Extraction of polytene chrmosome

PREPARATION OF SALIVARY GLAND CHROMOSOMES OF LARVA OF DROSOPHILA

AIM: To prepare temporary mount of salivary gland chromosomes of larva of Drosophila

MATERIALS REQUIRED: Rava, Jaggery, Agar-Agar, Bottles, Cotton plug, Glass rod, Propicnic acid, third instar larvae, Needle, Forceps, Slide, Ringers fluid, lacto acetocaramine ,HCl, dissecting microscope, blotting paper, coverslip and compound microscope

PROCEDURE:

1. PREPARATION OF MEDIUM

Ø Bottles should be thoroughly washed and rinse with detergents.

Ø Bottles have to be sterilized by autoclaving it at 80 degree Celsius for twenty–four hours.

Ø Take one litre of water in vessel and add 100g of Rava, 2g of agar-agar and Jaggery into it.

Ø Boil it for few minutes.

Ø Add 2 ml of propicnic acid to the mixture to avoid fungus attack.

Ø Boil till it becomes thick. (For 15 minutes).

Ø Transfer it to the bottles later.

Ø Close the mouth with the cotton and leave it for four for solidifying.

Ø After one day wipe the water droplets present inside the bottle with the help of blotting sheet.

Ø Transfer the fruit flies in the bottles.

Ø Label date, time and your name on bottle and add two drops of yeast solution into the bottle.

Ø Keep it in incubator at temperature 21 degree Celsius for three days.

2. MOUNTING POLYTENE CHROMOSOME

·        Take a clean slide and keep it on dissecting microscope.

·        Put a drop of ringer’s fluid onto the slide.

·        Take out biggest size third instar larvae onto the slide.

·        The third instar larva is held by a forcep and with the help of needle.

·        The jaws are squeeze out from the alimentary canal by holding middle body of the larvae with one needle; another needle is put on jaws and slowly pulls out the jaws in the opposite direction.

·        Separate the salivary gland from digestive system.

·        Take a clean slide, put a drop of lacto acetocaramine stain on it.

·        Carefully transfer the salivary glands on the slide with the help of needle or pointed brush.

·        Keep it undisturbed for 20 minutes.

·        After 20 minutes, using blotting paper, remove the stain, add a drop of 1 N HCl and put a coverslip on it.

·        Cover the slide with blotting paper and gently tap on coverslip using blunt end of the pencil to make squash of salivary gland.

·        Observe under the compound microscope, if polytene chromosomes are seen, immediately seal the slide with wax.