Day -1Edit

(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 0Edit

  • 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 1Edit

  • 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-endEdit

  • 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 presentEdit

(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