There are two bleaching methods for worms. Worms can be bleached to remove contamination from a strain or to obtain a large quantity of eggs. Below a protocol for both methods can be found.
BLEACHING EGGS FOR STERILIZATION
Worm Bleach Recipe
10 mL Bleach 4 mL 10 M NaOH 26 mL Distilled Water
1) Collect worms and eggs by washing plate with 5-10 mL distilled water and transferring worm solution to 15 mL conical tubes (at equal volumes per plate).
2) Centrifuge worm tubes for 2 minutes at 1200 rpm in a tabletop centrifuge.
3) Remove supernatant and re-suspend worms in 10 mL distilled water.
4) Centrifuge worms again for 2 minutes at 1200 rpm.
5) Remove supernatant and re-suspend worms in 5 mL Worm Bleach (recipe above).
6) Incubate at room temperature about 5 minutes. Monitor progress under dissecting scope, gently tilting the tube to mix every minute or so until all adult worms are dissolved.
7) When worms are dissolved, add 5 mL M9 Buffer to neutralize reaction.
8) Spin out eggs by centrifuge for 2 minutes on 2500 rpm. Remove supernatant.
9) Change all caps on centrifuge tubes.
10) Wash eggs 3 times with 10 mL M9 Buffer. Centrifuge for 2 minutes at 2500 rpm between each wash. Change caps after each wash.
11) Re-suspend each egg sample in 1 mL M9 Buffer and transfer to appropriately labeled NGM+OP-50 plates.
12) Incubate at 20 degrees Celsius until eggs hatch and sample can be proved to be uncontaminated.
BLEACHING WORMS FOR EGGS
Worm Bleach Recipe (this recipe has been used for two strain tubes in the past)
3 mL 5N KOH (may also be labeled 5M KOH) 18 mL 10 M NaOH 33 mL dd H2O
1) Make worm bleach in a 100 mL beaker with the materials listed above.
2) Determine the number of desired eggs to be obtained.
3) Allow plates to incubate for 2 - 3 days until there are lots of eggs and gravid adults.
4) Using a 10 mL tip, use the pipette aid to add 5 mL of M9 to one plate with worms and eggs.
5) Swirl the M9 on the plate, then suck it up and add the liquid to one 15 mL labeled confocal tube tube.
a) When using multiple plates per strain, suck up the 5 mL of M9 to one plate with worms and eggs, then swirl on the plate, suck up the liquid, and add it to the tube. b) Change the tip before getting more M9 and wash all plates adding the remaining liquid to one 15 mL confocal tube per strain.
6) Spin the tube at 1000 RPM for 30 seconds. Note: the tube needs to be balanced by another tube with the sane approximate volume of liquid on the opposite side of the centrifuge AND the centrifuge run can be ended if you are using a centrifuge that can only be set to whole minutes.
7) Aspirate off M9.
8) Add 7 mL worm bleach per tube.
9) Invert to mix.
10) Spin the balanced tube(s) at 1500 RPM for 30 seconds.
11) Aspirate off bleach.
12) Add 7 mL bleach again; this time, vortex until worms are not moving and most-if not all-are fragmented at least into halves.
13) Spin the balanced tube(s) at 1500 RPM for 1 minute.
14) Fill tube(s) to 15 mL line with M9; make sure the pellet is re-suspended by tapping bottom of tube on bench or vortexing it gently.
15) Spin the balanced tube(s) at 1500 RPM for 30 seconds.
16) Aspirate off M9.
17) Replace the tube cap.
18) Repeat steps 14-16 two more times.
19) Re-suspend the pellet in about 1 mL of M9.
19) For multiple tubes of the same strain, move with a p1000 pipette to one tube per strain.
20) Place tubes on the shaker while counting in the next steps.
21) Count about 3 uL per strain of liquid on a slide to estimate the eggs per mL.
22) Use the number from the last step to determine the approximate volume required to obtain your desired number of eggs.