted in Figure 2). The distance between Level II and Level I is about 0.8 in., while the distance between Level II and Level III is 3.5 in. (B) Taking photos using the LED light strip. The LED light is fixed 1 inch above the sample platform on the horizontal zinc-plated slotted angles. If the light is too strong, one can insert a white paper belt to diffuse and reflect the light. The black cloth near the camera is necessary to block reflections from the plates.


  3. Install the temperature and humidity control system

    1. Place the heat control module inside the tent, on the side at the bottom, such that it will not show up in the swarming photos when using the light shield. Adjust the temperature setting to the desired temperature for the swarming assay.

    2. Set the humidifier outside the tent and connect the power cord to the humidity controller outlet. Extend the extractable plastic mist tube through the hole on the tent wall into the tent beneath the sample platform.

      Note: Check the camera preview and make sure that the tube and the mist do not show up in the image.

    3. Fill the humidifier tank with water. Plug the humidity controller into the power strip, and adjust the humidity value with a tolerance range according to the controller manual. As an example, for Enterobacter sp. SM3 (see the corresponding main article), set the humidity to 40% ± 5% tolerance (tol).

    4. Place a 500-ml beaker under the mist tube to collect water droplets.

    5. Fix the AC fan on one of the slotted angle legs facing the beaker using the bolts and nuts that come with the fan. The fan is used not only to blow the mist from the humidifier to prevent fog from showing up in the photos but also to improve ventilation and uniformity of the temperature and humidity in the chamber.

    6. Tighten all the holes and seal the zip of the tent.


Part II: Perform the swarming assay

  1. Take the bacteria glycerol stock out of the -80°C freezer. Use a piece of sterilized wooden stick to scratch the bacteria-containing ice surface and then dip the stick into the LB broth. Place the glycerol stock back into the freezer and shake the inoculated LB broth overnight (~16 h) in a shaker. The temperature is set to 37°C and the shaking frequency to 200 revolutions per min (rpm). Start the overnight growth around 5 p.m. so that it will be ready for use around 9 a.m. the next morning.

  2. Use a micropipette to inoculate 2 μl overnight bacterial suspension on the center of each agar plate. Transfer the plates to the incubator after the inoculation drop has been absorbed by the agar (the spread of the circular drop has become nearly invisible).


Part III: Time-lapse photo taking and swarming rate quantitation

  1. To use the light shield, place one swarming plate inverted on the plate holder. Turn on the fluorescent light bulb and the camera. The LED light strip should stay off.

  2. In the preview of the camera, you should see the plate sitting in the center of the screen. Otherwise, move the light shield around to align the camera with the sample.

  3. Rotate the nuts on the threaded rod to adjust the position of the acrylic sheets. The distance between the sample platform and Level I is about 1.5 inches. The distance between Level I and Level II is about 0.8 inches, while Level II and Level III are separated by 3.5 inches. If you see a round light spot on the Petri dish, lift Level III slightly. If the Petri dish is too dark, lower Level III slightly. If you cannot get a good image by adjusting Level III, then adjust Level II slightly up or down (see Note 3).

  4. Set the camera focal length to 35-65 mm. Adjust the zoom ring so that the sample occupies the full screen but does not exceed the border. Use “M,” the manual mode, to focus on the bacterial colonies. Set the aperture to F5.6-F7.1 and adjust the shutter speed until the resulting exposure value is 0 or -, because overexposed images will result in loss of detail in the images (Figure 4A).

  5. For a multi-plate assay, remove the light shield, turn on the LED light strip, and place the uncut acrylic sheet on the sample platform. Place the swarming plates inverted on the acrylic sheet so that water will not form condensate on the lid. Check the camera preview to make sure that all the plates are within the range of the screen (Figure 4B).

  6. Set the frame rate and frame number of the LCD camera timer control according to the manual. For example, in the case of Enterobacter sp. SM3, we set the frame number to 50 and the time interval between frames to 15 min. Press the “start” button to start the time-lapse photo shooting.

  7. Zip the tent to form a good seal. The swarming assay may take about 10 h. You can leave the camera on overnight and collect the images the next morning. During the photo taking process, DO NOT shake the incubator; otherwise, the optical setup may be disturbed.

  8. Stop the timer controller when the swarming is finished. Download the images by connecting the camera to a laptop using a USB data wire or by pulling out the memory card of the camera and placing it into a computer to download the images.

  9. Check the images taken on your computer. If the brightness of the images varies, you can use the “Stack Deflicker” plugin in ImageJ to calibrate the brightness. If the sample position in the images varies over time, you can apply the “Image Stabilizer” plugin to fix it.

Data analysis

Representative one-plate and multi-plate swarming images are shown in Figure 4A and 4B, respectively. The time course for plates shown in Figure 4B was rendered to a video in Video 1.



Figure 4. Swarming images taken in the incubator. (A) Plates inoculated with Enterobacter sp. SM1 (left) and SM3 (right) were incubated for 6.5 h. This image was taken using the circular fluorescent bulb and the light shield assembly. (B) Swarms of SM3 vs. a swarm-deficient mutant of SM3, SM3_18 (plates 1, 2, 4), and SM3 vs. a swarm-deficient mutant of SM3, SM3_24 (plates 3, 5, 6). Each comparison was performed in triplicate, with SM3 inoculated on the right half of each plate. The image was taken after a 6.5-h incubation. A side LED light strip was used for illumination, with all six plates imaged together.


Video 1. Time-lapse video of Enterobacter sp. SM3 swarming assay taken using LED light strip illumination. SM3 and its mutant (SM3_18 or SM3_24, each in triplicate) overnight culture were inoculated on 0.5% LB agar plates and incubated in the photo chamber. Time-lapse images were taken for 10 h, and the images were rendered into a .avi video file using ImageJ (ver. 1.59 g) at 11 frames per second (fps).

To calculate the swarming area, open the image of interest in ImageJ. Click “Analyze” -- “Set Scale” to calibrate the ratio of pixels to the actual length. Then use the “freehand selections” tool to outline the colony edge and press “M” to calculate the area of the selected colony image by image (Figure 5). The swarming rates for Enterobacter sp. SM1 and SM3 are plotted in Figure 6 following calculation of the swarm areas.



Figure 5. Calculation of the swarm area using ImageJ. (A) ImageJ user interface. The “freehand selections” tool is selected. (B) Enterobacter sp. SM3 swarming colony (on the right) is outlined by the “freehand selections” tool. (C) The scale in pixels is set to the value of the real length. (D) By pressing “M,” the area of the swarm is calculated.



Figure 6. Quantitation of Enterobacter sp. SM1 and SM3 swarming motility. SM1 and SM3 were inoculated using 2 μl overnight bacterial culture on 0.5% LB agar plates and incubated in the swarming incubator (repeated a total of 10 times). Time-lapse images were taken every half an hour, and the swarm area was measured for each image. Data are represented as the mean, with error bars indicating the 95% confidence interval. When not visible, the errors are smaller than the size of the symbols.

Notes

  1. The humidity and temperature inside the incubator should reach the set values within 10 min after the power is turned on and remain stable for the duration of the overnight recording.

  2. Sometimes, certain models or specifications of products may be out of stock. If 18-in. slotted angles are not readily available, for instance, one can cut from longer angles using a hacksaw. Cutting a large circular hole out of a piece of acrylic requires some skill. To ensure safety, when using a hole saw or circle cutter, always clamp the acrylic sheet firmly on a workbench first. One alternative is to go to an engineering workshop or a carpenter’s shop to have these cuts done. They have more advanced tools such as saber saws or jigsaws to cut circular holes.

  3. Calibration of the light shield needs practice. There is a subtle distance relationship between each acrylic sheet to achieve the best image quality, depending on the camera setting. We want the light to shine through the transparent agar and be reflected by the swarming colony. Once you find the right position, tighten all the nuts so that the position is locked for subsequent imaging.

  4. If a swarming strain does not swarm but only forms a dense spot, the plate may be too dry; therefore, less medium may be readily available for swarm expansion. Always use fresh plates within two days from pouring them; beyond two days, the agar plate will become significantly drier and may not sustain swarming motility. The drying time in the hood should not be too long. When the lab humidity is below 30%, 10 min of drying is enough. Also, pour the plates when the agar solution is not too cold; otherwise, the agar poured on the plate may have a rough surface since solidification has already taken place in the bottle, which shows up on the plate in small clusters. Finally, double-check the tryptone or yeast. Occasionally, for certain Lot numbers, some chemicals do not dissolve thoroughly, which may change the texture of the agar surface, leading to suboptimal results.

  5. Sometimes, non-swarming strains also appear to “swarm,” and in exceptional cases, even faster than the swarming strains. In this case, the agar concentration may be too low or the drying time too short, so the cells are swimming as opposed to swarming on the plates. Different swarming species have different agar concentration tolerance. For SM strains, 0.5% agar is the preferred concentration to distinguish swarmers from non-swarmers, whereas for B. subtilis 3610, 0.7% has proven to be the optimal agar concentration. To fix the problem of false positives, one can try to increase the drying time or raise the agar concentration slightly.

  6. Condensation may occur on the plate lid if a different tent is used and the temperature of the lid is lower than that of the bottom. In this case, try to flip over the plates and the problem should be resolved. After flipping over the plates, adjust the camera lens accordingly to focus on the bacterial swarm.

  7. SM strains are newly isolated bacterial strains. To request these strains under a Material Transfer Agreement, please refer to the linked main article (https://doi.org/10.1053/j.gastro.2021.03.017).

Recipes

  1. LB broth, solution

    10 g/L tryptone

    5 g/L yeast extract

    5 g/L NaCl

    Note: The above medium recipe is designed for SM bacteria and is subject to change according to the necessities of other microbes one works with.

  2. 0.5% LB agar plates (swarm plates)

    10 g/L tryptone

    5 g/L yeast extract

    5 g/L NaCl

    0.5% agar

    1. Weigh 0.5 g yeast extract, 1.0 g tryptone, 0.5 g NaCl, and 0.5 g agar. Place these into a 100-ml Pyrex bottle. Measure 100 ml deionized water and pour it into the bottle.

    2. Put a magnetic stir bar of appropriate size into the bottle, loosely close the lid, and place the bottle on a hot plate magnetic stirrer. Turn on the heat and the magnetic stirrer. It takes roughly 10–15 min for the medium to boil.

    3. After the medium starts to boil, turn off the stirrer and the heat. Wear thermal-insulated gloves to transfer the bottle to an autoclavable tray containing a thin layer of water.

    4. Loosen the bottle lid and autoclave for 25 min under 15 psi at 121°C.

    5. Once the autoclave has finished, place the bottle of medium back on the magnetic stirrer, with the heating function off but the stirring function on. During this step, we want the medium to cool to 40-50°C; the constant stirring is to maintain uniformity in temperature.

    6. Once the target temperature is reached, use a pipet aid to transfer LB agar medium to 6 Petri dishes, with 15 ml in each. Perform this operation inside a laminar flow hood or near a flame to minimize the chance of contamination from the air.

      Note: 15-20 ml medium is ideal for one Petri dish. If the volume is too low, the agar plate will become too dry during the incubation; if the volume is too large, the image quality will be compromised due to poor transparency.

    7. When the agar has solidified, remove the lid of the swarming plates and dry in the hood.

      Note: The plates must be dried in the hood in order to remove excess moisture from the agar surface. If there is water on the agar surface, the bacteria may be swimming rather than swarming. If the room humidity is above 50%, dry the plates for 20 min; if the room humidity is below 30%, the drying time is about 10 min; and if the humidity is 30-50%, adjust the drying time accordingly to around 15 min. Do not over-dry the plates; otherwise, the bacteria may not be able to swarm on the agar. Alternatively, use pre-stored swarming plates, which may be stored inverted in a 4°C cold room for up to 2 days; parafilm or plastic bags are not required. Using pre-stored plates, the drying time should be reduced by ~5 min.

Acknowledgments

The instrument and studies presented here were built and conducted using funds at the Albert Einstein College of Medicine, INC and supported by the Broad Medical Research Program at CCFA (Crohn’s & Colitis Foundation of America; Grant# 362520) (to S.M.); NIH R01 CA127231; CA 161879; 1R01ES030197-01; and Department of Defense Partnering PI (W81XWH-17-1-0479; PR160167) (S.M.).

Competing interests

The authors declare that they have no conflicting financial interests.

References

  1. Harshey, R. M. (2003). Bacterial Motility on a Surface: Many Ways to a Common Goal. Annual Rev Microbiol 57: 249-273.
  2. Kearns, D, B. (2010). A field guide to bacterial swarming motility. Nat Rev Microbiol 8(9): 634.
  3. Steager, E. B., Kim, C. B. and Kim, M. J. (2008). Dynamics of pattern formation in bacterial swarms. Phys Fluids 20: 073601
  4. Morales-Soto, N. Anyan, M. E., Mattingly, A. E., Madukoma, C. S., Harvey, C. W., Alber, M., Déziel, E., Kearns, D. B. and Shrout, J. D. (2015). Preparation, imaging, and quantification of bacterial surface motility assays. J Vis Exp 98: 523-538.
  5. Peñil Cobo, M., Libro, S., Marechal, N., D'Entremont, D., Peñil Cobo, D. and Berkmen, M. (2018). Visualizing bacterial colony morphologies using time-lapse imaging chamber MOCHA. J Bacteriol 200(2): 413-417.

简介

[摘要]细菌集群是指带鞭毛的细菌在半固体表面上以协调运动快速传播( Harshey , 20 03 ) 。已经有关于motili的该特定模式广泛的研究TY,因为它的有趣生物和物理相关性,例如,增强的抗生素抗性(基恩斯,201 0 )和湍流集体运动(Steager等人,20 08 )。商业设备的 群扩展的现场记录很容易花费数万美元(Morales-Soto等人,20 15 );然而,条件往往没有得到准确控制,导致鲁棒性差和缺乏重现性。在这里,我们描述了一种可靠的设计和操作小号协议来执行再现的细菌蜂拥测定使用延时摄影。该协议包括三个主要步骤:1)构建“自制,”环境控制的拍摄培养箱 2)执行一个细菌蜂拥测定; 和3)计算从接管时间序列照片蜂拥率。一个有效的方法的CALCULAT荷兰国际集团的细菌蜂拥率是在执行蜂拥表型的关键-相关的研究,例如,筛选蜂拥缺陷等基因突变株。培养箱是经济的,容易操作,并且具有一宽的应用范围。事实上,这种系统可以应用于以许多缓慢演变过程,如生物膜的形成和真菌的生长,这需要由照相机监视下一个受控的温度和环境湿度。


[背景]细菌表现出不同的运动表型,例如游泳、蜂拥、滑翔、滑动和抽搐(Kearns,2010)。跨潮湿表面的协调多细胞迁移,称为细菌群落,是一种重要的运动表型,由于其与致病性、毒力和抗生素耐药性的相关性,已经研究了几十年(Kearns,2010 年)。℃的系统性研究olony膨胀速度和形态模式耦合到蜂拥可以提供深入了解我Ñ到细菌运动性和宿主健康之间的相关性。在这里,我们BUIL ð的环境控制的培养箱中执行一个蜂拥测定。培养箱内,数码相机我š安装在顶部采取蜂拥活动的时间推移的图像。在记录之后,图像一重新传送到笔记本电脑孔定量吨使用群膨胀通货膨胀的ImageJ软件。该系统通过记录超过 1,000 次蜂拥事件进行测试,以确保稳定性和可重复性。
乍一看,可以考虑一个群聚测定相当简单:在琼脂上填充在培养皿中接种只是细菌,然后取一个Ñ图像板的用于温育后一个一定的时间。然而,以可重复的结果进行蜂群检测并记录清晰的图像以进行进一步的定量分析可能具有挑战性。在这里,我们列出了一些可能遇到的技术挑战,并在我们的协议中提供了相应的解决方案:
1.一种典型的群聚事件可能需要10 - 20 ħ为细菌,以覆盖一个标准的9厘米的培养皿。除了覆盖范围时,有时我们需要知道的滞后期能持续多久,在什么时候分支形成的菌落边缘; 在微观层面,当细胞伸长时。因此,研究人员需要在显微镜的帮助下定期检查板。假设一个人开始在试验的白天; 一个可能需要扫描所述板每10 - 30分钟过夜。在我们的协议,延时拍摄,有助于捕捉图像,以便研究人员不必熬夜或检查每10 - 30分钟,为了不错失关键事件。
2.细菌群落对环境高度敏感。在环境温度和湿度的波动经常会引起大的差异的蜂拥率和集落图案; 吨herefore,一个稳定的湿度-和温度控制的环境是该测定的关键。在我们的系统中,我们利用一个隔热帐篷、一个湿度控制单元和一个温度控制单元来最大限度地减少检测过程中的环境波动。可容易地设置不同的humidit IES和温度小号为一个群聚应变屏幕。
3.由于琼脂凝胶通常含有超过 97% 的水(按质量计),因此板盖上很容易形成冷凝物,从而使图像拍摄变得模糊。我们设计培养箱的方式是,当平板倒置放置在平台上时,盖子的温度略高于琼脂的温度,从而防止冷凝。注意:琼脂表面结构可能因浇注方法、厚度和所用琼脂的百分比而异;这些需要针对特定细菌进行单独优化。
4.以清晰的照片的群荷兰国际集团板是棘手的。一大群ING板具有影响三个表面的同时成像光学质量:板盖,在琼脂表面,和板底部。使用相机手电或辅助前灯时,部分光线会被盖子和板底反射到相机上,在照片上形成不良光点。在我们的设计中,对于单板照片拍摄,我们使用圆形荧光灯管作为可调遮光罩下的背光。对于多板(最多9)测定法,一种LED灯条我š用于侧光照明。图像质量较好时使用,因为光屏蔽的光场设置; 然而,当使用效率更高一个LED灯,因为它照亮,可以在一个时间适应9个培养皿更大的面积。
5.孔定量吨蜂拥率的通货膨胀需要一个LO吨的EF堡; ř esearchers通常扫描板并测量群的半径ING使用尺子菌落。对于不规则形的殖民地,他们通常做的一个粗略估计的“有效半径。”当多个板被涉及在测定中,一个应绘制的只是计算平均蜂拥速率蜂拥面积与时间代替。大量的手动测量需要大量的工作。在我们的例子中,我们导入了数字图像蜂拥到ImageJ的软件和计算SWAR米ING通过概述群周边区域。这种计算机化的过程提高了效率并减少了错误。
与该领域的常规实践相比,我们的协议具有明显的优势:
(1)负担能力。在2015年,一个研究小组由Joshua Shrout博士领导的圣母大学描述的方案用于制备,成像和孔定量吨通货膨胀蜂拥的(莱斯-索托等人。,20 15 )。在他们的协议中,他们使用了商业设备“布鲁克体内成像站” 。“该公司不再提供该产品,使用过的产品成本超过 58,000 美元。大多数微生物实验室研究细菌蜂拥可能不需要许多的复杂的功能即第是特定设备提供,诸如x射线成像和体内荧光成像。日Ø SE实验室未必能买得起或舍得投入这么多我-n的OV erqualified细菌蜂拥室。相比之下,我们系统的总成本约为 1,000 美元,包括一切。
(2)准确性。对于该布鲁克成像站,将水的板旁边的每个群ING板我š建议为了保持湿度。但是,这样很难将湿度控制在特定范围内。在我们的设计中,腔室湿度得到很好的控制;o ne 可以在化验开始前设置参数,并使用数字传感器和反馈控制器动态控制环境。
(3)效率。在2018年,一个独立的研究者,Peñil科沃,开发了一种时间推移成像室,用于细菌菌落形态观察(Peñil科沃等人,20 1 8 ),它允许一个陪替氏培养皿的摄影在一个时间。由于该设计不控制温度,因此整个系统必须放置在温暖的房间内。在我们的案例中,多个 9 厘米培养皿可以安装在培养箱内,方便有效地利用空间和能源。有在我们的设计更大的箱体,必要举行多个板,改变了一种微妙的方式的光路,为摄影,但我们的孵化器的几何形状和光场一重以及校准,以确保在最佳的照明。
该协议分为三个步骤:(1)孵化器的组装;(2)的蜂拥测定; (3) 数据处理。通过以下详述的程序,我们保证您可以获得一台自制的细菌培养箱,该培养箱具有稳定的蜂群结果和高质量的图像。总成本取决于WH上ICH相机在系统中使用,但应在$ 1,000美元。实际上,任何具有“手动模式”的数码相机都足以满足此目的。A S升温测定需要研究人员将细致与某些细节,以确保可重复性。例如,细菌群落对条件的微小变化非常敏感,例如琼脂表面的粗糙度和湿度水平。在这里,我们试图阐述的程序和注意事项在尽可能多的细节尽可能为用户追踪。在照片拍摄部分,我们详细展示了如何调整相机设置。最后,我们解释了如何使用 ImageJ 处理数据来测量集群区域。

关键字:细菌群集, 细菌运动性, 菌落生长, 孵化, 延时成像


材料和试剂

 
A.到B uild拍摄培养箱        
1. Hydropo NIC室内花园成长帐篷(24英寸。× 24在。× 48在。,Yaheetech,米Odel等:YT-2801)      
2.摄影单位      
一种。d igital相机(松下,型号:DMC-FZ50)       
湾 LCD定时器遥控器(JJC,型号:TMD)      
C。AAA 电池(2 块,金霸王)       
d. 镀锌开槽角钢(4 个,1.5 in . × 14 Gauge × 36 in . ,皇冠螺栓)      
e. 镀锌开槽角钢(10 个,1.5 in . × 14 Gauge × 18 in . ,皇冠螺栓)       
F。铝制扁钢(0.75 in . × 36 in . × 0.125 in . , Everbilt)        
G。黑色涤纶布(20 in . × 20 in . , Dazian)      
H。螺栓和Ñ UTS(40对,M5,冠螺栓)      
一世。黑色丙烯酸片(2个,在18 。× 18在。× 0.125在。,国家安全镜像)        
j. LED灯带(3米,白色,国通)        
克。电源板(6 插座,贝尔金)      
3.背光屏蔽         
一种。黑色丙烯酸片(3片,在12 。× 12在。× 0.118在。,国家安全镜像)         
湾 荧光圆形吸顶灯(Sunlite,型号:FC12T9/CW)带启动镇流器      
C。锌-螺纹杆(4个,以0.25 。× 12在。,Everbilt)       
d. 六角螺母镀(24件0.25在。,Everbilt)      
4、温湿度控制单元      
一种。加热控制模块(Coy Lab,序列号:DC1807)       
湾 风扇(AC Infinity,型号:LS1225A-X)      
C。数字湿度控制器插座(Inkbird,型号:IHC200S)       
d. 爬行动物加湿器(2 L,Evergreen)      
e. 烧杯(500米升)       
注:其他品牌加湿器或控制器的可替代方案,如果IR组合Ç的好
                            控制室内湿度。
 
B.对于群荷兰国际集团板制备        
1.肠杆菌属。SM1 ,SM3,SM3_18,SM3_24 (对于细菌物种的蜂拥测定)      
2. LB肉汤(见食谱)         
3. 0.5% LB 琼脂平板(见食谱)         
 
设备
 
1. Labconco 净化器 II 类生物安全柜(Delta 系列)      
2.隼14 -毫升聚苯乙烯圆底管小号英寸(17毫米× 100mm时,Corning)中      
3.消毒木连接棒(2.5英寸。)      
4.移液器(Drummond, 1 - 100 ml) 用合适的移液器      
5.新不伦瑞克 Innova 4300 孵化摇床      
6.称重纸(4 in . × 4 in . , Fisher Scientific, c at alog number: 09-898-12B)      
7.实验室刮刀(4 件,6.5 英寸,不锈钢,家用科学工具)      
8.派勒斯玻璃瓶小号(250毫升,康宁,型号:1395)      
9. Pyrex 量筒(500 毫升,康宁,型号:3022)      
10.热板磁力搅拌器(Barnstead International,型号:SP46925)   
11.隔热手套   
12.高压灭菌器(AMSCO Scientific,型号:SV-120)带高压灭菌托盘   
13.培养皿ES英寸(100毫米× 15mm时,无菌,聚苯乙烯,Fisher Scientific公司,Ç在考勤数:FB0875712)   
14.微管(0.5 - 10微升,ê ppendorf,Ç在考勤编号:3123000020)   
15.亚克力刀具(弗莱彻)   
16.圆刀(博特)   
17.带合适钻头组的手钻/台钻   
18.钢锯   
 
软件
 
ImageJ (ver1.59g) ( https://imagej.nih.gov/ij/ )
 
程序
 
部分我:设置拍摄孵化器
 
A.组装相机支架      
1.按照说明书安装雅喜水培帐篷。该手册随产品包装一起提供。先组装骨架,然后用聚酯材料覆盖它。      
2.使用M5螺栓和螺母来组装相机帧,如所示图URE 1.连接第一镀锌开槽角,然后将铝条。开槽角钢的侧面有孔,但铝条没有。使用手钻或台钻在铝条的末端钻孔。由于铝棒用于稳定结构,因此它们的位置不必精确。      
 
 
 
˚F igure 1 。示意图显示了相机框架的结构。四个的36片-在镀锌槽的角度支架与地面垂直并且是由10片18的连接-在镀锌开槽角使用M5螺栓和螺母。铝条是没有特定位置的要求以稳定整个结构小号。当使用遮光,地面和样品平台之间的距离在18。W¯¯采用LED光照明母鸡,样品平台可以通过几英寸升高摄影。
 
3.使用的丙烯酸类切割器以切割SID 18的ES在×丙烯酸片材18略,以适应在样品平台。      
4.使用一个圆形刀具切割为9英寸的圆的直径从18中的一个的在× 18从中心压克力板“A” ; 保持另一张纸“B”未切割。      
5. 将板材“A”固定在样品平台上。钻的上侧适当的孔,使该螺栓经过一路走下来到孔的开槽角度。      
6.将相机固定在相机固定器上,镜头朝下。前后调整定影器,使相机与亚克力板上的圆圈对齐。      
7.在亚克力板上方 1 英寸的开槽角上,将 LED 灯条贴在样品平台周围。光的位置不能太高,因为我们要避免光从板反射。      
8.剪下两块黑布,大约 20 英寸× 20 英寸。将其中一块放在帐篷底部作为黑色摄影背景。对于另一块,在中心切一个孔让相机镜头通过,然后将布挂在相机平台上以遮挡顶部的反射。        
9.将相机支架放在帐篷内。      
10.负载的电池到所述LCD计时器遥控器和将其连接到通过在帐篷的顶部的孔的相机。使用周围的弹性绳将孔拧紧。   
 
B.制作遮光罩        
1.的切圆3.62中,在5.75 ,和9.50的直径ETER出三块丙烯酸片。钻0.25-在孔我n各自的角部的,1.2从两个边缘。根据图2组装遮光罩。将圆形荧光灯泡放置在 I 层和样品平台之间。使用胶带将启动镇流器粘在样品平台下。      
暂停点:Ť这里有照明的两种模式:ö NE US上课一个背光,而另一个我们上课一侧的光(图URE 3)。当拍摄接近-的图像š的一个一个板事件,它是优选使用的光屏蔽罩,其自己的光源光片下面。要拍照,请将遮光罩放在样品平台上并将其与相机对齐。使用遮光罩时关闭 LED 灯。在另一方面,在拍照时的一个以上的板,遮光被移除,和未切割的18 × 18在丙烯酸系片材为p股价上圆切割样品平台的顶部上。多达 9 个琼脂平板可以放置在未切割的薄片上进行成像。要拍照,请打开 LED 灯作为侧光源。
 
 
图2 。图像示出了一个光臂章d组件,其用于单-板成像。三片12中的× 12一中空丙烯酸系片材一个由连接重新4个在锌片12 -螺纹杆并用六角螺母镀固定。一级片材与地面之间的距离约为 1.5 英寸,只留下足够的空间来放置圆形荧光灯泡以适应。二级和三级的高度s可以调整以优化照明。
 
 
˚F igure 3 。使用遮光罩或 LED 灯带拍照。( A)使用遮光罩组件拍照。圆形荧光灯泡放置在层 I 和样品平台之间(如图 2 所示)。II 级和 I 级之间的距离约为 0.8 英寸,而 II 级和 III 级之间的距离为 3.5 英寸。 (B) 使用 LED 灯带拍照。LED 灯固定在水平镀锌槽角上样品平台上方 1 英寸处。如果光线太强,可以插入一条白纸带来漫射和反射光线。相机附近的黑布对于阻挡来自板的反射是必要的。
 
C 、安装温湿度控制系统        
1. P花边帐篷内的热控制模块,在底部侧,š UCH它不会使用遮光当蜂拥照片显示。调整温度设定为所需温度的蜂拥测定。      
2.将加湿器置于帐篷外,将电源线连接至湿度控制器插座。将可提取的塑料雾管通过帐篷壁上的孔延伸到样品平台下方的帐篷中。      
注:Ç赫克相机预览并确认该管和雾不会在图像中显示出来。
3.将加湿器水箱注满水。塞的湿度调节装置中的电源板,并与调节湿度值一个根据控制器手动公差范围。例如,对于肠杆菌属。SM3 (SE Ë的相应的主文章),设定湿度至40%±5%容差(TOL)。      
4.将500 -雾管收集水滴下毫升烧杯中。      
5.使用风扇随附的螺栓和螺母将交流风扇固定在面向烧杯的其中一个开槽角腿上。风扇不仅用于吹除加湿器中的雾气以防止照片中出现雾气,而且还用于改善室内温度和湿度的通风和均匀性。      
6. 将所有孔拧紧,并密封帐篷的拉链。      
 
部分II :执行的蜂拥分析
1.从 -80°C 冰箱中取出细菌甘油原液。用一块无菌木材的连接棒划伤细菌-含冰表面上,然后浸棒插入LB肉汤。P花边的甘油原液回在冷冻机和摇动接种LB肉汤过夜(〜16小时)一个振荡器。温度设置为 37°C,振荡频率设置为200 转/分钟(rpm)。下午 5 点左右开始过夜生长,以便第二天早上 9 点左右可以使用。          
2.使用一个微量吸移管接种2微升过夜细菌悬浮液上的中心每个琼脂平板上。转移板培养箱日以后Ë滴接种已经通过吸收在琼脂(传播的的圆形降已成为几乎不可见)。          
 
部分III :时间推移拍照和蜂拥率孔定量牛逼通货膨胀
1.为了使用遮光,对花边一个大群ING板倒置在板保持件。打开荧光灯泡和相机。所述LED灯条应保持关闭。      
2.在相机的预览中,您应该看到位于屏幕中央的盘子。否则,将光屏蔽周围与样品对准摄像头。      
3.旋转螺杆上的螺母,调整亚克力板的位置。样品平台与 Level I 之间的距离约为 1.5 英寸。一级和二级之间的距离约为 0.8 英寸,而二级和三级之间的距离为3.5 英寸。如果您在培养皿上看到一个圆形光点,请稍微抬起 III 级。如果培养皿太暗,请稍微降低 III 级。如果你不能得到通过调整级别III的良好形象,然后调整二级小幅上涨或下跌(见ñ OTE 3)。      
4.将相机焦距设置为 35 - 65 毫米。调节变焦环,使样品OCCUP IES全屏幕,但不会不超过边界。使用“M ,”手动模式中,将重点放在细菌结肠IES 。集合T他光圈F5.6 - F7.1和调节快门速度直到结果荷兰国际集团的曝光值是0或-⅓ ,因为曝光过度的图像将导致在图像细节的损失(图URE 4A)。      
5.对于一个多板测定中,除去遮光,点亮LED灯条,并放置在样品平台上的未切割的丙烯酸系片材。将蜂巢板倒置在亚克力板上,这样水就不会在盖子上形成冷凝水。检查相机预览,以确保其所有的板都卵石的范围内N(图URE 4B)。      
6、根据说明书设置液晶摄像头定时器控制的帧率和帧数。例如,在肠杆菌属的情况下。SM3,我们将帧数设置为 50,将帧之间的时间间隔设置为15 分钟。按下了“开始”按钮,开始时间推移照片拍摄。      
7.拉上帐篷的拉链以形成良好的密封。蜂拥检测可能需要大约 10小时。您可以让相机过夜并在第二天早上收集图像。拍照过程中,请勿摇晃培养箱;否则,光学设置可能会受到干扰。      
8.蜂群完成后停止定时器控制器。由c连接下载图像相机来使用USB数据线一台笔记本电脑或通过拉出相机的存储卡,并把它变成一个电脑上下载的图片。      
9.检查在您的计算机上拍摄的图像。如果图像的亮度变化,则可以使用“堆栈的去闪烁”插件ImageJ中校准的亮度。如果图像中的样本位置随时间变化,您可以应用“图像稳定器”插件来修复它。      
 
数据一nalysis
 
代表性的单板和多板蜂拥图像分别如图 4A 和 4B 所示。在所示的板的时间过程图URE 4B被渲染为视频在视频1。
 
 
˚F igure 4 。在孵化器中拍摄的蜂群图像。(A)P晚期š接种肠杆菌属。SM1(左)和SM3(右)瓦特ERE孵育6.5 ħ 。此我法师用的是环形荧光灯灯泡截取和遮光组件。SM3与(B)的成群的一个群缺陷型突变SM3,的SM3_18(板小号1,2,4) ,并相对于SM3一大群缺陷型突变SM3,的SM3_24(板小号3,5,6)。每个比较被执行以一式三份,用SM3上的每个板的右半接种。图像拍摄一后6.5 - ^ h孵化。侧面 LED 灯带用于照明,所有六个板一起成像。
 
肠杆菌属的延时视频。使用 LED 灯条照明进行的 SM3 蜂群检测。将 SM3 及其突变体(SM3_18 或 SM3_24,每个一式三份)过夜培养物接种在 0.5% LB 琼脂平板上并在光室中孵育。延时图像拍摄 10 小时,然后使用 ImageJ(版本 1.59 g)以每秒 11 帧 (fps) 的速度将图像渲染为 .avi 视频文件。
 
为了计算群ING区域,打开图像的兴趣ImageJ中。点击“分析”——“设置比例”,校准像素与实际长度的比例。然后用“徒手选择”工具来勾勒菌落边缘,然后按“M”通过图像(以计算所选择的集落的图像的区域图URE 5)。肠杆菌属的蜂拥率。SM1和SM3一个重新在图6中绘制的以下的计算的群区域。
 
 
图 5. 使用 ImageJ 计算群区域。(A) ImageJ 用户界面。选择了“手绘选择”工具。(B)肠杆菌属。SM3群荷兰国际集团的殖民地(右)由“写意选择”工具概述。(C)在S中的像素Cale的š我S设定到实际长度的值。(d)通过按下“M ,”种群的面积进行计算。
 
 
˚F igure 6 。孔定量牛逼的通货膨胀肠杆菌属。SM1 和 SM3 蜂拥运动。SM1 和 SM3用2 μl 过夜细菌培养物接种在 0.5% LB 琼脂平板上,并在蜂群培养箱中培养(共重复 10 次)。每半小时拍摄一次延时图像,并测量每个图像的群体面积。数据表示为在平均值,用误差条指示的95%置信区间。当不可见时,误差小于符号的大小。
 
笔记
 
1.孵化器内的湿度和温度应达到的电源被接通之后并且保持稳定的过夜记录的持续时间在10分钟内的设定值。      
2.有时,某些型号小号或规格的产品可能会脱销。如果18 -在开槽的角度不容易获得,例如,人们可以从再切割角度用钢锯。从一块亚克力上切出一个大圆孔需要一定的技巧。为确保安全,使用孔锯或切圆刀时,请务必先将亚克力板牢牢固定在工作台上。另一种选择是去工程车间或木匠店完成这些切割。他们有更先进的工具,如马刀锯或曲线锯来切割圆孔。      
3.光罩校准需要练习。每个亚克力板之间存在细微的距离关系,以实现最佳图像质量,具体取决于相机设置。我们希望光通过透明琼脂光泽和反射由所述群荷兰国际集团菌落。找到正确的位置后,拧紧所有螺母,以便锁定该位置以进行后续成像。      
4、如果一个群聚株不群聚而只是形成一个密集的斑点,可能是盘子太干了;因此,可用于群体扩展的培养基可能较少。始终使用中,从p2天新鲜板ouring他们; b eyond两天,琼脂平板将显著干燥器变得并且可能无法维持蜂拥蠕动。该d在引擎盖rying时间不宜过长。当实验室湿度低于30%时,干燥10分钟就足够了。另外,当琼脂溶液不太冷时倒入盘子;Ó therwise,琼脂倾倒在所述板可以具有一个粗糙的表面,因为凝固已在瓶,其上显示出在小簇板已经发生。最后,双-检查胰蛋白胨或酵母。有时,对于某些批号,某些化学品不会完全溶解,这可能会改变琼脂表面的质地,导致结果不理想。      
5.有时,非群ING菌株也出现“群,”在特殊情况下,甚至快于蜂拥株。在这种情况下,琼脂浓度可能太低或干燥时间太短,因此细胞在游动,而不是在平板上蜂拥而至。不同的蜂群物种对琼脂浓度的耐受性不同。对于 SM 菌株,0.5% 琼脂是区分蜂群和非蜂群的首选浓度,而对于枯草芽孢杆菌3610,0.7% 已被证明是最佳琼脂浓度。为了解决假阳性的问题,可以尝试增加干燥时间或稍微提高琼脂浓度。      
6.如果使用不同的帐篷,并且盖子的温度低于底部的温度,则板盖可能会出现冷凝。在这种情况下,尝试翻转盘子,问题应该得到解决。翻转盘子后,相应地调整相机镜头以专注于细菌群。      
7. SM菌株是新分离的细菌菌株。请求本身材料转让协议项下的应变,请参阅链接的主要文章(https://doi.org/10.1053/j.gatro.2021.03.017)。      
 
食谱
 
                             LB肉汤,溶液
10g / L的吨ryptone
5 g/L y东方提取物
5 克/升氯化钠
注:上述中介嗯配方是专为SM细菌和受根据其他微生物之一的生活必需品变化的作品有。
                             0.5% LB 琼脂平板(群平板)
10g / L的吨ryptone
5 g/L y东方提取物
5 克/升氯化钠
0.5%一加仑
称取0.5克酵母提取物,将1.0g吨ryptone,0.5克NaCl,和0.5克琼脂。P花边的SE中以100 -毫升的硼硅玻璃瓶。测量100ml去离子水中,并在倒到瓶中。 
把适当大小的磁力搅拌棒在向瓶子,松散地盖上盖子,然后将瓶子在热板上磁力搅拌器。打开热量和磁力搅拌器。介质沸腾大约需要 10 – 15 分钟。 
一压脚提升媒体开始s到沸腾,T瓮关闭stirr ER和的热量。戴上隔热手套,将瓶子转移到装有一层薄水的可高压灭菌的托盘上。
松开瓶盖和autoclav Ë下15psi下25分钟在121℃。
一旦高压釜公顷小号成品,对花边瓶子介质背面的上磁力搅拌器,具有加热功能关闭,但在上搅拌功能。在这一步中,我们希望介质冷却到 40 - 50°C ;不断搅拌是为了保持温度的均匀性。
达到目标温度后,使用移液器将 LB 琼脂培养基转移到 6 个培养皿中,每个培养皿中 15 毫升。执行个是在层流罩内或附近的火焰操作以最小化污染的来自空气的机会。
注意:15 - 20 ml 培养基非常适合一个培养皿。如果体积太小,在孵化过程中琼脂板会变得太干;我F中的体积过大,图像质量会受到影响,由于透明度差。
当琼脂已固化,除去的盖的蜂拥板和在引擎盖干燥。
注意:板必须在通风橱中进行干燥,以除去多余的水分从琼脂表面。如果琼脂表面有水,细菌可能会游动而不是蜂拥而至。如果室内湿度高于 50%,将板干燥 20 分钟;如果房间湿度低于30%,干燥时间约为10分钟;并且如果湿度是30 - 50%,相应地调整干燥时间约15分钟。不要过度-干板; 否则,该细菌可能不能一窝蜂琼脂上。可替代地,使用预先存储的蜂拥板,其可以被存储倒置在一个4℃冷室中静置2天; 不需要封口膜或塑料袋。使用预先存储的板,干燥时间应被减小由〜5分钟。
 
致谢
 
此处介绍的仪器和研究是使用Albert Einstein College of Medicine, INC 的资金建造和进行的,并得到 CCFA(美国克罗恩病和结肠炎基金会;Grant# 362520)(给 SM)的广泛医学研究计划的支持;NIH R01 CA127231;CA 161879;1R01ES030197-01 ;和国防部合作 PI (W81XWH-17-1-0479; PR160167) (SM)。
 
利益争夺
 
作者声明他们没有相互冲突的经济利益。
 
 
参考
 
1.哈尔希,R 。M. (2003)。表面上的细菌运动:实现共同目标的多种方式。年度修订微生物学57 :249 - 2 73 。      
2. Kearns, D, B. (2010)。细菌集群运动的现场指南。Nat Rev Microbiol 8(9) :634 。      
3.斯蒂格,E 。B.,金,C 。乙。和金,M 。J. (2008)。细菌群中模式形成的动力学。物理流体20 : 073601      
4.莫拉莱斯-索托,N.安彦,中号。乙。,很有趣,A 。乙。,Madukoma ,Ç 。小号。,哈维,C 。w ^ 。,阿尔伯,M 。,德齐尔,E 。,卡恩斯,D 。乙。和史劳特,J 。D. (2015 年)。制备,我maging,和q的uantification b acterial小号urface米otility一个ssays。Ĵ显示精通98 :523- 5 38 。         
5.佩尼尔科博,M 。,Libro ,S 。,马雷夏尔,N 。,D'Entremont ,D 。,佩尼尔科博,D 。和Berkmen , M. (2018)。使用延时成像室 MOCHA 可视化细菌菌落形态。Ĵ乙acteriol 200(2) :413-423 4 17 。      
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引用:Chen, W., Mani, S. and Tang, J. X. (2021). An Inexpensive Imaging Platform to Record and Quantitate Bacterial Swarming. Bio-protocol 11(18): e4162. DOI: 10.21769/BioProtoc.4162.
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