Biology Reference
In-Depth Information
Platinum wire pick: 2.5 cm of 30 gauge platinum or 90% platinum/10% irid-
ium wire inserted into a 6 00 Pasteur pipette and heated in a flame until the glass
melts around the wire. Flat-end hobby pliers or a small tack hammer can be
used to flatten the end of the pick.
Single-depression microscope slide (3 mm)
Syringes (1 cc) with 27 gauge
1 / 2 00 needles
Method
i. Use platinum wire pick to move approximately five gravid C. elegans her-
maphrodites from culture dish to single-depression microscope slide contain-
ing M9 buffer. The number of hermaphrodites needed will depend on several
factors, including the number of embryos of an appropriate age desired and the
gravidity of the worms being used.
ii. Holding one syringe and needle in each hand, place one on either side of a
hermaphrodite and draw flat sides of tips of needles across each other to cut the
worm in half transversely. The embryos will be released from the halves of the
hermaphrodite. Use eyelash brush to carefully prod halves to expel any remain-
ing embryos. It is important to cut as close to the vulva as possible to release
newly fertilized embryos in the uterus. This step can also be conducted by
cutting the worm in half with a #15 curved blade scalpel ( Fig. 1 ).
iii. Sort embryos using eyelash brush and brush together into group of approxi-
mately ten embryos. Embryos will tend to stick slightly to each other when
grouped. If one desires a certain stage of embryogenesis, it is at this point that
embryo stage should be assessed and sorted appropriately. Two-cell stage
embryos are the easiest developmental stage to collect.
iv. Using colored laboratory label tape, tape two microscope slides parallel and
one slide width apart on the laboratory bench. Place a third slide between the
two taped slides. Using a 6 00 Pasteur pipette, place three to four drops of molten
5% agar onto the middle slide. Immediately lay fourth slide perpendicular to
other three slides over agar and press over taped slides to flatten agar before it
cools.
v. Once agar has set up, use a razor blade to trim excess agar from edges of the
slides. Solidifed agar in the slide ''sandwich'' can be left assembled until
embryos are ready to be added to the pad. Carefully slide the untaped slides
apart so agar pad is left in center of one slide.
vi. Heat glass 50 m L pipette in flame. Once glass is soft and fluid, remove from
flame and quickly pull apart ends. Break two ends apart to create a pipette with
a tapered end with a diameter of approximately 40 m m. Place pipette in mouth
pipette aspirator.
vii. When ready, carefully slide the untaped slides apart so agar pad is left in the
center of one slide. Using mouth pipette, transfer the grouping of embryos
(from step iii) and approximately 20 m L of M9 to the corner of agar pad on the
microscope slide.
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