Growing worms in liquid First, make worm food -- grow liters of bacteria
- Inoculate each of several liters (4 is a good number) of LB in 2 liter flasks with 1 ml of an overnight culture of AMA1004. Grow overnight at 37&3176;C and shake at 250 rpm.
- Harvest by centrifugation in autoclaved 500 ml bottles at 4000 rpm for 10 min. (Use Sorvall and GS3 rotor in Montelone lab.) Pool cultures so that you have one liter's worth of bacteria in a single 500 ml bottle.
- Add 25 ml of S Medium to each bottle. Resuspend by shaking at 250 rpm from 15-60 min at room temp.
- Resuspended bacteria can be stored for several weeks at 4°C. You may want to pool pellets from several liters of bacteria to save on bottles and fridge space, label the pooled bottle how many liters-worth of bacteria it contains.
Note: after step 2, the pellet can be weighed to determine the yield. We should get 15-20 g of bacteria/liter.
Now, lets grow the worms...
For a culture of mixed staged worms (mostly adults and late larvae):
For staged cultures (i.e. to make RNA for a developmental Northern):
- Add 475 ml of S Medium and 25 ml of worm food (1 liter's worth of bacteria) to a 2 liter flask.
- Hypochloride treat a large plate of worms that have just starved and add the resulting eggs to the flask. Shake at ~160 rpm at 20°C. The worms should clear the bacteria in 4-5 days.
- Progress of the worm culture can be monitored by removing aliquots and examining them under the dissecting scope, but be careful not to contaminate the culture! This is useful determining the ages of staged cultures.
Harvesting the liquid culture...
- Chill the culture on ice. Tilt the flask so that the worms collect in the "corner". Once the worms have settled (15-60 min.), aspirate off most of the media.
- Collect the worms in 50-ml centrifuge tubes, as few as you can.
- Wash 1X with 0.1M NaCl, spin 2 min at 700 rpm. Use little or no braking so that the worm pellet is not disturbed. Keeping the worms cold also helps with this. If you had to use more than 50-ml tube for culture, pool them now.
Clean the worms by floating on sucrose...
- Resuspend worms in 20 ml (total) 0.1 M NaCl, mix with 20 ml ice-cold 60% (w/v) sucrose.
- Spin for 5 min at 700 rpm. Worms should end up on top, suck them out with at wide-mouthed pasteur pipet. Keep the tube on ice and work quickly -- once the worms start to eat the sucrose they get heavier.
- Wash 2X with 0.1M NaCl, spin 2 min at 700 rpm.
- Store washed pellet at -80°C.
- Grow a liter of mixed stage worms, harvest (don't need to float) and hypochloride treat.
- eggs: use what you now have
- L1s: let eggs hatch overnight in ~200 ml S Medium w/o bacteria. The next morning add a little food (1-2 mls), harvest after about 5 hrs.
- L2-L4s: same as L1s, but add more food and wait longer to harvest. Monitor progress by examining an aliquot under the scope. L2s should take about 20 hrs after feeding is started; about 30 hrs; L4s about 40 hrs.