Preparation of Polytene Chromosome

Object:- To prepare Polytene Chromosome

Preparation of Polytene Chromosome 

Material Required :- Larva, Equipment and other supplies, Solutions (Poels' salt solution,Aceto-methanol,50% Glacial acetic acid, Aceto Orcein (2%), Transparent nail polish, Cleaning of slides and coverslip

Procedure:- 

1. Take late third instar larvae (about 5 day old if grown at 24°C) from a healthy culture (late third instar larvae crawl out of the food medium and move actively on surface of food or food-free surface of the Petri dish/ vial/ bottle), wash them free of adhering food particles etc with water and transfer to a clean slide in a drop of Poels' salt solution.

2. Keep one pair of fine forceps and/or dissecting needle at the middle of the larval body and the other at the mouth region. Pull them in opposite directions so that all internal organs are forced out of the larval cuticle. Salivary glands are seen as a pair of whitish translucent elongated structures connected at their anterior ends with a common salivary duct. Remove fat bodies adhering to glands very carefully, Persistence of some small regions of fat bodies with salivary glands however would not seriously affect the quality of the final chromosome preparation.

3. Using tips of the dissecting needles, transfer the cleaned salivary glands (typicallyone pair) to a drop of Poels salt solution on a clean slide. Drain out the salt solution by keeping the slide in a slanting position without letting the glands dry. Continuing to keep the slide in slanting position, add drops (drop-by-drop) of freshly prepared fixative over a period of 45-60 sec. Wipe out excess fixative with a piece of filter blotting paper

4. Immediately, thereafter, keep the slide fiat on the table and, before the remaining small volume of fixative dries, add two small drops of Aceto-Orcein and one drop of Aceto- Carmine stain and cover the slide with a watch glass to avoid drying of the stain solution. Let the glands be stained for about 5 min (only Aceto-Orcein stain can also be used).

5. Drain out the stain and add a few drops of 50% acetic acid to remove excess stain. Remove the coloured acetic acid solution by slanting the slide so that the acetic acid drains down in a waste-collection dish Finally place a drop of fresh 50% acetic acid on the glands, which should appear brick-red in colour, and cover them with a clean 22 mm² coverslip.

6. For squashing, keep the slide with its coverslip between folds of a clean filter blotting paper and lightly tap the paper immediately above the coverslip area either with the rubber- end of a pencil or with the blunt end of the needle-holder or with the needle tip, while holding the coverslip in position with thumb and a finger of one hand placed over the filter paper such that they press on two diagonal corners of the coverslip. After the tapping continue to hold the coverslip in position over the slide and apply firm pressure with thumb of the other hand on the coverslip. This act of squashing spreads the polytene chromosome arms of a nucleus and makes them flat in one plane. The "optimum thumb-pressure is learnt with experience.

7. After squashing, seal the coverslip with nail polish and observe under a microscope. Note: These preparations will be usable for a few days only, temporary preparations will last longer if after the 50% acetic acid washing step, a drop of Lacto-Aceto-Orcein is placed over the stained glands and then squashed as above).

Observation :-

1. Scan the slide under low power objective and count the number of polytene nucle visible on the slide.

2. Examine the well spread polytene nuclei under 20 X or 40 X objective to count the number of long chromosome arms in each. 

3. Identify regions that are puffed