Theory of practical
Lecture 1
The life cycle of a C. elegans worm takes 4 days. One worm can have 300 children.
- The eggs develop inside the worm, but develop further outside the worm
- L1; By bleaching (starvation) in this stage, all the worms can synchronize
NGM agar – Nematode Growth
Medium
- Agar,
- Bactopeptone
- Nacl
- dH2O
After autoclaving and melting in the microwave,
the last chemicals need to be added:
- MgSO4
- CaCl2
- Phosphate potassium buffer
- Cholesterol
E. coli OP50
- Is an uracil-deficient strain of bacteria (uracil tekort)
- Is streptomycin resistant
- liquid cultures, are stored at 4 °C
- Seeden met ± 150 uL bacterial culture, needs 1-2 days to dry
Bleaching – Protocol
* use glass pipettes to prevent worms sticking to plastic pipettes
* Bleach the gravid worms to release eggs – The bleach dissolves the cuticle but leaves the
eggs behind as the eggshell is thicker (Prepare bleaching solution freshly)
* Overnight hatching of eggs in M9 buffer – L1 diapause (Wash 3 times with M9 after
bleaching) (Shake vigorously in the solution for no more than 3 minutes)
* Transfer L1 worms to a plate with food – transfer the L1’s into an Eppendorf, spin down and
remove the supernatant
Waste
- Pipette tips and worm plates in the biohazard bags – send for autoclaving at the end of the
day
- All glass – even if they have worms on/in it – should go in the glass waste bin. (Gets
autoclaved eventually)
Lecture 1
The life cycle of a C. elegans worm takes 4 days. One worm can have 300 children.
- The eggs develop inside the worm, but develop further outside the worm
- L1; By bleaching (starvation) in this stage, all the worms can synchronize
NGM agar – Nematode Growth
Medium
- Agar,
- Bactopeptone
- Nacl
- dH2O
After autoclaving and melting in the microwave,
the last chemicals need to be added:
- MgSO4
- CaCl2
- Phosphate potassium buffer
- Cholesterol
E. coli OP50
- Is an uracil-deficient strain of bacteria (uracil tekort)
- Is streptomycin resistant
- liquid cultures, are stored at 4 °C
- Seeden met ± 150 uL bacterial culture, needs 1-2 days to dry
Bleaching – Protocol
* use glass pipettes to prevent worms sticking to plastic pipettes
* Bleach the gravid worms to release eggs – The bleach dissolves the cuticle but leaves the
eggs behind as the eggshell is thicker (Prepare bleaching solution freshly)
* Overnight hatching of eggs in M9 buffer – L1 diapause (Wash 3 times with M9 after
bleaching) (Shake vigorously in the solution for no more than 3 minutes)
* Transfer L1 worms to a plate with food – transfer the L1’s into an Eppendorf, spin down and
remove the supernatant
Waste
- Pipette tips and worm plates in the biohazard bags – send for autoclaving at the end of the
day
- All glass – even if they have worms on/in it – should go in the glass waste bin. (Gets
autoclaved eventually)
