Investigating Plant Root Tips

Staining: Because DNA and chromosomes are naturally transparent, a stain like acetic orcein or toluidine blue must be applied. These stains bind specifically to the negatively charged phosphate groups in DNA, coloring the chromosomes a deep purple or blue so they can be clearly distinguished from the cytoplasm.

The Squash: A coverslip is placed over the stained root tip and firm, vertical pressure is applied using a paper towel. This 'squash' spreads the cells out into a single, thin layer, which is essential for light to pass through the specimen and to prevent cells from overlapping, which would obscure the view of individual nuclei.

Definition: The mitotic index is a quantitative measure of the proliferation rate of a tissue sample. It represents the proportion of cells that are actively undergoing nuclear division compared to the total number of cells in the sample.

Calculation: The index is calculated using the following formula: $\text{Mitotic Index} = \frac{\text{Number of cells with visible chromosomes}}{\text{Total number of cells in the sample}}$

Significance: A high mitotic index indicates a rapidly growing tissue, such as a healthy root tip or a cancerous tumor. In experiments, changes in the mitotic index can be used to study the effects of environmental factors or chemicals on the rate of cell division.

Magnification Equation: To determine the actual size of the cells observed under the microscope, the standard magnification formula is used: $\text{Actual Size} = \frac{\text{Image Size}}{\text{Magnification}}$

Unit Conversion: Since cells are measured in micrometres ($\mu m$) but image sizes are often measured in millimetres ($mm$), it is critical to convert all values to the same unit. To convert $mm$ to $\mu m$, the value must be multiplied by $1000$ ($1 mm = 1000 \mu m$).

Systematic Counting: When calculating the mitotic index from a micrograph, use a grid or a 'Z' pattern to count cells. This prevents the common mistake of double-counting the same cell or missing cells at the edges of the field of view.

Identifying Stages: Always look for the most distinct features: visible threads for prophase, a central line for metaphase, V-shaped structures moving apart for anaphase, and two distinct clusters for telophase. If no chromosomes are visible and the nucleus is a solid circle, the cell is in interphase.

Sanity Checks: Ensure the mitotic index is always a value between $0$ and $1$ (or $0\%$ and $100\%$). If your calculated index is greater than $1$, you likely swapped the numerator and denominator in the formula.