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wiki:protocols:foraging:protocol

Lawn-entry assay

Strain

Microscope settings

32.0x magnification
YFP filter
Fluorescent bulb turned on and brightness set to 100%
30fps

pylon Viewer settings

For worms

Binning: 1
Gain: 0.0
30,000 exposure

For lawns

Binning: 1
Gain: 20.0
300,000 exposure

Saving recording:

As sequence of images, 1 Frame at a time, stop after 5 minutes

Conversion settings

For 1x magnification 1px = 2.349 um.

Protocol

Maintaining control and mutant animals between recordings

  • Ideally the worms should not be crowded and definitely not starved, as these factors can affect C. elegans behavior and we want to control for that as much as possible
  • Worms should be incubated at 20C unless taken out of the incubator for experiments - temperature also affects behavior so we want to keep them at room temperature only for as much time as necessary and 15C only for long-term storage
    • If left at 15C or thawed from frozen stock –> 4 generations (around 2 weeks of maintenance) should pass before the worms can be used for behavioral experiments
  • Smell definitely impacts behavior, and as we're looking at the impact of smell on foraging behavior this is especially important:
    • This should go without saying but no popping popcorn in the lab microwave - the smell chemical released by the popcorn is enough to disrupt C. elegans behavior for a long time
    • Let Jun or Monika know if there is a strange smell in the behavior room (something like the inside of a small animal cage) - this means that smell could be coming from the mouse labs nearby and should be noted
    • Try not to let them near higher order alcohols, aromatics or bleach - if they're unpleasant for us then they're unpleasant for them
  • We don't use bleaching as a synchronization method because of the potential adverse effects bleaching can do to the eggs - as a result if contamination is unmanageable enough to require bleaching then allow for 4 generations (around 2 weeks of maintenance) to pass before using worms in behavioral experiments

Preparing adults for synchronization

  • To be able to record one strain per week:
    • 5 worms generally make it to the lawn per video - to get a decent sample size (100 worms) this means 20 recordings are needed
    • 20 worms x 20 recordings =
      • 400 worms in total =
      • 200 worms per day =
      • 100 worms during morning recordings and 100 worms during afternoon recordings
    • That's a lot of worms!!
  • To reach these numbers of worms requires some preparation 1 week in advanced
    • Monday -
      • Mutants: pick ideally 5x 1-2 day old adults onto a 10cm plate - repeat until you have 4x 10cm plates of adults - if you don't have enough for 4 plates or 5 adults per plate don't panic! - usually 1 plate of 5 adults is sufficient, but it's always better to be safe than sorry, especially if some plates grow contamination!
      • GRU101 control: 1x 10cm plate with 3x adult worms is usually sufficient but make an extra in case of contamination
    • Tuesday - repeat steps from Monday
    • Thursday - take adults from Monday's plate and repeat the steps for both mutant and control strains
    • Friday - repeat steps from Thursday
  • By next week you should have plenty of egg-laying adults to choose from without the plate being starved!
    • The benefit of doing this twice per week is that adults should only be about 1-2 days old when picking for synchronization i.e. Thursday worms are good for Monday syncing, Friday worms are good for Tuesday syncing!

Syncing GRU101 adults

  • GRU101 takes ~66-67 hours until laying of first eggs - ideally looking for 65hr adults.
  • Set up one set of syncs Monday evening and one set of syncs Tuesday evening for data collection on Thursday and Friday morning-afternoon respectively.
  1. Take two seeded plates - one is used as a lawn to remove unwanted worms while picking and the second is used for egg-laying.
  2. From a healthy plate of GRU101, pick 20 gravid hermaphrodites and allow to crawl on the lawn before transferring onto the egg-laying plate (ELP). Preferably pick adults of the same age i.e. day 1-2 adults.
  3. Allow the hermaphrodites to lay eggs for 1 hour.
  4. Remove all hermaphrodites from the ELP.
  5. Incubate at 20C.
  6. Check the progress of the ELP on day 2.
  7. Pre-egg laying adults should be grown after 65hrs.

Syncing mutant adults with GRU101 as control

  • Each mutant strain has a slightly different time it develops into a pre-egg laying adult - to minimize stress all mutants should reach adulthood after incubating at 20C for 72 hours - to ensure that all strains and worms are roughly within the same developmental period during recording, there should be one synchronized batch in the morning (for morning recordings) and one synchronized batch in the afternoon (for afternoon recordings)
  • See above instructions for syncing
  • For GRU101 - also incubate for 72 hours along with the afternoon batch

Seeding testing plates

  • 6cm NGM plates should be made fresh at the beginning of each week and put into 4C storage (if necessary) after drying overnight.
  • OP50-RFP should be cultured before seeding:
    • OP50-RFP requires gentamycin and strptomycin in at least 150ml LB:
      • To make 10ug/ml gentamycin + 100ug/ml streptomycin in 200ml LB, add 40ul and 400ul of 50mg/ml gentamycin and streptomycin respectively when inoculating
    • Take 1ml of culture and spin down at 13,000RMP for 1 minute - remove supernatent and resuspend in 100ul LB with antibiotic resistance.
      • 20mL of LB with 50mg/ml gentamycin and 50mg/ml streptomycin can be created and stored as 1mL aliquoted at -20C to then resuspend the bacterial pellet in antibiotic resistant LB when needed
    • Due to the volume of LB necessary for culturing OP50-RFP, making one 10x per week and storing it overnight at 4C is acceptable.
  • Dip the tip of a 100ul pipette tip into 10x OP50-RFP and gently touch onto the agar, ideally without poking a hole, to seed a lawn exactly in the center of the 6cm plates.
  • Allow to dry for 24hrs.

Starving worms

  • Worms should be starved for 1 hour before recording - we've determined that this makes the distinction between foraging and feeding pumping more defined
  • Take unseeded 6cm plates and pick as many worms as necessary (+ extra!!) for recordings - e.g. recording 6 videos during a morning session:
    • 20 worms x 6 videos = 120 worms minimum + 80 worms extra = 200 worms
    • 50 worms per plate = 4 plates of starved worms
  • Be careful to pick plates with as few rips/bubbles in the agar as possible - worms CAN and WILL disappear into the holes/agar and you won't be able to find them again!

Setting up recording

  1. Put a 5ml drop of M9 0.5cm away from the lawn and opposite each other. Measurement should start from the center of the lawn outwards.
  2. Pick 10 pre-egg laying hermaphrodites and immerse them in one drop of M9. Replenish the M9 if the drop is beginning to dry. Put another 10 worms in the other drop.
  3. Replenish both M9 drops before setting up the microscope camera. Place the plate under the microscope and find the focus of the pharynx as best as possible.
  4. Center on the bacterial lawn, making sure the outside is completely visible.
  5. Before recording, take a picture of the bacterial lawn.
  6. Begin recording once the first pharynx comes into view and the pharynx is clearly focused.

Camera settings

Compressing video recordings before uploading to soma

Videos can take a huge amount of space when stored as individual .tiff files, so it's a good idea to keep an eye on how much space you've been using. When space starts to run out, you can compress the video files on nif3004 and nif3011 and then transfer them to soma (storage server @ caesar). Before transferring to soma, be sure to check how data should be called and organized to keep things as tidy as possible.

  1. Open a command prompt.
  2. Type compressBehavior.sh followed by the directory of the folder you want to compress (input), then the directory of the new folder you want the compressed data to be saved into (output).e.g. compressBehavior.sh '/media/Nicolina/video_big' '/media/Nicolina/video_compressed' There will now be an original uncompressed folder (video_big) and a compressed folder (video_compressed).
  3. WARNING: if the input and output folders are EXACTLY the same then the compressed data will REPLACE the uncompressed data!!! Do this only if you're sure the compression will happen correctly. It's recommended to practice with different input and output names to begin with.
  4. WARNING: if the compression process is stopped while the input and output names are the same, this will cause a corrupted image file to form and PharaGlow is not able to handle this, which will throw an error. Be sure to check the data thoroughly if this happens before attempting to analyze.
  5. You will know a compression has been successful if the original file size is larger than the compressed file size. The code in the command prompt window will be able to tell you this.

Ideally you would want to compress after PharaGlow analysis, as compressed files take longer to analyze, but when pushed for space this is also fine.

wiki/protocols/foraging/protocol.txt · Last modified: 2020/07/30 09:16 by nzjacic

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