Part 1: What this practical is about

In this required practical, you prepare a stained squash of cells from a plant root tip, observe the stages of mitosis using an optical microscope, and calculate the mitotic index. The key idea is simple: the root tip contains a meristem, and meristematic cells are dividing more actively than cells further up the root.

The practical helps you do three things:

1. prepare a temporary microscope slide safely and correctly
2. recognise cells in interphase, prophase, metaphase, anaphase, and telophase
3. calculate the mitotic index from observed cells

Only the tip of the root is used because that is where the meristem is found. Cells further from the tip are usually elongating or differentiating and are less useful for observing mitosis.

Part 2: Method and safety

Equipment

Safety

Method

1. Grow onion, garlic, or shallot roots before the lesson so that fresh root tips are available.
2. Place 1 mol dm^-3 hydrochloric acid in a water bath at 60 degrees C.
3. Cut a root tip and place it in the warm hydrochloric acid for 5 minutes.
4. Remove the root tip and rinse it in cold distilled water.
5. Cut off the terminal 1 to 2 mm of the root tip. This is the meristem.
6. Place the meristem on a microscope slide and add a few drops of toluidine blue O stain.
7. Use a mounted needle to tease the tissue apart gently.
8. Lower a cover slip carefully onto the tissue.
9. Press vertically down on the cover slip through filter paper to squash the tissue into a thin layer. Do not slide the cover slip sideways.
10. Start with the lowest power objective lens on the optical microscope, then move to a higher magnification once the tissue is in focus.
11. Identify cells in different stages of mitosis and count the total number of cells and the number of cells in mitosis.

Teachers may use slightly different versions of this method. Some centres warm a more dilute hydrochloric acid for a short time, while others use a more concentrated acid for longer. The important idea is that the treatment kills and softens the tissue so the meristem cells separate more easily when squashed.

Hydrochloric acid is used because it helps break down the middle lamella, so the cells can be separated in the squash.

Part 3: Recognising mitosis under the microscope

Most cells in a root tip squash will be in interphase, because cells spend most of the cell cycle there. These cells have a visible nucleus but no separate visible chromosomes.

Use the features below to identify the stage you are looking at.

Root-tip meristem cells are usually small and fairly square with the nucleus near the centre. Cells from above the meristem are more elongated, so they are less useful for observing mitosis.

If you mainly see large elongated cells, you have probably used tissue from above the meristem rather than the root tip itself.

Part 4: Mitotic index and measuring cell size

To calculate the mitotic index, count all cells in the field of view and then count only the cells in mitosis.

If 62 cells out of 250 are in mitosis:

Mitotic index = 62 / 250 = 0.248

This means 24.8% of the observed cells were dividing.

Measuring the size of root-tip cells in this practical

The named measurement skill in this practical is to measure the apparent size of a root-tip cell and calculate its actual size.

If you are measuring from a photomicrograph or microscope image, use:

Workflow 1: Measure from an image of a root-tip cell

1. Choose one clear cell from the meristem, not an overlapping cell.
2. Measure the image of the cell with a ruler in mm.
3. Use the total magnification written on the image.
4. Divide image size by magnification.
5. Convert the answer into micrometres if needed.

For example, if a root-tip cell image measures 12 mm on a photomicrograph at x600 magnification:

Actual size = 12 mm / 600 = 0.02 mm = 20 micrometres

Workflow 2: Measure directly under the microscope using a graticule

1. Put the eyepiece graticule in the microscope eyepiece.
2. Place a stage micrometer on the microscope stage and focus at the magnification you will use for the root-tip cells.
3. Line up the graticule scale with the stage micrometer scale to work out how many micrometres one graticule division represents at that magnification.
4. Replace the stage micrometer with the stained root-tip slide without changing the magnification.
5. Count how many graticule divisions span the width or length of a cell.
6. Calculate actual cell size using:

1. Recalibrate if you change objective lens, because one graticule division represents a different actual distance at a different magnification.

For example, if 1 graticule division has been calibrated as 2.5 micrometres and a root-tip cell spans 16 divisions:

Actual size = 16 x 2.5 micrometres = 40 micrometres

Part 5: Interpreting results and improving the method

In a successful root tip squash:

• most cells should be in interphase
• fewer cells should be in mitosis
• of the mitotic stages, prophase is usually seen more often than anaphase because it lasts longer

Common problems and improvements are shown below.

If counts differ between fields of view, this does not automatically mean one result is wrong. It may simply mean different fields contained different numbers of meristematic cells, so using several fields improves reliability.