TITLE: CeMbio Screen 96WP
AUTHORS: Saul Moore

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Protocol for behavioural screening of </span><span style = "font-style:italic;">Caenorhabditis elegans </span><span>response to bacteria from its natural microbiome (CeMbio)</span></div></div>

[STEPS]
?. [Preparing worms]
Using an eyebrow hairpick, pick 10 L4-stage N2 worms onto each of 10 OP50-seeded 90mm petri plates 4 days prior to bleaching (e.g. on Monday if bleaching on Friday).
?. [Preparing worms]
On day of bleaching (e.g. Friday 2pm) follow the protocol for Bleach synchronisation of C. elegans:
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?. [Preparing worms]
Re-feed arrested L1 larvae approximately 72 hours prior to tracking (e.g. on Monday if tracking on Thursday), following the steps outlined in the above protocol for Bleach synchronisation of C. elegans.
?. [Preparing worms]
Store the refed L1 plates in 20°C incubator.
?. [Making LB NGM agar imaging plates (96WP)]
2 days prior to tracking (e.g. on Tuesday if tracking on Thursday), make 500ml LB NGM agar following the protocol Making LB NGM Plates:Post-autoclave salts:
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[Sodium Chloride (Sigma-Aldrich-71376-1KG)]
[Bio Agar (Biogene-400-050)]
[LB Broth Powder (Fisher. BioReagents-BP-1426-2)]
[Cholesterol, 5mg/ml in EtOH (Sigma-C1145-250MG)]
[Sterile Water]
[Calcium Chloride, 1M (CaCl2; Sigma-C3881-1KG)]
[Magnesium Sulphate, 1M (MgSO4; Fisher-M/1050/53)]
[Potassium Sulphate, 1M, (KPO4, pH 6.0; Sigma-Aldrich-P0662-500G-M)]
?. [Making LB NGM agar imaging plates (96WP)]
Using the Integra ViaFill, dispense 200ul of LB NGM agar into 96-well imaging plates, following the protocol Dispensing agar into multiwell plates:
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?. [Making LB NGM agar imaging plates (96WP)]
Leave the plates on the lab bench (with lids on) until the agar is dry.
?. [Inoculating bacterial cultures (96WP)]
3 days prior to tracking (e.g. on Tuesday if tracking Thursday), inoculate overnight cultures of bacterial strains in 96-well from 96-well CeMbio frozen stock plates, following the protocol for Growing overnight bacterial culture in 96WP: Place inoculated cultures in 96WP in a shaking incubator at 25°C, 200rpm.
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?. [Seeding bacterial lawns with ViaFlo (96WP)]
The next day (e.g. Wednesday, 1 day prior to tracking), remove overnight cultures from the shaking incubators and replace the LB in each of the wells of the inoculated 96WPs (that are reserved for the control bacterial strain) with 200ul OP50 bacterial culture that was inoculated separately.
?. [Dispensing Day1 worms onto seeded plates using COPAS (96WP)]
On the day of tracking (e.g. Thursday, if re-feeding was performed on Monday), remove seeded plates from 4°C, and dry for 30 mins under a hood.
?. [Tracking using Hydra rigs]
After acclimation under the Hydra rigs, record worm behaviour on the bacterial food for 15 minutes (25fps, exposure 25000msec, bluelight stimulation).
?. [Tracking using Hydra rigs]
Record 15 minute videos at the following timepoints: 1, 3, 5, and 24 hours on food.
?. [Tracking using Hydra rigs]
After tracking, discard the plates in a biological waste bin.
?. [Inoculating bacterial cultures (96WP)]
Separately inoculate 100ml E. coli OP50 bacteria in an Erlenmeyer flask and place in a shaking incubator at 37°C, 200rpm.
?. [Seeding bacterial lawns with ViaFlo (96WP)]
Remove imaging plates from 4°C storage and record the average weight of 3 of the plates.
?. [Seeding bacterial lawns with ViaFlo (96WP)]
Place seeded plates to dry under a hood for about 30 minutes.
?. [Seeding bacterial lawns with ViaFlo (96WP)]
Store the bacterial cultures at 4°C until seeding bacterial lawns later that day.
?. [Seeding bacterial lawns with ViaFlo (96WP)]
Once imaging plates are dry, remove bacterial cultures from 4°C and seed 10ul bacterial culture into 96-well imaging plates, following the protocol Seeding bacterial lawns using Integra ViaFlo (96WP)
?. [Making LB NGM agar imaging plates (96WP)]
Measure the weight of 3 plates (with lids on) and record average plate weight on day of pouring.
?. [Making LB NGM agar imaging plates (96WP)]
Store the plates upside-down at 4°C (plates can be stored for up to 1 week prior to use).
?. [Seeding bacterial lawns with ViaFlo (96WP)]
Place the imaging plates in a drying cabinet for approximately 2-3 hours (until the plates have lost approximately 3-5% of their original weight).
?. [Dispensing Day1 worms onto seeded plates using COPAS (96WP)]
Dispense Day1 worms onto seeded plates for imaging, following the protocol Using the worm sorter (COPAS):
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?. [Seeding bacterial lawns with ViaFlo (96WP)]
Leave seeded plates on the lab bench (with lids on) for 4 hours to allow lawns to grow.
?. [Preparing worms]
Keep the tube with bleached worms on a rotator in an incubator at 20°C until re-feeding (maximum 5 days post-bleaching, as behaviour is not consistent after this time frame).
?. [Dispensing Day1 worms onto seeded plates using COPAS (96WP)]
Remove worms on 90mm Petri plates from 20°C incubator, and follow the protocol Preparing worms for the COPAS (wormsorter):
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?. [Seeding bacterial lawns with ViaFlo (96WP)]
Store seeded plates at 4°C overnight.
?. [Dispensing Day1 worms onto seeded plates using COPAS (96WP)]
Leave plates to dry under a hood for 30 minutes.
?. [Dispensing Day1 worms onto seeded plates using COPAS (96WP)]
Place plates in the imaging cave under the Hydra rigs to acclimate for 30 minutes prior to imaging (+1hr timepoint).