Biology Reference
In-Depth Information
6. Days 11-13: Growth of the culture should be carefully followed (see Section
III C.) and worms harvested when majority are adults. The culture will take
about 3-3.5 days depending on age of worms on starter plates and the desired
state of the final culture.
Note: The bacterial food for liquid culture can be obtained from fermentor facil-
ities. However, we have had contamination as well as growth problems with exter-
nally supplied food and, consequently, prefer growing up our own bacterial cultures.
C. Monitoring Worm Cultures
Worm liquid cultures should be monitored under a dissecting microscope once a day.
This allows assessment of the developmental stage and health of the worms, ensures
that worms have enough food, and confirms that there is no significant contamination.
1. In a laminar flow hood transfer 1 mL of the culture with a sterile pipet into 1.5 mL
microcentrifuge tubes.
2. Spin in microcentrifuge 600g for 3 min.
3. Carefully pipet 20 m L worm slurry using a cut-off tip onto a glass slide. Place
cover slip on top and look at the worms under dissecting microscope.
D. Harvesting Worms from Liquid Cultures
Worms are harvested by settling under gravity and cleaned by flotation on a
sucrose cushion, which separates healthy worms from bacteria and debris ( Fig. 4 ).
The sucrose floated worms are washed and used to isolate embryos.
1. When the majority of worms in the culture have 10-15 embryos, transfer the
cultures into 500 mL graduated cylinders. Settle worms in ice water bath for 1 h.
2. Aspirate off media using a sterile 5 mL pipet and transfer slurry (brown film at
bottom) to two 50 mL conicals per L culture.
3. Pellet in centrifuge at 600g for 3 min with slow deceleration.
4. Aspirate off supernatant, collect worms into 50 mL conical, and add cold M9.
5. Pellet in centrifuge at 600g for 3 min and aspirate off supernatant.
6. Resuspend pelleted worms by adding cold M9 to the 25 mL mark. To this add
25 mL cold 60% (w/v) sucrose. Mix and centrifuge immediately at 1500g for 5 min.
7. After the spin, adult worms will form a layer at the top of the tube. Remove adults
down to the 35 mL mark with 5 mL pipet and transfer to a new conical.
8. Wash worms by adding cold M9 to 50 mL mark. Pellet the worms at 600g for
3 min and carefully remove supernatant.
Note: It is important to work rapidly during the sucrose flotation step. Do not leave
the worms for too long in sucrose and wash them out of sucrose rapidly after
collection from the top of the cushion.
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