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. Practical work.........71 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 72.........Practical work 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)
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