TITLE: Microbiome Assay with 96WP Updated April 2020
AUTHORS: Priota Islam

[STEPS]
?. [-10 days]
Pick 10 L4s onto 10x90mm OP50 seeded platese.gon Monday (10 L4 per plate so total 100L4s)
?. [-6 days]
At 2pm on day of bleaching (eg Friday) follow the protocol Bleach synchronization of C elegans
?. [-6 days]
Keep the tube with bleached N2s on a rotator at 20C incubator until refeed (make sure not to exceed 5days as the worm behaviour is not consistent post this time frame)
?. [-3 days]
If tracking is intended to be performed on the following Thursday, then refeed the arrested L1s onto 4 OP50 seeded 150mm plates on the Monday (+72hrs post bleaching, at 2pm following the protocol Bleach synchronization of C elegans
?. [-3 days]
Store the refed L1 plates at 20C incubator
?. [-2days]
At least 2days prior to tracking day (e.g. Tuesday if tracking is planned on Thursday) make about 250ml no peptone NGM
?. [-2days]
Dispense 200ul of agar into each well of the 96WP using the integra viafill following the protocol Dispensing agar into multi well plates
?. [-2days]
Let the agar dry and store the plates at 4C (lid side down) until used (plates can be stored for up to a week)-Note:
?. [-2days]
Measure the weight of the plates with lids on and record average plate weight on day of pouring
?. [-2days]
Grow an overnight culture of the bacterial strain from the (shuffled) 96WP library stock plates, 2days prior to tracking (eg on a Tuesday afternoon if tracking is to be intended on Thursday) following the protocol Growing overnight bacterial culture in 96WP.
?. [-2days]
Separately inoculate OP50 and incubate at 37C overnight in a shaking incubator at 200 rpm
?. [-1day]
Take the bacterial cultures out of the incubators in the morning, and store at 4C until used for seeding later that day
?. [-2days]
Cover the plates with breathable seals and shake at 180-200rpm in a shaking incubator
?. [-1day]
Take agar-filled 96WPs out of the cold room and dry under in the drying cabinet for about 2-3hrs (Measure the weight of 3 plates before drying without the lid)
?. [-1day]
After drying measure the weight of the 3 plates again. Ensure that the plates lose between 3-5% of their original weight following drying.
?. [-1day]
Seed the dried plates with the bacterial culture grown the night before using the ViaFlo
?. [-1day]
Turn the ViaFlo ON (Make sure to book it beforehand)
?. [-1day]
Edit the protocol SAUL to have the mixing stage consisting of 15 cycles to initially mix the bacterial cultures and to resuspend each strain
?. [-1day]
Load the tips (long 12ul tips), Adjust the Z height to the one that worked best in the test runs (See lab notes)
?. [-1day]
Mix the bacterial plate using the ViaFlo
?. [-1day]
Change the tips
?. [-1day]
Edit protocol SAUL again to have mixing stage be of 1 cycle when actually seeding plates
?. [-1day]
Run the protocol- To dispense 10ul volume in each well
?. [-1day]
Change tips every time a new plate is seeded
?. [-1day]
After all the plates are seeded dry them under the hood for another 1hr
?. [-1day]
After drying put the plates in a closed box upside down in the cold room (4C)
?. [Day 0: Tracking day]
On the day of tracking prepare the worms following the protocol Preparing worms for the COPAS (wormsorter)
?. [Day 0: Tracking day]
Dispense the worms using the COPAS following the protocol Using the worm sorter (COPAS)
?. [Day 0: Tracking day]
Dry the plates for 30minutes under the hood
?. [Day 0: Tracking day]
After drying, place the plates under the Hydra rig, leave for a further 30mins to acclimatize, and then record for 15mins, following the protocol Tracking on the Hydra rigs
?. [Day 0: Tracking day]
Record each plate at four time points: 1hr, 3hrs, 5hrs, and 23.5hrs (next day). Between 1hr, 3hr & 5hr the plates are kept in the cave outside the rigs
?. [Day 0: Tracking day]
Store the plates in 20C incubator overnight between tracking 5hrs and 23.5hrs timepoint
?. [Day 0: Tracking day]
Discard the plates in the biological waste bins post tracking
?. [Day 0: Tracking day]
Record with the lids on and lid side facing up inside the rig. Makesure to wipe the lids with lint-free tissues before placing them under the rig. Also use the script that involves using blue light in the recording