5.08 Practicals: Mitosis

Purpose

• Objective: Study and observe stages of mitosis in plant cells, specifically within the root tip meristem where active cell division occurs.

Background

• Meristems: Growth regions in plants where cell division is concentrated.
• Root Tip Meristem:
  • Located just behind the root cap.
  • Contains a zone of small, actively dividing cells suitable for observing mitosis.

Materials

• Plant Options: Garlic, onion, broad bean, sunflower.
• Tools and Chemicals:
  • Root tips (about 1 cm in length).
• Stain: Acidified acetic orcein (chromosomes stain deep purple).
• Glass slide and blunt instrument (e.g., mounted needle handle).

Procedure

1. Grow Plant Roots:

• Place bulbs or seeds over water (suspended by a pin) for 1–2 weeks to produce root tips.

1. Prepare Root Tips:

• Cut root tips (about 1 cm length).
• Place in a warm solution of acidified acetic orcein stain to color chromosomes purple.

1. Squash Technique:

• Place the stained root tip on a glass slide.
• Gently squash it with a blunt instrument to spread cells into a thin layer.
• This method allows individual cells and stages of mitosis to be visible.

1. Observation:

• Use a microscope to observe and identify stages of mitosis (refer to Figure below).
• Draw and label observed stages: prophase, metaphase, anaphase, and telophase.

Observations and Notes

• Expected Visuals:
  • Chromosomes should be visible due to staining, displaying clear stages of mitosis.
• Prophase: Chromosomes condense and become visible.
• Metaphase: Chromosomes align at the equatorial plane.
• Anaphase: Chromatids separate and move to opposite poles.
• Telophase: Chromatids reach poles, and nuclei begin to reform.

Tips for Success

• Ensure the root tips are properly stained to enhance chromosome visibility.
• Apply gentle but firm pressure during squashing to avoid cell damage and obtain a clear view of dividing cells.

Application

• Visualization Aid: Use annotated drawings based on observations and Figures as a guide for identifying mitotic stages.
• Educational Value: Observing real cells at various mitosis stages enhances understanding of cell division in living organisms.

Practical: Observing Mitosis in Onion Root Tip Cells

1. Preparation of Onion Root Tip Slide:

• Materials Needed: Onion root tips, microscope, glass slide, cover slip, stain (e.g., acetic orcein or toluidine blue), forceps, scalpel, and water bath.
• Steps:
  1. Obtain root tips: Cut 1–2 mm from the growing tip of an onion root (this region has actively dividing cells).
  2. Fix the sample: Immerse root tips in a fixative (e.g., acetic acid) to preserve the structure.
  3. Stain: Add stain to make chromosomes visible under the microscope.
  4. Soften tissue: Heat the stained root tips in a water bath for easier squashing.
  5. Squash and mount: Gently squash the root tip on a glass slide with a cover slip for a single-cell-thick layer.

1. Observation under the Microscope:

• Low Power Objective: Locate the region of active cell division (meristem).
• High Power Objective: Observe individual cells and identify stages of mitosis.

Identifying Stages of Mitosis

1. Prophase:

• Appearance:
  • Chromosomes condense and become visible as thick threads.
  • Nuclear membrane begins to break down.
• Tips:
  • Look for clumped, dark-staining chromosomal material.
  • No aligned chromosomes or clear poles yet.

1. Metaphase:

• Appearance:
  • Chromosomes align along the equatorial plane (metaphase plate).
  • No nuclear membrane visible.
• Tips:
  • Look for a straight line of chromosomes at the cell’s center.
  • No separation of chromatids.

1. Anaphase:

• Appearance:
  • Sister chromatids separate and move towards opposite poles.
  • Chromatids appear as V- or U-shaped structures.
• Tips:
  • Look for clear movement of chromatids.
  • Check for empty space between chromatids and equatorial region.

1. Telophase:

• Appearance:
  • Chromatids reach the poles and decondense into chromatin.
  • Nuclear membranes reform around each set of chromosomes.
• Tips:
  • Look for reappearance of a nuclear outline and less distinct chromosomal material.
  • Cytokinesis (cell splitting) may be visible as a developing cell plate in plants.

Practical Tips for Success:

• Staining:
  • Ensure chromosomes are clearly stained for better visibility.
  • Avoid overstaining, which may obscure details.
• Squashing:
  • Apply even pressure to avoid breaking the slide or creating uneven layers.
  • Aim for a single layer of cells for clarity.
• Region to Focus On:
  • Look at the meristematic region near the root tip, as it contains actively dividing cells.
• Recognition Tips:
  • Identify the absence or presence of the nuclear membrane to distinguish between stages.
  • Observe chromosome arrangement to determine specific mitotic phases.

Key Diagrammatic Features to Practice:

• Prophase: Chromosomes condensing, no alignment.
• Metaphase: Chromosomes in a single line at the equator.
• Anaphase: Chromatids moving towards poles, forming V-shapes.
• Telophase: Chromosomes at poles, cell plate forming (in plants).

Key Terms:

Stain: Dye used to make cellular structures visible (e.g., chromosomes).

Meristem: Area of active cell division (root tips).

Chromosomes: Structures made of DNA visible during mitosis.