Finding Ideal bacteria concentration for 50uL per well in a 384 well plate

• looking for sparse speckles of bacteria on the well bottom when pipetted at 1.5uL
• weight of bacteria: 0.0878g in 9mL
• Exp conditions:
  • Col. 1-4, Control: 500 µL stock → mix (9.744 mg/mL)
  • Col. 5-8, Tube 1: 500 µL stock + 500 µL diluent → mix (4.872 mg/mL)
  • Col. 9-12, Tube 2: 500 µL Tube 1 + 500 µL diluent → mix (2.436 mg/mL)
  • Col. 13-16, Tube 3: 500 µL Tube 2 + 500 µL diluent → mix (1.218 mg/mL)
  • Col. 17-20, Tube 4: 500 µL Tube 3 + 500 µL diluent → mix (0.609 mg/mL)
  • Col. 21-24, Tube 5: 500 µL Tube 4 + 500 µL diluent → mix (0.3045 mg/mL)
• Will be plated with L1 worms
• group that reaches L4 the most uniformly by 10am M will be the ideal concentration to use.
• 8/12 S-complete contaminated with bacteria
  • added 200mg of strep

Adjusting plates to 50uL of worms for echo compatibility

100uL is too close to max of plate, so when flipped over it may spill which would fail the experiment, volumes must be halved

• Worm growth normal for the most part, few wells are lagging behind
• Bacteria volume added at 3uL may be slightly too much
  • most worms L3, some are late L2 early L3
    • maybe too much bacteria
  • see if worms reach L4 by tmr
    • if not lower bacteria added to 2uL
• Since volume is halved, FudR concentration may have to be lowered to half
  • Created 25x FudR and 12.5x FudR
  • testing both 7/18
  • if both sterilize and don't kill worms we are good, convert to 12.5x bc its more efficient
  • if 25x kills worms use 12.5x FuDr

Increasing liquid culture worm shelf life (success, nystatin in S-complete)

now that we have worms growing and have narrowed down the issue to the s-complete messing everything up we can identify the best place to add nystatin

• Upon next age sync, add nystatin to one of the 4 tubes and leave it on neutator over night to eggs can hatch
• plate these worms and see if they grow
• if they grow as normal we can have nystatin in our s-complete media
  • if they can hatch and grow to maturity normally when hatched in nystatin+ s-complete media that means we can add nystatin directly to s-complete when making the media
• this will increase the time we can have worms in the nutator without risking fungal contamination