Exercise II Fruit Fly Salivary Gland Chromosomes
Each student will remove the salivary glands from Drosophila melanogaster and prepare amicroscope slide presentation of the polytene chromosomes. Obtaining a good salivary gland chromosome squash requires skill, patience, perseverance and luck. You may repeat your attempts to remove the salivary glands and make a chromosome preparation until successful (or until there are no more larvae or class time remaining). Your instructor does not have to dothis exercise but, as usual, reaps the benefit of the students' efforts and successes. Prior to doing this lab exercise you should watch the video presentation.
Materials Needed
Drosophila video
Living Drosophila melanogaster larvae
Dropper bottles of
0.7% saline
aceto-orcine stain
Probes
Forceps
Clean slides and cover slips
Compound light microscope
Stereoscopic (dissecting) microscope
Procedure
This procedure has been adapted from, Life Science: A Laboratory Manual, by Glenn Powell,
Bellevue Community College
1. Prepare a clean slide with 2-3 drops of 0.7% NaCl (saline) and put the slide (without a
cover slip) on a stereoscopic (dissecting) microscope.
2. Select a large Drosophila melanogaster larva and place it on the slide.
3. While looking through the microscope use probes or forceps to grasp the larva by its
midsection just behind its jaws.
4. Gently stretch the larva by pulling on it until its head separates from the rest of its body.
5. Look for the salivary glands in the head section. The glands are very small, fairly
transparent, usually paired and have dark fat particles attached. Remember, this takes patience, luck and skill; qualities Biology 211 students have in abundance!
6. When you have located the salivary glands, separate them from the rest of the fruit fly tissues. Once you are certain that you have successfully done this, you may dispose of the rest of the larva appropriately. Keep the salivary glands moist with saline at all times.
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7. Add 2-3 drops of aceto-orcein stain to your Drosophila salivary glands. If the slide is too "messed up" from the separation of the salivary glands from the rest of the larva, you might transfer the glands to a clean slide, using a forceps or probe, prior to adding the aceto-orcein stain.
8. Without a coverslip, put the slide on the stage of the compound microscope. Use the scanning objective lens. Have your instructor verify that you have the salivary glands if you have not previously done so.
9. Remove the slide from the microscope and set it on the table. Allow the glands to stand in the stain for 10-15 minutes. Do not let the slide dry out. Add more stain if needed.
10. After the stain has set, get two paper towels and place your slide on one of them. Put a
coverslip on the slide (on top of the salivary glands). Fold the second paper towel and place it on top of the coverslip.
11. Place your thumb on the paper towel over the coverslip and press down slowly and firmly, rocking your thumb back and forth a few times. Use sufficient pressure but do not allow the coverslip or slide to slip or move.
12. Examine your stained, squashed salivary glands using the medium power objective lens. Look for nuclei and chromosomes. After you have located chromosomes, use the high Power objective lens to see details of the chromosomes.
Removing the salivary glands:
- Remove a large larva from the stock. Larger larvae are easier to dissect. However, select an active larva and one that has not started to pupate.
- Using the stereomicroscope, dissect the larva by placing one teasing needle on the posterior aspect of the larva and the other needle at the anterior end, near the black mouth parts. (Diagram 1)
Drosophila larva: Internal Structures
- Carefully pull outward with the anterior needle. (Diagram 2)
Procedure for removing Drosophila salivary glands
- There are two transparent salivary glands located anteriorly in the larva. The glands are characterized by a granular, bead-like appearance. A narrow, white ribbon of fat surrounds the glands and should be torn away.
- Discard all of the larva except for the salivary glands.