Modified from The Cell Lab Cookbook, by Susan Fleming, Science Museum of Minnesota, 2002
For nearly a century, researchers have used Drosophila (or the "fruit fly") to study the rules of genetic inheritance and to understand the development of complex organisms. Many of us are familiar with the genetic crosses carried out in college biology classes producing stubby-winged, white-eyed or leg-headed progeny. Recently, the entire Drosophila genome sequence was determined and vast molecular similarities were found between flies and humans. Many of the genes implicated in human disease are closely related to fruit fly genes.
The genetics of Drosophila were first studied in 1910, when Thomas Hunt Morgan adopted the fruit fly as one of his research subjects. It wasn't until the1930s however, that polytene chromosomes were characterized and detailed genetic maps identifying locations of specific genes were constructed. Polytene chromosomes, especially those found in the salivary glands of larva, are massively thick chromosomes, which can easily be seen under a microscope. As the fly larva grows, the number of cells remains the same, but nuclei continue to replicate the DNA hundreds and hundreds of times, in anticipation of needing more gene product. The replicated DNA strands do not separate but stay attached to each other forming a thick, polytene chromosome. These chromosomes have a pattern of light and dark bands, like a bar code, which correspond to specific gene sequences. Any large genetic deletions, mutations or rearrangements can be identified by observing the polytene chromosomes and correlating this information to observed genetic traits. Fruit fly genes can be easily manipulated and analyzed and could prove to be powerful tools in medical science.
Polytene chromosomes are prepared from salivary glands dissected from live (squirming) larva cultures. The glands are stained, squashed to release the chromosomes and observed using a clinical light microscope. The procedure is not technically difficult but a certain level of manual dexterity is required to complete the experiment.
- larval culture of Drosophila virilis
- petri dish
- 0.7% saline solution
- dissecting microscope
- glass slides
- glass cover slips
- chromosome stain (2% aceto-orcein)
- plastic droppers
- paper towels
- light microscope
- prepared chromosome slides
- dissecting needles
- Place one drop of saline solution on the inside cover of a petri dish.
The salt in the saline causes the larva cells to swell, making the chromosomes easier to see.
- Check to make sure that there are no flies in the Drosophila culture tube by turning it around and examining the inside.
The larvae are the white worm-like organisms feeding on the culture medium and crawling up the side of the tube.
- Remove the foam plug from the culture tube.
- Using a tweezers, remove a large larva from the Drosophila culture tube.
For best results, select an active larva that has moved up the side of the culture tube but has not started to pupate or turn brown. Large larvae are easier to dissect.
- Place the larva in the drop of saline on the petri dish.
- Return the foam plug to the culture tube.
- Put the petri dish on the dissecting scope. Move the dish around with your hands until the larva is in view. The larva is still alive and may move.
- Orient yourself with the head, which contains the black "cone-like" mouthparts. The salivary glands are located just behind the mouthparts.
- Dissect the larva by using a tweezers to pinch the head, just below the mouthparts, and another tweezers to pinch the middle.
- Carefully separate the head from the body by pulling the two tweezers apart and dragging the body out of your way.
The salivary glands are two clear, bead-like structures attached to the head and may be surrounded by dark globs of fat.
- Try to remove as much fat as possible without injuring the salivary glands.
- Hold the head in place with a tweezers and use a dissecting needle to tease away the fat.
- Use a plastic dropper to transfer the head and the glands to a glass slide. Try to minimize the amount of saline transferred with the sample.
You should be able to see the larva head and glands as a white mass on the slide.
- Place two or three drops of chromosome stain on top of the sample and wait three minutes.
- While you wait you can observe the cell nuclei as they take up the stain. Carefully place a glass cover slip over the sample and place the slide on the dissecting microscope stage.
- Once the stain has been absorbed, the salivary glands will appear dark red and you may see speckles.
The speckles are the chromosomes inside the nuclei.
- After the glands have absorbed the stain, place the slide onto a clean paper towel.
- Fold the paper towel over the slide, covering it completely.
- With moderate, even pressure, roll the pencil back and forth over the slide several times.
The pressure exerted when rolling the pencil over the slide squashes the salivary gland cells, releasing the tightly coiled chromosomes from the nuclei like a spring. If too little pressure is exerted the cells will not be broken, but too much pressure will fracture the DNA.
- You are now ready to view the chromosomes using the compound light microscope. Start at low magnification.
Be patient and scan slowly through the slide. Look for dark staining spots corresponding to cell nuclei that have not been squashed completely. The chromosomes will look like very thin threads at this magnification.
- Once you find an area that appears to contain chromosomes, increase the magnification.
The chromosomes have a pattern of light and dark bands, which, like a bar code, are unique for each section of the chromosome.
- Scanning slowly, you should find several chromosomes on your slide.
- Do not be discouraged if your slide does not come out perfectly the first time. This activity can take some practice. Have fun and be patient. This activity is very rewarding.
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