Part 1, set up the photography incubator
Time: 1 d
i) Assemble the camera stand
Time: 3 h
1. Setup 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. A rough estimation of the position of the aluminum bars is enough since they are just used to stabilize the structure. The slotted angles have holes by the side, but the aluminum bars don’t. Drill holes by the ends of the aluminum bars with hand drill or table drill.
3. Use acrylic cutter to cut the sides of 18’’ x 18’’ acrylic sheets slightly to fit in the sample platform.
4. Use the circular cutter to cut a circle 9’’ in diameter out of one of the 18’’ x 18’’ acrylic sheets “A” from the center and leave the other one “B” uncut.
5. Fix the sheet “A” on the sample platform. Drill appropriate holes on the side to allow bolts to go through all the way down to the holes of slotted angles.
6. Fix the camera on the camera fixer with its lens facing downwards. 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 unnecessary reflections of the light from the plates.
8. Cut two pieces of black cloth around 20’’ x 20’’ in size. Place one of them on the bottom of the tent as black photography background. For the other piece, cut a hole in the center to let the camera lens go through and hang the cloth onto the camera platform to shield any reflection from the top.
9. Place the camera stand inside the tent.
10. Load batteries for the LCD timer remote control and connect it with the camera through the hole on the top of the tent. Tighten the hole using the elastic cord.
ii) Make the light shield.
Time: 3 h
11. Cut circles of diameter 9.2 cm, 5.75’’ and 9.5’’ respectively out of three pieces of acrylic sheets. Drill ¼’’ holes on each of the corners of the sheets 3 cm 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 tapes.
12. (Optional) Sometimes, the light bulb comes with metal spring clamps. You can fix the clamps on the sample platform and clamp the bulb.
Pause point: There are two modes of illumination. One is using backlight and the other is using side light (Figure 3). When taking high quality image of one-plate event, the light shield is used, and it has its own backlight light source. 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 for more than one plate. The light shield is removed and the uncut 18’’ x 18’’ acrylic sheet is put on top of the circularly cut sample platform. To take photos, turn on the LED light as side light source.
iii) Install the temperature and humidity control system
Time: 2 h
13. Put the heat control module inside the tent on the side of the bottom so that it will not show up in the swarming photos when using the light shield. Adjust the temperature setting to the desired temperature for swarming assay.
14. 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. Notice: check the camera preview and make sure the tube and the mist do not show up in the image.
15. Fill the humidifier tank with water. Plug in the humidity controller to the power strip and adjust the humidity value with tolerance range according to the controller manual. For Enterobacter sp. SM3, set the humidity for 40% RH ± 5%RH tolerance (tol).
16. Place a 500 ml beaker under the mist tube to collect water droplets from the mist tube.
17. 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 avoid the fog showing up in the photos but also to improve ventilation and uniformity of the temperature and humidity in the chamber.
18. Tighten all the holes and seal the zip of the tent.
Part 2, perform swarming assay
Time: 2 d
i) Grow bacteria suspension
Time: 16 h
19. Weigh 0.5 g yeast extract, 1 g tryptone, and 0.5 g NaCl, placed in a 100 mL Pyrex bottle. Measure 100 mL deionized water and pour in the bottle.
20. Close the lid and shake the bottle until all the powders dissolve in water. You should see a clear yellow solution at this point. The solution is called LB broth (Lysogeny Broth).
21. Loose the bottle lid and put in an autoclavable tray with a thin layer of water inside. Autoclave the LB broth for 25 minutes under 15 psi pressure and 121 °C.
22. After autoclave is finished, take out the tray and wait for the broth to cool down to room temperature. Transfer 5 mL LB broth to a 14 mL tube using pipet aid in the hood.
23. Bring out the bacteria glycerol stock out of the -80 °C freezer. Use a piece of sterilized wood stick to scratch the bacterial containing ice surface and then dipping the stick into the LB broth. Put the glycerol stock back to the freezer and shake the inoculated LB broth overnight (~16 h) in the shaker. The temperature is set to 37 °C for SM3 and shaking frequency 200 rpm (revolutions per minute). Prepare the overnight suspension around 5 pm so that it will be ready for use around 9 am the next morning.
ii) Prepare the swarm plates
Time: 5 h
24. Weigh 0.5 g yeast extract, 1.0 g Tryptone, 0.5 g NaCl, 0.5 g Agar and put them in a 100 mL Pyrex bottle. Measure 100 mL deionized water and pour it in the bottle.
25. Put the magnetic stir bar of appropriate size in 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 minutes for the medium to boil.
CAUTION: DO NOT LEAVE THE HOT PLATE UNATTENDED.
26. Turn off the stirring and heating after the medium start boiling. Wear thermos-insulate gloves to transfer the bottle to an autoclavable tray with a thin layer of water.
27. Loosen the bottle lid and start autoclaving for 25 minutes under 15 psi, 121 °C.
28. Once the autoclave is done, put the bottle of medium back to the magnetic stirrer with heating function off but stirring function on. In this step, we want the medium to cool down to 40~50 °C and the constant stirring is to avoid non-uniformity in the agar.
29. Once the target temperature is reached, set a flame and use a pipet aid to transfer LB agar medium to 6 petri dishes, with 15 ml on each petri dish.
30. Turn off the flame and wait for the agar to solidify. This will take about 2 hours.
31. (Optional) Use the freshly made plate or store the swarming plates in 4 °C cold room with them inverted for up to 2 days. Whenever you plan to use the swarming plates, you need to dry the plates in the hood first. This is to remove water on the agar surface. If there’s water on the agar surface, the bacterial motility may be swimming rather than swarming. Thus, the next step is crucial.
32. Remove the lid of swarming plates in the hood. When the room humidity is above 50%RH, dry the plates for 20 minutes. When the room humidity is below 30%RH, the drying time is about 10 minutes. When the humidity is between 30-50%, you can adjust the drying time accordingly to around 15 minutes. Do not over dry the plates; otherwise, bacterial cells may not be able to swarm, due to either surface friction or dryness.
33. Use micropipette to inoculate 2 μL overnight bacteria suspension on the center of a swarm plate. Transfer the swarm plates into the incubator after the inoculation drop dried (3D hemisphere turns to a 2D circle).
Part 3, time-lapse photo taken and swarming rate quantification
Time: 1 d
34. To use the light shield, put one swarm plate inverted on the plate holder. Turn on the fluorescent light bulb and the camera. LED light strip stays off.
35. In the preview of the camera, you should see the plate sitting in the center of the screen. Otherwise move around the light shield to align the camera with the sample.
36. 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 slightly under 1 inch (about 2 cm) while Level II and Level III are separated by 3.5 inches. If you see a round light spot on the petri dish, slightly lift Level III. If the petri dish is too dark, slightly lower Level III. If you cannot get a good image by adjusting Level III, then adjust Level II slightly up or down.
Caution: Calibration of the light shield takes practice. There is a subtle distance relationship between each 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 from the swarm colony. Once you find the right position, tighten all the nuts and the position is locked for later imaging.
37. Set the camera focal length to 35 mm - 65 mm. Adjust the zoom ring to have the sample occupy the full screen but not exceed the border. Use “M” manual mode to focus on the bacteria colony. The aperture is set to F5.6 -F7.1 and adjust the shutter speed until the resultant exposure value is 0 or -⅓. This is to make image processing easier because overexposed images will lose information details.
38. For 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 swarm plates inverted on the acrylic sheet so that water will not condensate on the lid. Check the camera preview to make sure all the plates are within the range of the screen.
39. (Optional) The mode of the scene can be “Auto” or “fluorescent bulb”. If you find the image too yellowish, you can adjust the white balance to add more blue tone. You can save the images as raw format if the memory card space allows.
40. Set the frame rate and frame number of the LCD camera timer control according to the manual. In the case of SM3, we set the frame number to 50 and time interval between frames as 15 min. Press “start” button to start time-lapse photo shooting.
41. Seal the zip of the tent. The swarming assay may take about 10 h. You can leave the camera on for overnight and collect the images the next morning. During the photo taking process, DO NOT shake the incubator; otherwise the optical setup may be perturbed.
42. Make sure there is enough water in the humidifier water tank. If you want to check the condition inside the chamber, you may gently open the window on the top of tent to see the camera screen without disturbing the photographing.
43. Stop the timer controller when the swarming is finished. Connect the camera with a laptop using a USB data wire and download the images to the designated folder. Detach the laptop from the camera.
44. Check the images taken in your computer. If the brightness of the images varies, you can use the “Stack Deflicker” plugin in ImageJ to calibrate brightness. If the sample position in the images varies over time, you can apply the “Image Stabilizer” plugin to fix it.
45. (Optional) Use a common software (Photoshop CC, ImageJ, etc.) to render a time lapse video of the images. Or you can just use ImageJ. Go to “File” -- “Import” -- “Image Sequence” to Import the images. Go to “Image” -- “Stacks” -- “Animations” -- “Animation options” to set the frame rate you want. Then, save the image sequence as an AVI file.
46. To manually calculate the swarm area, drag the image that you are interested in to the ImageJ control bar. Go to “Analyze” -- “Set Scale” to calibrate the ratio of pixels to 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.
47. To calculate the swarming speed automatically, open the python script and change the file directory to the directory where you store your image sequences. Running the script will calculate the swarming colony area over time and save the data in a .txt file. The python script utilizes the subtraction edge detection algorithm to identify the edge of the swarming colony and then calculate the enclosed area value in each frame uploaded.