26. Preparation of giant chromosomes squash

70.........Practical work
26. Preparation of giant chromosomes squash
Chromosomes of certain insects, notably those
in the salivary glands of bloodworm
(Chironomid larvae), are usually large, and if
appropriately stained can be seen to be
banded. Relations between crossover data and
the positions of the bands suggest that the
bands correspond to specific gene loci.
Procedure
1.
Identification of the head and tail regions
of the bloodworm
•
Place a bloodworm on a slide with a
drop of water or saline solution.
•
Observe the bloodworm under the
lowest power of the microscope
(preferably a stereomicroscope). The
small head is brownish in colour with
a few simple eyes and movable
mouthparts. The tail region is
characterized by the presence of tufty
gills.
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2.
Removal of salivary glands
•
Hold the bloodworm firmly at a short
distance behind the head with a pair
of extra fine forceps.
•
Then, with a downward and forward
movement of another pair of forceps,
pull the head end and separate it from
the rest of the body.
•
The salivary gland will generally be
drawn out completely with the head
(Fig.1) and can easily be recognized
as two flat and whitish structures,
attached to the anterior of the gut,
Fig.1 Salivary glands (x25)
with large cells and prominent nuclei
(Fig.2).
Fig.2 Salivary glands (x100)
3.
Staining and squashing of salivary
glands
•
Add a drop of aceto-orcein stain on
the slide.
•
Leave the gland in the aceto-orcein
stain without covering for about 20
minutes. If the gland begins to dry
up, add another drop of stain.
•
Place a cover slip over the gland in
the drop of stain.
•
Lay a paper towel on the desk, turn
the slide over, and lay it face down on
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the towel.
•
Press firmly on the back of the slide
with the tip of the finger, so as to blot
thoroughly.
•
The gland can then be squashed as
follows:
Place the slide on the desk with cover
slip on top, hold the edge of the slide
steadily with one hand, and press on
the cover slip intermittently with the
wooden handle of a dissecting needle.
4.
Observation of the giant chromosomes
under the microscope
•
Place the slide under the
microscope. Increase the contrast of
the field of view by adjusting the
diaphragm of the condenser and
observe the chromosomes under
high power magnification (Fig.3).
Fig.3 Giant chromosomes in five different nuclei of the salivary gland (x400)