TITLE: Behavioural phenotyping of C. elegans response to Keio single-gene E. coli mutants
AUTHORS: Saul  Moore

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Protocol for screening </span><span style = "font-style:italic;">Caenorhabditis elegans</span><span> behavioural response to </span><span style = "font-style:italic;">E. coli </span><span>BW25113 single-gene deletion mutants from the 'Keio Collection', to identify behaviour-modifying mutations for follow-up analyses.</span></div><div class = "text-block">Duration of experiment: 6 weeks total (6 replicates over consecutive weeks)</div></div>

[STEPS]
?. [Seeding Petri plates + worm maintenance]
Prior to each experiment replicate, inoculate 10ml LB broth media with E. coli BW25113 in an Erlenmeyer flask for overnight culture (used as negative control and for raising worms) following the protocol, Inoculating a Liquid Bacterial Culture:(Friday, -11 days)
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?. [Seeding Petri plates + worm maintenance]
Remove the BW culture from the shaking incubator and place in 4°C fridge until seeding.(Saturday, -10 days)
?. [Seeding Petri plates + worm maintenance]
Remove 10 maintenance plates from storage and the BW culture from the fridge and leave to acclimate to room temperature for approximately 30 minutes.(Monday, -8 days)
?. [Seeding Petri plates + worm maintenance]
Leave under hood until dry (with lids on, timing depends on humidity). (Monday, -8 days)
?. [Seeding Petri plates + worm maintenance]
Using a platinum pick, gently pick 30-35 adult N2 Bristol C. elegans onto each of the 10 seeded maintenance plates, and store in an incubator at 20°C.(Monday, -8 days)
?. [Seeding Petri plates + worm maintenance]
Remove the adult worms after 24 hours, leaving the eggs behind to hatch into L1 larvae.(Tuesday, -7 days)
?. [Seeding Petri plates + worm maintenance]
Inoculate a further 25ml LB broth media with BW25113 bacteria for overnight culture and place in a shaking incubator at 37°C, 200 rpm.(Tuesday, -7 days)
?. [Seeding Petri plates + worm maintenance]
Remove the culture from the incubator and 15 nursery plates from storage, and leave to acclimate on bench top for approximately 30 minutes.(Wednesday, -6 days)
?. [Seeding Petri plates + worm maintenance]
Wash the worms off the 10 BW-seeded maintenance plates, into two 15ml Falcon tubes.(Friday, -4 days)
?. [Seeding Petri plates + worm maintenance]
Perform an egg prep on worms in the Falcon tubes, by following the protocol for Egg Prep for Bleach Synchronization (Cabreiro Lab):(Friday, -4 days)
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?. [Seeding Petri plates + worm maintenance]
At around noon the next day, wash L1 larvae off the empty plate and re-feed onto 15 BW-seeded nursery plates using a glass Pasteur pipette. Aim to dispense around 1000 worms per plate. Incubate at 20°C.(Saturday, -3 days)
?. [Inoculating from frozen stocks (96-well)]
Fill all 96 wells in a total of 55 culture plates (for the 45 Keio library plates + 1 plate control + 9 spares) with 200μL LB broth media and 50μg/ml Kanamycin.(Wednesday, -6 days)
?. [Inoculating from frozen stocks (96-well)]
Remove the Keio frozen library stock plates (in batches) from -80°C, gently remove the aluminium film, and then leave the wells to defrost for a few minutes.(Wednesday, -6 days)
To avoid damaging the bacterial stocks through repeated freeze-thawing, do not let the wells completely defrost. Just enough to be able to pick up some cells with the replicator.
?. [Inoculating from frozen stocks (96-well)]
Inoculate the 96-well culture plates from the Keio stock plate, following the protocol for Growing overnight bacterial culture in 96WP (Cabreiro Lab):(Wednesday, -6 days)
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?. [Inoculating from frozen stocks (96-well)]
Wet some tissue with MilliQ water, and wrap the culture plates in the tissue.NB: The tissue provides humidity that aids growth, while the presence of Kanamycin should prevent contamination.(Wednesday, -6 days)
?. [Inoculating from frozen stocks (96-well)]
Incubate cultures overnight at 37°C (no shaking).(Wednesday, -6 days)
?. [Seeding imaging plates (96-well)]
Remove the imaging plates from 4°C storage and record the average weight of 3 randomly chosen plates.(Friday, -4 days)
?. [Seeding imaging plates (96-well)]
Ensure that the imaging plates have lost approximately 3-5% of their original weight. Place under a hood (or drying cabinet) until this is the case.(Friday, -4 days)
?. [Seeding imaging plates (96-well)]
Remove overnight cultures of Keio strains from 4°C storage.(Friday, -4 days)
?. [Seeding imaging plates (96-well)]
Place seeded plates under a hood to dry for 20 minutes, then place in an incubator at 25°C (no shaking) for 7 hours 40 minutes (total lawn growth time: 8 hours).(Friday, -4 days)
?. [Seeding imaging plates (96-well)]
After 8 hours, remove the plates from the incubator and store at 4°C.(Friday, -4 days)
?. [COPAS worm-sorting and Hydra tracking (96-well)]
On the day of tracking, first thing in the morning, ensure that the imaging cave air conditioning is turned on (and there has not been a power-cut) and also the dehumidifier waste water tray is empty. Normal temperature range: 19 - 21°C,  humidity: 35 - 45%. (Pre-imaging checklist)(Tuesday, Tracking Day)
?. [COPAS worm-sorting and Hydra tracking (96-well)]
Turn on the COPAS machine by following Steps 1 - 16 of protocol COPAS wormsorter, to prepare and clean the machine for dispensing Day1 N2 worms:(Tuesday, Tracking Day)
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?. [COPAS worm-sorting and Hydra tracking (96-well)]
Fill up the tubes to 15ml with PBS 'A' and centrifuge at 1000rpm for 2 minutes.(Tuesday, Tracking Day)
?. [COPAS worm-sorting and Hydra tracking (96-well)]
Remove the supernatant using a Pasteur pipette. (Tuesday, Tracking Day)
?. [COPAS worm-sorting and Hydra tracking (96-well)]
Repeat steps  to  four more times to thoroughly rinse off any remaining BW25113 bacteria. (Tuesday, Tracking Day)
?. [COPAS worm-sorting and Hydra tracking (96-well)]
Re-suspend the worms and divide them equally into two 50ml Falcon tubes (for the COPAS), and fill them both up to approximately 40ml with PBS 'A'. (Tuesday, Tracking Day)
?. [COPAS worm-sorting and Hydra tracking (96-well)]
Dispense 3 Day 1 N2 worms into each well of the imaging plates with the COPAS following the protocol COPAS wormsorter:(Tuesday, Tracking Day)
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?. [COPAS worm-sorting and Hydra tracking (96-well)]
Leave the plates to dry under a hood for 30 minutes to 1 hour (until dry, timing depends on humidity), then place in incubator at 20°C until tracking (at +4 hours on food).(Tuesday, Tracking Day)
Check that worms are crawling (not swimming) on plates, and lawns look matt and not shiny
?. [COPAS worm-sorting and Hydra tracking (96-well)]
Repeat steps  to  to prepare all 46 imaging plates (in batches of 10) with 3 worms in each well for tracking.(Tuesday, Tracking Day)
?. [COPAS worm-sorting and Hydra tracking (96-well)]
Record worm behaviour on the bacterial food for 15 minutes at the +4 hour timepoint (25 fps, exposure: 25000 msec, with blue-light stimulation).(Tuesday, Tracking Day)
?. [COPAS worm-sorting and Hydra tracking (96-well)]
After tracking, discard the plates in a biological waste bin.(Tuesday, Tracking Day)
?. [COPAS worm-sorting and Hydra tracking (96-well)]
40 minutes prior to each tracking run (every 20 minutes, 10 runs in total), remove 5 imaging plates from the 20°C incubator and leave to acclimate in the imaging cave.(Tuesday, Tracking Day)
?. [Prepare NGM agar Petri plates]
Prior to the screening the Keio library, prepare the materials needed for 6 replicate weeks of experiments:- 330 Whatman square-well flat-bottom 'imaging' plates - 330 Nunc™ round-bottom microwell 'culture' plates        (45 Keio library plates + 1 control plate of E. coli BW25113 + 9 spares = 55 plates per replicate)- 90 x 150mm Petri 'nursery' plates for rearing N2 worms (15 plates per replicate)- 60 x 90mm Petri 'maintenance' plates (10 plates per replicate)
?. [Prepare NGM agar Petri plates]
Make a total of 7L of normal Nematode Growth Media (NGM) agar (enough to fill 55 imaging plates x 6 replicates = 330 imaging plates) following the protocol, Making normal NGM for imaging plates (Cabreiro Lab)
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This is best done over 2 days, in two separate batches, to avoid problems with agar cooling during dispensing.
?. [Prepare NGM agar Petri plates]
Using the Integra ViaFill, dispense 200μL of NGM agar into each well of all of the imaging plates following the protocol, Dispensing agar into multiwell plates
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?. [Prepare NGM agar Petri plates]
Leave the plates on the lab bench (with lids on) until the agar has cooled and solidified (approximately 1 hour, timing depends on humidity).
?. [Prepare NGM agar Petri plates]
Measure the weight of 3 plates (with lids on) and record average plate weight on day of pouring.
?. [Prepare NGM agar Petri plates]
Dry the plates under a hood (or drying cabinet) until the plates lose between 3-5% of their original plate weight (with lids on).
?. [Prepare NGM agar Petri plates]
Store the imaging plates upside-down at 4°C until used for experiments.
Imaging plates can be stored for up to 8 weeks at 4°C.
?. [Prepare NGM agar Petri plates]
Pour, following the protocol for Plate pouring (dx.doi.org/10.17504/protocols.io.6bhhaj6):- 35ml NGM agar into each of 60 maintenance (90mm) plates- 50ml NGM agar into each of 90 nursery (150mm) plates
?. [Prepare NGM agar Petri plates]
Leave under a hood until dry, then store upside-down at 4°C until used for experiments.
?. [Seeding Petri plates + worm maintenance]
Place the inoculation in a shaking incubator at 37°C at 200 rpm and leave to grow overnight. (Friday, -11 days)
?. [Seeding Petri plates + worm maintenance]
Using aseptic technique, seed the 10 maintenance plates each with 400μL of BW25113 culture(Monday, -8 days)
?. [Seeding Petri plates + worm maintenance]
Seed 15 nursery plates each with 1mL of fresh BW25113 culture. Leave under hood until dry.(Wednesday, -6 days)
?. [Inoculating from frozen stocks (96-well)]
Remove overnight cultures from incubator and store at 4°C until seeding imaging plates later in the day.(Friday, -4 days)
?. [Inoculating from frozen stocks (96-well)]
Remove overnight cultures from the incubator and repeat steps   to  to to inoculate a second round of overnight cultures from yesterday's culture plates. NB: These 'twice removed' cultures from frozen stocks will be used to seed imaging plates for the experiment. (Thursday, -5 days)
?. [Seeding imaging plates (96-well)]
Seed 10μl of bacterial culture from the wells of each of the 46 overnight culture plates into the corresponding wells of each of 46 imaging plates using the Integra ViaFlo.(Friday, -4 days)
Ensure correct plate orientation under the Integra ViaFlo, with well A1 in the top left corner!
?. [COPAS worm-sorting and Hydra tracking (96-well)]
Remove 10 seeded imaging plates from 4°C, and dry for 30 minutes under a hood.(Tuesday, Tracking Day)
?. [COPAS worm-sorting and Hydra tracking (96-well)]
Remove 3 nursery plates from the incubator and wash the worms off the plates into two 15ml Falcon tubes using approximately 10ml sterile PBS 'A' buffer. (Tuesday, Tracking Day)
?. [COPAS worm-sorting and Hydra tracking (96-well)]
Perform the following steps in 5 consecutive batches of 10 plates at a time:(Tuesday, Tracking Day)
?. From here on, perform the following steps weekly for each experiment replicate.
?. [Repeat x 5]
Repeat steps  to  five more times to carry out a total of 6 replicates of the screening experiment.
?. [COPAS worm-sorting and Hydra tracking (96-well)]
Check tracking checklist to ensure that all videos have been saved correctly: '/Volumes/behavgenom$/Documentation/Protocols/analysis/tracking-checklist-20210210.docx'
?. [Prepare NGM agar Petri plates]
Make a further of 7L of normal Nematode Growth Media (NGM) agar following the protocol, Making normal NGM for imaging plates (Cabreiro Lab)