LAB 3

Squashing

  • This technique is used for chromosome counts and examination of mitotic structures.
  • For this purpose onion bulbs are grown in bottle filled with water. The root tips of the roots grown in bottle are used for the squash preparation.

Squash by Feulgen method:

The method consists of following steps:

(1) Root germination:

  1. Select a large onion bulb with undamaged disc-like stem.

2. Under a running tap, gently rub the stem to remove old, withered roots and adhering soil particles.

3. Place the bulb on a glass jar of suitable size filled with tap water so that the base is completely immersed in water or alternatively Onion bulbs arerooted in moistened vermiculite at room temperature until 3- 4 cm long.

  1. Change the water in the jar twice daily.
  1. When the adventitious roots have grown to 2-3 cm long, cut and fix the distal 1-2 cm of the roots in a vial containing freshly prepared fixative such as acetic alcohol.

(2) Killing and Fixaton:

  1. Acetic alcohol is prepared by mixing 1 part glacial acetic acid and 3 partsabsolute ethanol. It should be mixed just before using for fixing purpose. Acetic acid has a swelling action and the alcohol has a shrinking action on the protoplasm.
  1. Shake the contents of the vial. Replace the used fixative with equal volume of freshly prepared fixative. Allow it to stand at room temperature for 24-48 hours. After 24-48 hours pour off the fixative, and add 70% alcohol to the vial and shake.
  1. Replace with 70% alcohol, repeat twice.

(3)Hydrolysis:

Roots are then transfer to 1N HCl for 10-12 minutes at 60 ˚C. HCl

is important in aiding the penetration of the stain by macerating the tissues. Rinse the root tips with distilled water and transfer to a clean vial.

(4)Staining:

  • Add the Feulgen stain and place the root tips in the dark. Staining will take between 10-20 minutes.
  • Once the root tips have stained dark purple excise the tip and place in a drop of 45% acetic acid on a slide.
  • Macerate carefully and apply a cover slip. Feulgen stain will fade over time so be sure to place your remaining root tips back in the dark.

Procedure:

  1. Dissect root tips of the onion bulb in a drop of 45% acetic acid.
  1. Chop the material with a scalpel; place a cover slip over the tissue/material.
  2. Place paper towels on the cover slip.
  3. Tap and apply vertical pressure through thumb.
  4. The squash preparation is ready.
  5. Check your slide carefully for a good metaphase cell. Remember the more detail at 40X the easier it will be to draw.

Mitosis:

Mitosis results in two new cells with genetic material that is identical to that of the originalcell.

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Exercise 3

Prepare the squash of the given material.

Observe the slide under the microscope and draw the diagram on the observation sheet.

Exercise 3

Observations and Results

Name:______

I.D No.______

Date:______Group______

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