GRADES: 9-12

TIME NEEDED: 1-2 hours

Today we will examine polytene ("many copy") interphase chromosomes of giant cells in the salivary glands of Drosophila (fruit fly) larvae. You should be aware of the distinction between these type of chromosomes and the chromosomes of normal cells undergoing mitosis. Diploid Drosophila cells have 8 chromosomes (2 copies each of 4 different chromosomes). In the larvae of 2-winged (dipteran) insects, some diploid cells stop dividing and instead grow to giant size as the larva grows. These giant cells form much of the digestive system of the larva. We will look at the salivary gland cells, which secrete digestive juices and silk proteins to form cocoons. In the nuclei of these giant cells, DNA replication continues despite the absence of cell division, in order to maintain a normal ratio of cell cytoplasm to DNA. The many copies of each chromosome remain together, lined up parallel, closely packed. Up to 1600 copies of each chromosome can be laid out parallel. Only 4 chromosomes can be seen in these cells because the 2 copies of each chromosome are also plastered together. When the larva forms a pupa, the giant cells die and normal diploid cells (with only 2 copies of each chromosome) grow to form the adult fruit fly.

In the interphase part of mitosis, chromosomes of normal cells are not visible in the light microscope. As the chromosomes condense in preparation for mitosis, we can easily observe them by staining either for DNA or for basic protein. The polytene chromosomes of Drosophila giant cells are partially condensed all the time, but sections of them are more or less extended. By staining for DNA or basic protein, we can see bands along the chromosomes - dark where there is a high concentration of DNA and proteins, light where the DNA is more extended. There appear to be about 5,000 to 10,000 bands which can be seen in the electron microscope. The bands are characteristic in appearance, and some have been identified with specific genes using the same techniques that we will use here to identify the bands. Try to estimate how many bands you can distinguish on the chromosomes that you prepare.

Drosophila larva (purchase large 3^rd instar larvae from Wards or other vendor)

microscope slides & cover slips

dissecting needles & forceps

Ringer’s solution (Wards)

aceto-orcein stain (Wards)

compound microscope

dissecting microscope

clear nail polish

1. Select a large white larva from a Drosophila culture tube and pick it up on a dissecting needle. Don’t stab it! It will stick to the dissecting needle if gently pushed against it. Place it on a clean slide and examine it under the dissecting microscope. The black mandibles identify the head end - this is the end we want. You can try to see in which direction the larva is moving.

2. Place a drop of Ringer’s solution on the larva. Using two needles or a needle and forceps, press the back end and the head of the larva against the slide and gently pull the front needle (and head end) away to "dissect" the larva. (See diagram) The larva must be wet with Ringer’s solution or the glands will tear when you pull. When you have dismantled the larva, try to find the salivary glands. With luck they float out to either side as the larva comes apart. They are almost transparent and featureless. Don’t confuse the gut or the fat bodies with the salivary glands. The unwanted parts look like they are full of tiny glistening beads (which are droplets of fat). The salivary glands are just dull gray sacs. Use a dissecting needle to move the glands away from the carcass. Adding small drops of Ringer’s solution may make it easier to move things on the slide. Remove the carcass and try to clean off large bits of fat from the glands. Try several dissections. Some larva are more accommodating than others, and practice helps. Do not let the glands dry out at any time or the cells will burst.

3. Put a small drop of aceto-orcein stain (stains basic proteins) on the glands. Leave the stain on for 10 minutes for it to penetrate. Add more stain if the slide begins to dry out at any time. After 10 minutes, wick away any excess stain using a tissue, then place a cover slip on top of the stained glands.

4. Place a folded paper towel over the slide. To break open the nuclear membrane and spread out the chromosomes, you need to "squash" the salivary glands between the slide and the cover slip. Squash the glands by pressing firmly on top of the slide with your thumb. Be careful not to move the cover slip. If a low-power scan under the compound microscope shows only a mass of clear bubbles, try again. This means that you squashed a fat body, not a salivary gland!

5. Use the compound microscope to observe your slide under low and high magnification. You may also want to use oil immersion to see the chromosome banding more clearly. You can follow the twists of the chromosomes inside the nucleus by changing focus very slightly. Use fine focus adjustments only when you are viewing under oil immersion.

6. If your slides are too light and show poor banding, stain them a bit more. If your slides are too dark, then stain for a shorter time. This takes some practice! To make your slides semi-permanent, you can seal around the cover slip with clear nail polish.

7. Observe the pre-prepared slides of Drosophila polytene chromosomes, and compare them to your own preparations.