Biology Required Practical: Investigating Mitosis

Understanding mitosis is fundamental to grasping how organisms grow, repair tissues, and maintain their cellular integrity. This required practical allows you to visualise this dynamic process firsthand, moving diagrams in a textbook into a tangible reality under the microscope. Mastering the technique of slide preparation and analysis is a core skill for A-Level Biology, directly assessing your ability to handle biological equipment, make accurate observations, and perform quantitative calculations related to the cell cycle.

The Theoretical Foundation: Why Root Tips?

To investigate mitosis, you need a source of actively dividing cells. The root tip meristem, particularly from plants like garlic or onion, is ideal for this purpose. This region is a site of rapid growth, containing a high proportion of cells undergoing the various stages of mitosis. The practical involves treating these tips to arrest cells at their current stage, softening the tissue, and using stains that bind specifically to genetic material, making the chromosomes visible. The goal is to produce a thin, monolayer root tip squash slide, which spreads the cells out so individual nuclei and chromosomes can be clearly observed and counted.

Slide Preparation: From Root Tip to Microscope Slide

The procedure is a sequence of precise steps, each crucial for a successful observation. First, you must prepare root tip squash slides. This typically involves cutting approximately 5 mm from the tip of several freshly grown roots. These tips are then placed into a pre-warmed vial of hydrochloric acid. The acid serves two key functions: it hydrolyses the pectin in the middle lamella that holds adjacent plant cells together, and it breaks down the proteins associated with the chromosomes, allowing the stain to access the DNA more effectively.

After rinsing to remove the acid, the tips are transferred to a watch glass containing a suitable stain. Acetic orcein is a common choice, as it stains chromosomes a dark red or purple and the cytoplasm a lighter shade, providing excellent contrast. Toluidine blue is an alternative basic dye that binds to acidic components like DNA, staining chromosomes blue. The staining process requires careful heating gently, without boiling, to intensify the colouration. Once stained, a single tip is placed on a clean microscope slide, a drop of stain added, and then carefully squashed under a coverslip using firm, vertical pressure from your thumb over a folded paper towel. This separates the cells into a single layer.

Identifying Stages and Recording Evidence

With a well-prepared slide, you can now systematically identify and photograph cells in different stages of mitosis. You should scan the slide methodically under low power to find regions of well-spread, stained cells, then switch to high power (e.g., 400x) for detailed observation.

• Prophase: Look for condensed, visible chromosomes appearing as dark, tangled threads within the nucleus. The nuclear envelope will be breaking down.
• Metaphase: The most distinctive stage. Chromosomes are lined up single-file along the equator (centre) of the cell, attached to spindle fibres. This is the best stage for counting chromosome numbers in a species.
• Anaphase: Identified by the separated sister chromatids (now called chromosomes) being pulled apart to opposite poles of the cell by the shortening spindle fibres.
• Telophase: Two distinct groups of chromosomes are at opposite poles, beginning to de-condense. A new nuclear envelope reforms around each set, and a cell plate begins to form in plant cells, marking the start of cytokinesis.

Using a microscope with a digital camera or a smartphone adapter, you should photograph clear examples of each stage. This provides permanent evidence for your analysis and allows for more accurate drawing and labelling.

Quantitative Analysis: The Mitotic Index

A key quantitative skill in this practical is to calculate the mitotic index. The mitotic index (MI) is a measure of the proportion of cells actively undergoing mitosis in a tissue at a given time. It is calculated using the formula:

$$\text{Mitotic Index (MI)}=\frac{\text{Number of cells in mitosis}}{\text{Total number of cells observed}}$$

To perform this calculation, you must systematically count cells in several fields of view. Tally cells that are in any stage of mitosis (prophase, metaphase, anaphase, telophase) separately from those in interphase (the non-dividing stage, where the nucleus appears as a stained, uncondensed blob). For reliability, you should count a minimum of 100-200 cells across multiple fields of view. A higher MI indicates a faster rate of cell division, which can be influenced by factors like temperature, growth hormones, or the presence of mutagenic chemicals.

Furthermore, you can relate the duration of each stage to the proportion of cells observed in that phase. Assuming all cells are progressing through the cycle at the same rate, the time a cell spends in a particular stage is proportional to the number of cells you find in that stage. For example, if you count 100 cells in mitosis and 20 of them are in metaphase, you can estimate that metaphase accounts for roughly 20% of the total time spent in mitosis. This is because you are effectively taking a snapshot of a dynamic process; stages that last longer will have more cells "frozen" in them at any one moment.

Common Pitfalls

1. Poor Root Tip Selection and Squashing: Using the wrong part of the root (not the meristematic tip) or applying too little pressure during squashing will result in a thick, multi-layered preparation where cells overlap, making identification impossible. Apply firm, vertical pressure to create a monolayer of cells.
2. Inadequate Staining or Over-Heating: Under-stained chromosomes are faint and difficult to see. Over-heating the stain can destroy the tissue or cause the stain to precipitate, creating dark crystals that obscure the view. Heat gently until fumes are just visible.
3. Misidentification of Stages: The most common error is misidentifying late prophase for metaphase, or early telophase for two separate cells. Remember the key identifiers: metaphase has chromosomes aligned centrally; anaphase has chromosomes moving apart; telophase has two reforming nuclei.
4. Inaccurate Mitotic Index Calculation: Failing to scan the entire slide and only counting from one dense cluster of dividing cells will bias your count. You must sample multiple fields of view randomly to get a representative average. Also, ensure you can confidently distinguish a cell in early prophase from a densely stained interphase nucleus.

Summary

• The root tip squash technique, using stains like acetic orcein, is a standard method for preparing slides to visualise chromosomes and the stages of mitosis in plant cells.
• Accurate identification of prophase, metaphase, anaphase, and telophase relies on key morphological features such as chromosome position and the state of the nuclear envelope.
• The mitotic index is calculated as the ratio of dividing cells to the total number of cells observed, providing a quantitative measure of cell division activity in a tissue.
• The proportion of cells found in a specific mitotic stage can be used to estimate the relative duration of that stage within the cell cycle.
• Success in this practical depends on meticulous technique in slide preparation and systematic, unbiased cell counting for quantitative analysis.