Chromosome Smears
SCIENTIFIC
Introduction
BIO FAX!
Chromosomes contain all of the coded information needed by organisms to make any protein it
needs. However, they are usually visible only during mitosis of the cell cycle. Chromosomes
located in the cells of the salivary glands of Drosophila virilis fruit fly larvae are large and much easier to see.
The purpose of this activity is to make a slide and examine these large chromosomes using a microscope.
Concepts
• Drosophila larvae
• Chromosome banding
Background
The salivary-gland chromosomes of Drosophila virilis larvae begin much smaller in size. However, repeated rounds of DNA
replication without the cells dividing causes the centromeres of the chromosomes to become bundled together resulting in
“giant” chromosomes. The large reliable size of these chromosomes has made them ideal structures for study. With practice, the consistent preparation of well-stained chromosomes can be obtained. If done well, the characteristic banding patterns, commonly seen in textbook photographs, will be recognizable.
Materials
Drosophila virilis culture with 3rd instar larvae
Dissecting needle
Aceto-carmine stain, several drops
Microscope, compound
Vaseline®/Petroleum
Microscope, dissecting
jelly
Microscope slides
Probe
Saline solution, several drops
Small paint brush
Paraffin, melted
Toothpicks
Coverslips
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.
Please review current Material Safety Data Sheets for additional safety, handling, and disposal information. Wear chemical splash
goggles, chemical-resistant gloves, and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory.
Preparation
Special care is required in culturing Drosophila larvae. The most critical variable to the success of this activity is to ensure
that the larvae are kept at a temperature between 60–64 °F (16–18 °C). Also, to avoid overcrowded containers, adult flies
should be transferred to new containers with media every two days so that no single bottle contains too many eggs. Remove
salivary glands only from larvae that are fully-grown and preparing to pupate. Larvae preparing to pupate will usually crawl
out of the food and onto the sides of the container where they can be easily collected. (However, if paper is present inside
the container, the larvae will not do this.)
Procedure
1. Obtain the culture of D. virilis. Select a larva whose cuticle has not yet hardened. These larvae will generally be found
out of the food and on the sides of the container. Put a drop of saline on a microscope slide and place the the larva in
the drop.
2. Place the slide on the stage of a dissecting microscope and view the larva under low power. While looking through the
microscope, grasp the anterior end of the larva (see Figure 1) with fine-point forceps and hold down the posterior por© 2016 Flinn Scientific, Inc. All Rights Reserved.
Publication No. 10378
061616
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Chromosome Smears continued
tion with a dissecting pin or needle. Gently decapitate the larva by pulling off the head with the forceps. Discard the rest of
the body in the garbage.
A. Lateral View
Salivary Ganglion
Gland
(Brain)
B. Dorsal View
Salivary
Gland
Stomach
Intestine
Trachea
Fat Body
Scale
1 mm
Figure 1. Mature larvae of Drosophila
3. Locate the salivary glands and the fat bodies attached to them. The glands are semitransparent and attached by ducts to the
digestive system. The fat bodies are white and opaque. Carefully separate the fat bodies from the glands and discard.
4. Put a drop of aceto-carmine stain on the slide next to the saline and move the salivary glands into the stain using the dissecting needle. Blot away any excess saline solution. Stain the glands for about three miutes. Add a drop of stain if the tissue
sample begins to dry out.
5. Use the forceps to transfer the glands to a clean glass slide in a drop of fresh stain.
6. Place a coverslip over the preparation. Gently squash the stained glands in the following way:
a. Place the slide between several layers of towels.
b. Place a thumb on top of the towel immediately over the cover slip and gently roll it, like a fingerprint, while exerting a
small amount of pressure.
c.Remove the slide from the towel, carefully hold down the coverslip with the dissecting needle, seal the edges using petroleum jelly and a toothpick.
d. Examine the slide with a compound microscope starting with low power.
e. Adjust the light carefully to maximize the contrast in the chromosome banding pattern. Diagram the chromosome’s
appearance and patterns.
f.Estimate the chromosome’s size and the band’s width by putting a clear plastic ruler under the slide. Record estimations
on the drawing page.
g. Repeat the above procedure as many times as necessary in order to locate and view visible chromosomes.
Disposal
Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review
all federal, state and local regulations that may apply, before proceeding. Aceto-carmine solution can be disposed of according to
Flinn Suggested Disposal Method #24a.
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© 2016 Flinn Scientific, Inc. All Rights Reserved.
Chromosome Smears continued
Connecting to the National Standards
This laboratory activity relates to the following National Science Education Standards (1996):
Unifying Concepts and Processes: Grades K–12
Evidence, models, and explanation
Form and function
Content Standards: Grades 5–8
Content Standard B: Physical Science, properties and changes of properties in matter
Content Standard C: Life Science, structure and function in living systems
Content Standards: Grades 9–12
Content Standard B: Physical Science, structure and properties of matter
Content Standard C: Life Science, the cell
Tips
• The technique of teasing the salivary glands from a mature Drosophila larva is not easily described. With practice and familiarity with larval 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.
Materials for Chromosome Smears are available from Flinn Scientific, Inc.
Catalog No.
LM1129
C0259
Description
Drosophila virilis Culture
Aceto-carmine Solution
Consult your Flinn Scientific Catalog/Reference Manual for current prices.
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© 2016 Flinn Scientific, Inc. All Rights Reserved.