RP02 - Mitosis in root tips

Finding dividing cells in a root tip

Not every plant cell still divides, so this practical depends on sampling the right tiny piece of root.

Read the diagram from the right-hand end. The root cap protects the tip but is not the meristem. Just behind it lies the meristem, a plant region containing cells that divide repeatedly by mitosis. Further back, in the elongation zone, cells mostly lengthen and differentiate, so fewer are in mitosis.

So take the short terminal region containing the meristem, commonly about 1–2 mm. This sample includes the root cap, so not every cell in it is meristematic.

A squash spreads this tissue thinly, so individual stained nuclei and chromosomes can be distinguished under an optical microscope. Mitotic index is the proportion of observed cells that are in mitosis, so you count cells, not chromosomes. A poor squash can hide cells and distort the index even when the arithmetic is correct.

Making a squash and using the microscope

Each method step fixes a specific problem: cells stuck together, pale chromosomes or tissue too thick to see through.

Treatment times and stain choice depend on the specimen and reagent, so follow your teacher's risk-assessed protocol. One school method uses fresh onion or garlic root tips, $1\,\mathrm{mol\,dm^{-3}}$ hydrochloric acid warmed to 60 °C, and toluidine blue O.

Preparing a thin stained squash

1. Soften. Treat the root tip in the warm acid for about 5 minutes, or your centre's validated time. Acid softens the pectin-rich middle lamella, the layer that holds neighbouring cell walls together, so the cells separate. It also kills them, so you get a snapshot, not a live film of mitosis.
2. Rinse and trim. Rinse off excess acid with water. Keep the 1–2 mm tip and remove most of the elongation zone.
3. Stain. Add the prescribed stain on a clean slide. Positively charged toluidine blue binds to the negatively charged phosphate groups of nucleic acids such as DNA, so tightly packed chromosomes stand out dark. The acid is not the chromosome stain.
4. Spread. Gently tease the tissue apart and lower a coverslip carefully to reduce trapped air. Press vertically through folded filter paper, following your centre's safe pressing procedure. Sliding sideways can distort or damage cells.
5. Blot. Absorb excess stain. A thin layer lets light through and reduces overlapping cells, which greater magnification can't correct.

Microscope use and safety

1. Secure the slide and start with a low-power objective, whose wider view makes the tissue easier to find. Centre a clear area, then adjust illumination and focus.
2. Watch from the side as the objective nears the slide, so it can't hit the coverslip.
3. Move to higher power and use the fine focus, because coarse focusing risks driving the lens into the slide.
4. Record the objective and eyepiece magnifications. Their product is the total magnification.

In a method answer, tie each precaution to its hazard: “wear eye protection because acid can injure eyes”, not just “be careful”.

Identifying stages and drawing cells

Name a stage from what you can see, then explain that appearance from what the chromosomes are doing.

Evidence for each stage

Telophase and cytokinesis can overlap, so a cell with a cell plate may also be in telophase.

Real specimens vary in orientation, staining and focus, so some cells can't be assigned confidently. A view along the spindle axis can show metaphase chromosomes as a cluster rather than a neat side-on line. Use several features and fine focus, and don't invent spindle fibres or centromeres that the preparation cannot resolve.

Scientific drawing from observation

Choose a clear cell actually visible in your preparation and record its stage with a reason.

• Outlines: draw large, with a sharp pencil and single clear lines. Avoid shading and sketchy repeated lines.
• Accuracy: match relative sizes and positions. Don't copy an idealised diagram or add invisible organelles.
• Labels: use straight ruled lines that meet the structure and don't cross.
• Title and scale: identify the specimen and view, and record the magnification or a calibrated scale bar.
• Annotations: an annotation adds biological meaning to an observation, for example “two chromosome groups moving apart: anaphase”.

In your notebook, draw and annotate an observed dividing cell and an interphase cell, recording the stain, magnification and date.

Mitotic index and stage duration

A squash is one frozen moment, yet counting its cells lets you estimate how long each stage lasts.

Calculating the index

Use a tally for interphase and each mitotic stage.

The numerator includes prophase, metaphase, anaphase and telophase. The denominator includes all counted cells, including interphase.

Prophase is also $24/40=0.60$ of the cells in mitosis, so check which total a question means.

From cell counts to stage duration

Meristem cells divide asynchronously, not in step. So when the acid kills them they are scattered across the whole cycle, and a longer stage holds more cells at that moment. The proportion of cells at a stage therefore approximates the proportion of cycle time spent there.

With a 20-hour cycle, the example gives prophase $0.15\times20=3$ hours and interphase $0.75\times20=15$ hours. Cells spend most of the cycle in interphase, so most cells in your squash will not be dividing. Among the mitotic stages, the largest count probably marks the longest stage: here, prophase.

This assumes the cells are actively cycling at a steady rate and the sample is representative. It fails if many cells have stopped cycling or a treatment arrests them at one stage. It is also a population average, because times vary between individual cells. A snapshot measures occupancy, not duration directly.

Measuring cell size

A cell's image can be almost any size, so compare cells by actual size, not by how big they look.

The length you measure on an image is its apparent size. Measure a clear non-overlapping cell across a defined dimension, such as its width.

You met this formula in 3.2.1.3.

Measure the image at the size for which its magnification was specified, and convert using 1 mm = 1000 μm.

A photomicrograph, a photo taken through a microscope, may have been resized, so use a scale bar that resized with it. A cell 3 times the length of a 10 μm scale bar is 30 μm long regardless of screen zoom.

Calibrating the eyepiece graticule

You calibrated an eyepiece graticule in 3.2.1.3: its divisions have no units until compared with a stage micrometer.

1. At your chosen objective, align the graticule with the micrometer and count divisions over a long span, which reduces the effect of a reading error.
2. Divide the known distance by the number of divisions. If 40 divisions span 100 μm, one division is 2.5 μm.
3. Replace the micrometer with the root-tip slide without changing the objective.
4. Multiply the cell's span by the length per division. A 16-division cell is $16\times2.5=40\,\mathrm{\mu m}$.

Recalibrate after changing objective: the eyepiece scale stays the same while the specimen image changes size, so each division represents a different real distance. Record the calibration alongside your measurements.

Reliable counts and valid conclusions

A large count can still be biased, and a high index doesn't always mean faster growth.

What a mitotic index can and can't show

A high mitotic index can reflect more cells entering mitosis, but it can also reflect cells remaining in mitosis longer. For example, a spindle-blocking drug may prevent anaphase, so metaphase cells accumulate even though fewer divisions finish.

The index shows where cells are at one moment, not how many divisions finish per hour, so don't automatically equate a high index with faster growth.

Sampling and comparing fairly

Choosing only fields with dramatic mitotic figures pushes the index up, however many cells you count. Instead:

• Move through the meristem using a predefined non-overlapping pattern or randomised fields.
• Count all suitably visible cells using the same rule in each field. For example, count cells touching the top and left borders but exclude those touching the bottom and right, to avoid counting them twice.
• Keep the criterion for identifying mitosis consistent, and record the number of cells sampled as well as the final index.
• Record cells that overlap or can't be classified, then improve the preparation, because quietly omitting difficult cells may bias the stage frequencies.

Repeat across several independently prepared root tips: more fields improve sampling within one root, while more roots test biological repeatability.

For a treatment comparison, use comparable plant material and root regions, a suitable untreated control, and the same exposure time, temperature, preparation and counting procedure. Record variation rather than claiming a difference from two isolated indices. To test for faster growth, measure root length over time in treated and control roots.