This document outlines the procedure for observing polytene chromosomes in Drosophila melanogaster larvae. The key steps are: 1. Dissecting the larva and separating out the salivary glands 2. Staining the salivary glands with acetocarmine stain 3. Crushing the glands under a coverslip to expose the cells 4. Viewing the polytene chromosomes under a microscope at 40x and 100x magnification. The polytene chromosomes will appear as light and dark bands that can be used to study genetics.

1. Observing Polytene Chromosomes
in Drosophila Melanogaster
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2. Methodology
and protocol to
observe
polyetene
chromosome in
Drosophila
Melanogaster

3. Protocol Overview
• This procedure involves the dissection of a Drosophila melanogaster larva
and separating the salivary glands to stain and view the polytene
chromosome present within their cells.
• In lab, we performed a similar experiment using the larvae of Chironomus.
• The methodology used is similar.

4. Key Terms
• Drosophila melanogaster is a fruit fly that has been studied extensively by
Thomas Hunt Morgan to gain deeper insights into genetics. He chose this
species because of the relative ease in extracting salivary glands for
chromosomal study.
• The polytene chromosome is a gigantic chromosome found in larvae of many
insect species that forms by repeated stages of genetic multiplication without
cell division (endomitosis). This aggregation of genetic material helps in
excessive protein synthesis needed during metamorphosis of insects when
they develop from their larval form.
• The chromosomes show light (More RNA, less DNA) and dark (more DNA ,
less RNA) regions in addition to puff( uncoiled regions which are active genes
during protein synthesis).

5. Materials required for viewing polytene
chromosome in salivary glands of Drosophila
Materials
Drosophila larvae stock
Dissecting Needle
Forceps
Microscopic slide
Coverslip
Acetocarmine stain
Microscope

6. Procedure Summary
Step 1
• Dissect the larvae and
bring out the salivary
glands
Step 2
• Stain the salivary glands
with a few drops of
acetocarmine
Step 3
• Place a coverslip over
the aggregate of
glands and crush
them. Wait for 5-10
minutes.
Step 4
• Observe under the
microscope at
objective
magnification of 40x
and then 100x

7. Step I: Dissecting the larvae
• Take a minimum of 4 and maximum of 8-10 Drosophila
larvae on a clean microscopic slide
• One by one, place hem on a drop of water using the forceps
• Hold one with forceps such that it’s tracheal tubes are facing
upwards, use a dissecting needle to separate a portion from
the top equal to about one third of the body length. You may
have to look for two transparent salivary glands in the
dissected material. Discard the rest.
• Repeat with other larvae. When you have 4-8 salivary glands
on the slide, proceed to the next step.

8. Step 2: Applying stain on the glands
• If you have enough, separate into 1-2 glands per region to
make three regions on the slide.
• Carefully place 1-2 drops of acetocarmine or aceto-orcein
stain on each of the regions.
• Wait for a couple of minutes
• Proceed to the next step.

9. Step 3: Crushing glands to expose cells
• Place a cover slip carefully in each of the regions of the slide
where salivary glands were placed.
• Crush each cover slip against the slide with the help of your
thumb. It is easier if you keep two fingers on either side of
the coverslip, but that is tedious if you have three coverslips
per slide.
• If the crushed region appears too thick, you can flatten it
further by tapping the coverslip multiple times with the back
of a dissecting needle or cap of a pen.
• Allow the exposed cells to absorb the stain for 5-10 minutes
before proceeding to the next step.

10. Step IV: Locating the polytene chromosome under a microscope
• Place the slide under the microscope
• Locate the squashed regions under 4x and 10x objective
magnification to see where to focus.
• Once those are determined change the objective to 40x to
locate individual cells. If certain fibrous projections appear,
switch to 100x.
• With some patience and skill, it is possible to observe the
light and dark bands as shown.

11. Conclusion
• This concludes the procedure for viewing the polytene chromosome in larvae
of the fruit fly Drosophila melanogaster.
• A picture of a stained salivary gland cell of Chironomus larvae is given below.
A similar procedure was adopted for this.
Polytene Chromosome

12. Works Cited
• Lab manual: Lab 4A. Preparation of Drosophila Polytene Chromosome
Squashes.
http://web.as.uky.edu/biology/faculty/kellum/bio315/Lab%204A,%204B%20Ex
ercise-Spring%202015.pdf
• https://en.wikipedia.org/wiki/Polytene_chromosome
• https://www.scribd.com/presentation/242652985/lab3-polytene-chromosome-
ppt

13. Thank you