Name: Setting up the Imaging Apparatus for C. elegans Lawn Avoidance Assay
Uploaded: 2023-05-19T16:00:00
Description: Watch this Scientific Journal Video about Setting up the Imaging Apparatus for C. elegans Lawn Avoidance Assay at JoVE.com

The video outlines instructions for setting up an imaging apparatus using a smartphone and LED light box for conducting time-lapse experiments. It details requirements for equipment, placement of components, and settings for optimal video recording to capture assays within an incubator environment.

setting up the imaging apparatus for c. elegans lawn avoidance assay

Smartphone-Based Imaging for C. elegans Lawn Avoidance Assay

Among the many advantages of studying C. elegans, its simple nervous system offers the opportunity to study how changes at the genetic and cellular level impact network function and behavioral output. Despite having a limited number of neurons, C. elegans display a wide range of complex behaviors. One of these is lawn avoidance, in which the bacterivorous nematode responds to a harmful food source by leaving the bacterial lawn. C. elegans avoid lawns of pathogenic bacteria1,2,3, lawns of bacteria that produce toxins or are spiked with toxins1,4, and even RNAi-expressing bacteria whose target gene knockdown is detrimental to the health of the worms4,5. Studies have shown that worms respond to external cues such as metabolites produced by the pathogenic bacteria1,6, or internal cues that indicate that the food is making them sick4,7. These cues are processed through conserved signaling pathways, such as the mitogen-activated protein kinase (MAPK) pathway and the transforming growth factor beta (TGF&#946;) pathway, and require communication between the gut and the nervous system4,6,7,8.
Although the assay is simple, the learned behavior develops over many hours, often overnight. While there are mutants that are incapable of leaving, in which case scoring avoidance at just one time point is sufficient to demonstrate the defect, many mutants do leave eventually but are slower to come out. For these, the movement of the worms needs to be tracked every few hours, which can be difficult to do overnight. Counting itself also takes time, creating a lag time between the plates, and thus limits the number of plates that can be tested at the same time. Using an imaging setup to record many plates simultaneously for the whole duration of the assay would be very useful, but the cost of setup can be prohibitive, depending on the funding situation of the research lab.
To address this, we devised a very simple method that uses smartphones to record avoidance assays. Each phone can record time-lapse videos of up to six assay plates. To provide transmitted light, we use a light emitting diode (LED) light box that can be easily purchased online. Assay plates are placed on an elevated platform, supported by hollow rectangular tunnels, that focus the incoming light, creating contrast. We also provide a Python script that converts the videos into audio video interleave (AVI) files showing 10 s clips of each hourly time point. The videos are then cropped to individual plates and saved in separate files to use for manual counting.
The method provides a low-cost procedure that is also extremely easy to use, using items that are readily available to most people. Here, we describe the method using the well-established lawn avoidance assay against the human pathogen Pseudomonas aeruginosa (PA14), whose protocol has been previously described2,9. Finally, we also review the considerations and limitations of the imaging method for those that want to apply it to other C. elegans behavior experiments.

Author Spotlight: A Smartphone-Based Imaging Method for C. elegans Lawn Avoidance Assay  

A Smartphone-Based Imaging Method for C. elegans Lawn Avoidance Assay

Among the many behaviors displayed by C-elegans, pathogenic lawn avoidance is the worm's defense strategy to minimize its exposure to harmful pathogens. In our lab, we use this behavior as a tool to identify genes involved in the proper functioning of the neurons that mediate this behavior. The lawn avoidance behavior is a simple assay, however it develops over many hours.Hence, it is convenient if the assay can be recorded to observe behavior over long time periods. Also, if multiple assays can be recorded at the same time, that's even better. Although there are many imaging solutions described in the literature, even a few modestly priced cameras may be out of reach for labs with limited resources.Also, some may want to try the assay, but do not plan on doing it long term. For such cases, short-term solutions such as this protocol might help. Our amazing protocol offers an easy, low cost way to record worm behavior.We use smartphones to record the assay, then process the video through a Python script that makes it suitable for counting.

Watch this Scientific Journal Video about Setting up the Imaging Apparatus for C. elegans Lawn Avoidance Assay at JoVE.com

Setting up the Imaging Apparatus for C. elegans Lawn Avoidance Assay  

Setting up the Imaging Apparatus for C. elegans Lawn Avoidance Assay

To set up the imaging apparatus, use a smartphone with a minimum specification of 12 megapixel camera 1080 pixel resolution, five gigabyte storage space, in a timeLapse video app from the application store. Place the LED light box on the bottom rack of an incubator set at 25 degrees Celsius where the assay will take place. To hide the dotted pattern on the LED light surface, spread two sheets of tissues to cover the entire surface of the LED box.Make an elevated stage for the specimen comprising of a clear plastic sheet supported by hollow rectangular tunnels. Ensure that the tunnel's walls are somewhat dark to minimize light scatter. Place another rack above the stage to place the phones for recording.Each phone will record three to six plates, so adjust the rack height accordingly. Put a power strip inside the incubator to plug in the phones during overnight recording. To prepare for the recording, plug in the smartphone to the power strip connected to a power outlet.Ensure to disable the auto lock setting to prevent the phone from returning to the lock screen while recording. Open the timelapse camera app, and set the video quality to 1080 pixels at 30 frames per second. Set the timelapse interval to two seconds.Place the smartphone with the screen facing up to record with the back camera and ensure that the paper box tunnels fit within the field of view.

Research

JoVE Journal

Behavior

When exposed to toxic or pathogenic bacteria, the nematode Caenorhabditis elegans displays a learned lawn avoidance behavior, in which the worms gradually ...