Squash Preparation of Polytene Chromosomes

 Title: Squash Preparation of Polytene Chromosomes 

Objective: 

To prepare and observe polytene chromosomes from the salivary glands of Drosophila  melanogaster (fruit fly) or Chironomus larvae, and to study their unique banding patterns and  structure. 

Theory: 

Polytene chromosomes are giant chromosomes found in the salivary glands of dipteran insects  like Drosophila melanogaster. They result from repeated rounds of DNA replication without cell  division, leading to the formation of large, multi-stranded chromosomes. These chromosomes  exhibit distinct banding patterns due to variations in chromatin condensation, which can be  observed under a microscope. Studying polytene chromosomes provides insights into  chromosome structure, gene activity, and chromosomal aberrations. 

Materials Required: 

∙ Drosophila melanogaster larvae or Chironomus larvae 

∙ Forceps 

∙ Needles 

∙ Microscope slides 

∙ Cover slips 

∙ Aceto-orcein stain/ Aceto-carmine stain

∙ Distilled water 

∙ Dropper or pipette 

∙ Light microscope 

Procedure: 

1. Obtain third-instar larvae of Drosophila melanogaster or Chironomus from a culture and place a larva on a clean microscope slide

2. Head portion of the larvae was dissected using a needle and remaining portion was discarded.

3. It was squashed well with a needle.

4. Carefully add a few drops of stain to the salivary glands on the slide and incubate for 5-10 minutes. This will stain the chromosomes and  enhance the visibility of the banding pattern. 

5. Gently place a cover slip over the stained salivary glands. 

6. Use the blunt end of a pencil or another smooth object to gently press down on the cover  slip, squashing the glands to spread out the chromosomes. 

7. excess stain was blot dried using a blotting paper

8. Observe the slide under a light microscope at high magnification (400x or higher).

9. Focus on the polytene chromosomes, which should appear as large, banded structures.  The bands represent regions of tightly packed DNA, while the interbands are less  condensed. 

Observations: 

∙ Polytene Chromosomes: Look for the distinct, banded pattern of polytene  chromosomes. The chromosomes may show puffing, which is an indication of gene  activity. 

Results: 

∙ The preparation should reveal polytene chromosomes with clear, alternating dark and  light bands. Any puffs or swelling on the chromosomes indicate active transcriptional  regions. 

Conclusion: This experiment allows for the observation of polytene chromosomes, providing  insight into their structure and function. The banding pattern observed is crucial for  understanding chromosomal organization and gene activity.

Precautions: 

∙ Handle the larvae carefully during dissection to avoid damaging the salivary glands. ∙ Ensure the squash is done gently to avoid breaking the chromosomes. ∙ Use fresh stains and reagents for clear visibility of the chromosome bands.