Materials Included In Kit

Additional Materials Required

Prelab Preparation

Special care is required in growing the Drosophila larvae. One of the variables critical for success in obtaining large, firm chromosomes is the selection of well-fed larvae raised at a low temperature (16–18 °C). Crowded culture bottles should be avoided—it is best to transfer the parents every second day, so that no single bottle will contain too many eggs. Salivary glands should be taken from fully grown larvae. (If no paper has been placed in the bottles, the larvae preparing to pupate will crawl out of the food onto the sides of the bottle, where they can then be collected.)

It is suggested that many culture bottles of Drosophila be prepared and made available for this laboratory. At lower temperatures it will take a week to ten days to get 3rd instar larvae.

Disposal

Aceto-carmine solution can be disposed of according to Flinn Suggested Disposal Method #24a. Please consult your current Flinn Scientific Catalog/Reference Manual for proper disposal procedures.

Teacher Tips

• Enough materials are provided in this kit for 30 students working in pairs or for 15 groups of students. The laboratory can be completed in one 50-minute class period.

• The technique of teasing the salivary glands from a mature Drosophila larvae is not easily described. With practice and familiarity with larvae anatomy, the ability to isolate the salivary glands will improve. Let students practice until they are successful in locating chromosomes.
• Consult a histology manual for techniques to prepare “permanent” slides of salivary-gland chromosomes.

Introduction

Chromosomes are very small structures and are visible only during certain phases of the cell cycle. The salivary gland chromosomes of Drosophila virilis are large and relatively easy to see. Make a slide and examine these large chromosomes.

Concepts

• Drosophila larvae

• Chromosome banding

Background

The salivary gland chromosomes of Drosophila virilis are large and incapable of division. Their centromeres are united in a region called a chromocenter. The maternal and paternal chromosomes in each pair are so closely appressed that a pair of arms appears as one and makes them more visible. The large and consistent size of salivary gland chromosomes has made them a regularly studied genetic structure. With practice, the consistent preparation of well-stained chromosomes can be obtained. Though not as clear as textbook photographs, salivary gland chromosomes can be recognized by their characteristic banding patterns.

Materials

Safety Precautions

Aceto-carmine stain is a vital stain and will stain clothing and skin. It is corrosive to body tissue and moderately toxic by ingestion. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron.

Procedure

1. Select a third instar larva, for which the cuticle has not yet hardened, from a culture of Drosophila virilis. Place it in a drop of saline solution on a slide.
2. Place the slide on the stage of a dissecting microscope and view the larva with low power. Grasp the anterior of the larva with a fine-point forceps and pin down the posterior portion of the larva with a dissecting needle. Gently decapitate the larva by gently pulling off the head with the forceps. Discard the remainder of the larva.

3. Locate the salivary glands and their attached fat bodies. The glands are semi-transparent and attached by ducts to the digestive system. The fat bodies are white and opaque. Tease away the fat bodies and discard.
4. Place a drop of aceto-carmine stain on the slide next to the saline and move the salivary glands into the stain using a dissecting needle. Blot away any excess saline solution. Stain the glands for about 3–5 minutes. Add a drop of stain if the tissue begins to dry up.
5. Transfer the glands to a clean glass slide in a drop of fresh stain.
6. Place a coverslip over the preparation. Gently squash the gland preparation as follows: Place the slide between several layers of paper toweling. Place your thumb on top of the towel immediately over the cover slip and gently roll your thumb (like taking a fingerprint) while exerting a small amount of pressure.
7. Remove the paper towels and seal the edges of the coverslip using a toothpick and petroleum jelly. Doing this will prevent the preparation from drying out too quickly.
8. Examine the slide with a microscope starting with low power. Regulate the microscope light carefully to maximize the contrast in the chromosome banding pattern. Diagram the appearance of the chromosomes and their patterns. Measure the size of the chromosomes and the bands.
9. Repeat the procedures as necessary in order to locate visible chromosomes.
10. Consult your instructor for appropriate disposal procedures.