Required Practical: Identifying Mitotic Stages

Step-by-Step Preparation

• Sample Collection: Cut a small section (approx. 1cm) from the tip of a growing root, ensuring the meristematic region is included.
• Fixation: Submerge the sample in ethanoic alcohol to preserve the cellular structures.
• Softening: Place the root tip in a test tube of dilute hydrochloric acid and heat in a water bath (typically $60^\circ C$) to soften the cell walls.
• Staining: Transfer the tip to a slide and add a drop of stain; allow several minutes for the chromosomes to take up the pigment.
• Squashing: Place a coverslip over the sample and press down firmly and vertically with a thumb or paper towel, avoiding any lateral movement that could smear the cells.

Microscopic Analysis

• Initial Search: Use the lowest power objective lens to locate the area of the meristem where cells are small, square-shaped, and densely packed.
• High Power Identification: Switch to a high-power objective lens to identify individual cells in prophase, metaphase, anaphase, or telophase based on chromosome arrangement.

• Visual Identification of Stages:

• Mitotic Index vs. Growth Rate: While the mitotic index measures the proportion of dividing cells at a single point in time, it serves as a proxy for the overall growth rate of the tissue.

• Formula Mastery: Always remember the formula for the Mitotic Index: $\text{Mitotic Index} = \frac{\text{Number of cells with visible chromosomes}}{\text{Total number of cells observed}}$.

• Unit Conversions: Be prepared to convert between millimeters ($mm$), micrometers ($\mu m$), and nanometers ($nm$). Remember that $1 mm = 1000 \mu m$ and $1 \mu m = 1000 nm$.

• Magnification Calculations: Use the formula $\text{Actual Size} = \frac{\text{Image Size}}{\text{Magnification}}$. Ensure that the image size and actual size are in the same units before dividing.

• Identifying Stages: In exam questions featuring micrographs, look for the 'equator' to identify metaphase and 'V-shaped' chromatids moving apart for anaphase.

• Lateral Movement: Moving the coverslip sideways during the squash creates 'smearing' rather than a monolayer, making it impossible to distinguish individual cells.

• Artefacts: Dust, air bubbles, or damaged cells caused by excessive pressure can be mistaken for cellular structures; these are known as artefacts.

• Denominator Error: A common mistake in calculating the mitotic index is dividing by the number of non-dividing cells (interphase) instead of the total number of cells in the sample.

• 3D to 2D Interpretation: Remember that cells are 3D objects; if a cell is cut at an unusual angle during preparation, its mitotic stage might look atypical on a 2D slide.

• Cancer Research: The mitotic index is used in clinical settings to determine the aggressiveness of a tumor; a higher index often correlates with a more rapidly growing cancer.

• Cell Cycle Regulation: This practical provides visual evidence of the cell cycle stages discussed in theory, linking the physical appearance of DNA to the molecular checkpoints of the cycle.

• Plant Physiology: Comparing the mitotic index of roots grown under different conditions (e.g., temperature, nutrient availability) allows for the study of environmental impacts on plant development.