Growth and Reproduction Assay

Day -1
(solutions can be made earlier)
 * prepare S-media (add the following to a 100mL bottle of S-basal)
 * 1mL Potassium Citrate
 * 1mL Trace Metals Solution
 * 300uL Calcium Chloride
 * 300uL MgSO4
 * Bleach 8-10 NGM plates full of gravid N2s and grow overnight in S-media to stage L1s
 * Also grow 2 bottles of OP50 in LB

Day 0

 * Concentrate 200mL OP50 culture
 * spin at 4000rpm for 5 minutes in 50ml conical tubes in tabletop centrifuge
 * pour off supernatant and resuspend in 20ml S-media for 10xOP50
 * dilute 1:5 for 2xOP50 and again 1:5 for 0.4xOP50
 * Add 2ml of appropriate OP50 concentration to wells in a 12 well tissue culture plate
 * Store remaining OP50 at RT, but best to make fresh each day
 * add approximately 2000 L1 N2 worms to each well
 * spin L1s at 2000rpm for 2 minutes, remove supernatant, resuspend in 5mL fresh S-media
 * spot 4x 2uL onto slide and count; calculate # L1s/uL
 * add appropriate volume to each flask
 * incubate in plastic box on orbital shaker tray (100-120 rpm) at 20°C
 * Photograph 20-50 leftover L1s
 * place 20-50 individuals on unseeded NGM plate
 * spread out individuals for better images
 * photograph with dissecting scope for day 0 measurements
 * also photograph a 1mm scale bar each time the zoom is changed
 * rename files to avoid forbidden characters
 * convert to 8-bit grayscale .tif files (in Fiji: Image -> Type -> 8-bit)
 * Measure with WormSizer (remember to pass/fail as needed)
 * Convert: average mass (nanograms) = 1.08 * average volume (picoliters)

Day 1

 * Wash a sample of 20-50 worms 2-3x with M9 in a 1.7ml Eppendorf tube to remove OP50
 * spinning at 3600rpm for 30 seconds (or in smaller benchtop centrifuge briefly)
 * Photograph as above for day 1 measurements

Day 2-end

 * Photograph as above
 * Count 2-hour progeny production
 * prepare 10+ unseeded NGM plates per group by adding 100uL appropriate OP50
 * place 10+ individuals from 12-well plate onto NGM plate
 * pick single individuals into droplets of liquid
 * replenish appropriate OP50 as needed, about every 30 minutes
 * after 2 hours, pick parents off plate
 * incubate plates for 2-3 days, then count progeny

Filter daily after progeny are present
(suggestion: separate protocol for filtration)
 * Place a clean 30 micron nylon filter into filter holder (and then autoclave them together?)
 * pass some M9 through filter to moisten it
 * draw up a population into a clean 3ml (or 6ml) syringe; attach to filter holder
 * place filter over 15 ml conical tube, and slowly pass the worms through the filter
 * use a few ml of M9 to rinse worms from the well and syringe into the filter holder
 * pass additional M9 through filter to wash worms through
 * connect tubing to outlet
 * place filter holder upside down over a new 15 ml conical tube
 * pass M9 through to release larger worms from filter
 * centrifuge the product to concentrate adult worms
 * check for presence of L1s and other small worms in the “adult” fraction


 * check yield and look for trapped adult worms on filter
 * if backflow is insufficient, may need to wash adult worms off filter by vortexing the filter in a 50ml conical tube half-full of M9