V-Lab @ ANDC

Aim

Preparation of permanent mount of Allium cepa (Onion root tip) to study different stages of mitosis using acetocarmine staining technique.

Material

Onion root tips (Allium cepa)

Glassware

  1. Slides
  2. Coverslips
  3. Forecep
  4. Blade
  5. Microscope

Chemicals

  1. Acetocarmine
  2. 45% Glacial acetic acid

Principle

To study the stages of mitosis, the tissues where there are many cells in the process of mitosis would be most appropriate. The cells at the growing tip of the roots are actively dividing, and thus many cells will be in stages of mitosis (The region of mitosis is more properly called the apical meristem). It is easy to obtain, as even from one onion the roots grow in large numbers and many roots can be generated in a few days simply in a dish of water. The mitotic region (apical meristem) is easy to cut for viewing the cells which are large and distinct (real size of a typical onion root tip cell is about 0.075mm). The chromosomes of onion are large and number is sixteen which includes metacentric, submetacentric and acrocentric chromosomes and all can be observed easily.

Theory

Description of Mitotic Stages

PROPHASE

  • Chromatin begins to condense and chromosomes appear as thin threads.
  • Centrioles move to each pole of the cell.
  • Spindle apparatus is assembled.
  • Nuclear envelope begins to dissolve.
  • Nucleoli disappear.
  • Chromosomes appear distinct at the end of prophase.

METAPHASE

  • Chromosomes align themselves at equatorial plate.
  • Chromosomes become attached to the microtubules (spindle apparatus) by their kinetochores.
  • Microtubules begin to pull each chromosome toward the center of the cell.
  • Metaphase plate: imaginary plane through the center of the cell where the chromosomes align.

ANAPHASE

  • Microtubules pull apart the kinetochores of sister chromatids toward the poles.
  • The sister chromatids of the chromosomes move towards opposite poles.
  • Chromosomes assume various shapes, i.e., V-shaped in metacentric, J-shaped in submetacentric and rod-shaped in telocentric.

TELOPHASE

  • Spindle apparatus disassembles.
  • Nuclear envelope forms around each set of sister chromatids.
  • Chromosomes begin to uncoil.
  • Nucleolus reappears in each new nucleus.

CYTOKINESIS

  • Cytoplasmic division, cells divides in to two daughter cells.
  • During cytokinesis there is a formation of cell plate (Phragmoplast) in plant cells whereas in animal cells cytokinesis proceeds by formation of cleavage furrow.

Procedure

  1. Remove the old roots from the base of onion.
  2. Put onion on a couplin jar or a flask filled with water (base of onion should dip in water). New roots appear after 3-4 days.
  3. When roots become about 2-3 cm in length, cut the roots and fix in carnoy’s fixative for 30 minutes (For long term storage wash the fixed roots and store in 70% alcohol).

    4. Acetocarmine Staining

  4. Take a few roots and downgrade to water.
  5. Stain them in acetocarmine for 40 minutes.
  6. Take out one root on a slide and with the help of surgical blade cut the darkly stained root tip for further processing (Remove the lighter stained part).
  7. Add a drop of 45% acetic acid.
  8. Put a coverslip and make a squash.

Animation

Simulator

Precautions

  • Avoid under/over staining.
  • Avoid under/over squashing.
  • Avoid formation of any air bubble.