TITLE: Bleach synchronisation of C. elegans
AUTHORS: Ida Barlow

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is for obtaining synchronised populations of </span><span style = "font-style:italic;">C. elegans</span><span>. Eggs are harvested from gravid hermaphrodites and allow to grow to L1 arrest for 12-120 hours (0.5-5days). L1 larvae are then fed and left to develop to the appropriate developmental stage</span></div></div>

[STEPS]
?. [Washing]
Wash hermaphrodites off plate with several ml of M9 solution and transfer to 15ml falcon tube (Fisher Scientific-Falcon 352096)
?. [Washing]
Fill falcon tube up to 15ml with M9 solution
?. [Washing]
Centrifuge for 2 minutes at 1500 rpm (RCF:210, ascending 9; descending 7) – program 1Program 1 retains the worms as pellets and the bacteria is suspended as the supernatantThe descending is slow as the worm pellet is lose at this stage which we don't want to break
?. [Washing]
Remove supernatant using a plastic Pasteur pipette taking care not to disturb pelletLeave atleast 0.5ml M9 to avoid disturbing the pellet
?. [Washing]
Fill the tube with M9 upto 15ml
?. [Washing]
Spin program 1
?. [Washing]
Repeat steps 4-6
?. [Washing]
On final wash remove as much supernatant as possible and add M9 upto 4ml
?. [Bleaching]
Add 4ml 2X Bleach solution  (From here onwards try to work as quickly as possible to avoid over-exposure of the worms  to the bleach)USE FRESHLY PREPARED BLEACH EVERYTIME
2X Bleach solution:5% Sodium hypochlorite solution - 4mlSterile water - 3.5 ml1M NaOH solution - 2.5 mlTOTAL - 10 ml
?. [Bleaching]
Vortex on maximum setting for 4 min (no more as this will damage the eggs) Makesure the vortex forms After vortexing, top up the tube with M9 till 15ml
?. [Bleaching]
Centrifuge for 2 mins at 2500rpm (RCF:590, ascending 9; descending 7) – program 2(Always check the program on the centrifuge before using it)
?. [Bleaching]
Remove supernatant by pouring into waste bottle – pellet should be compact and yellow in colour at bottom of falcon, but be careful not to lose
?. [Bleaching]
Add 15ml M9
?. [Bleaching]
Centrifuge at program 2
?. [Bleaching]
Repeat steps 12-14 four more timesThe number of washes is crucial here as we need to get rid of all the bleach
?. [Bleaching]
After final wash add 15ml M9 and store eggs/larvae in the falcon on the rotator that is constantly spinning at 20oC, until feeding
L1 arrested larvae can be starved for up to 5 days before refeeding
?. [Feeding]
Centrifuge larvae on program 2 to pellet
?. [Feeding]
Remove supernatant with plastic Pasteur pipetteThe pellet is lose here so makesure not to disturb it
?. [Feeding]
Add 15ml M9, spin to wash
?. [Feeding]
On final wash leave 0.5ml M9 in falcon
?. [Feeding]
Resuspend the pellet by gently tapping the tube/flicking it
?. [Feeding]
Place droplet containing larvae onto seeded plate and allow to grow to desired developmental state (ie. 2 days for L4s, 2.5 days for young adults)  Use glass pipette to place the droplet onto seeded plate, avoid using plastic pipette as larvae will stick to it
Development times at 20oC:2 days for L4s2.5 days for young adultsNote:If you feed larvae within 12hrs of bleaching then they develop faster than the longer arrested onesIt is a good practice to bleach in two tubes in parallelIf you drop the tube at any point of the process, makesure to transfer the contents into a new tube as the dropped tube may get cracked resulting in loss of worms during centrifugation/vortexingAny unused larvae can be topped up with M9 and stored spinning in the rotator to be re-usedUse clean autoclaved rubber bulbs for the refeeding everytime to avoid contaminationPut the used bulb in the box labelled 'Used Teets'Note:If you feed larvae within 12hrs of bleaching then they develop faster than the longer arrested onesIt is a good practice to bleach in two tubes in parallelIf you drop the tube at any point of the process, makesure to transfer the contents into a new tube as the dropped tube may get cracked resulting in loss of worms during centrifugation/vortexingAny unused larvae can be topped up with M9 and stored spinning in the rotator to be re-usedUse clean autoclaved rubber bulbs for the refeeding everytime to avoid contaminationPut the used bulb in the box labelled 'Used Teets'