Required practical 2

What is the aim of Required Practical 2?

• To prepare stained squashes of plant root tip cells.

• To use an optical microscope to identify the stages of mitosis.

• To calculate a mitotic index.

What is the first step in preparing the root tip sample?

• Use a scalpel to cut a thin slice (approx. 5mm) from the tip of a plant root.

• Mount it on a microscope slide.

Why is the root tip soaked in hydrochloric acid?

• To soften / separate the middle lamella between cells.

• This allows cells to be spread more easily during squashing.

• It stops mitosis by killing the cells.

• It helps allow stain to diffuse into the cells.

Why is a stain (e.g., toluidine blue) applied?

• To stain the chromosomes (which contain DNA).

• Chromosomes are not visible without staining.

How should the coverslip be lowered onto the slide?

• Place it at a 45° angle using a mounted needle.

• Lower it gently to avoid trapping air bubbles.

How and why is the coverslip squashed?

• Firmly press straight down on the coverslip.

• Purpose: To spread the cells into a single layer so light can pass through, making chromosomes visible.

• Do not push sideways to avoid rolling cells together or breaking chromosomes.

Why are root tips specifically used for observing mitosis?

• The root tip meristem is a region where rapid cell division (mitosis) occurs.

What are the first two actions when setting up the microscope?

• Clip the prepared slide securely onto the stage.

• Turn on the light source (lamp or adjust the mirror).

What magnification should you start with, and how do you initially focus?

• Start with the lowest power objective lens (e.g., x4).

• Use the coarse focusing dial: first move the stage close to the lens, then slowly move it away until the image comes into focus.

How do you achieve a sharp, clear image?

• Use the fine focusing dial to adjust and clarify the image.

What do you do to view the specimen at a higher magnification?

• Rotate the nosepiece to select a higher power objective lens (e.g., x10 or x40).

• Refocus using the fine focus dial only (the coarse dial should not be used at high power).

What formula is used to calculate the actual size of a cell?

• Actual size = Size of image / Magnification

What is the first rule for making a scientific drawing of a cell?

• It must look similar to the specimen / image.

• Draw all parts to the same scale / relative size.

What are the rules regarding lines in a scientific drawing?

• Use clear, continuous lines.

• No sketching, shading, or hatching.

What two pieces of information must be included with the drawing?

• A magnification scale (e.g., x400).

• Labels with straight, uncrossed lines pointing to structures.

How can you distinguish a cell in interphase from a cell in mitosis?

• Interphase: Chromosomes are not visible; you see one or more distinct nuclei.

• Mitosis: Chromosomes are visible (condensed).

What does a cell in prophase look like?

• Chromosomes are visible and distinct (appear as long, thin threads).

• They are randomly arranged in the cell.

Why do chromosomes appear this way in prophase?

• They are condensing.

• They are not yet attached to the spindle fibres.

What is the defining feature of a cell in metaphase?

• Chromosomes are lined up along the equator / centre of the cell.

How can you identify a cell in anaphase?

• Chromatids (V-shaped) are seen in two separate groups at opposite poles of the cell.

Why are the chromatids V-shaped and at the poles?

• They are being pulled apart at their centromeres by shortening spindle fibres.

What does a cell in telophase look like?

• Two sets of chromosomes, one at each pole of the cell.

• Nuclear envelopes may be reforming.

What is the mitotic index (MI)?

• The proportion of cells in a sample that are undergoing mitosis (have visible chromosomes).

What is the formula for calculating the mitotic index?

• MI = (Number of cells undergoing mitosis) / (Total number of cells in the sample)

How should you count cells to ensure consistency?

• Count only whole cells.

• Use a standard method (e.g., count cells on the top and right edges only to avoid double-counting).

How do you obtain a representative and reliable MI value?

• Repeat counts for many (at least 5) fields of view.

• Ensure fields are selected randomly.

• Calculate a mean from these repeat values.

How do you calculate the time a cell spends in a specific phase (e.g., metaphase)?

1. Find the proportion of cells in that phase: (Number in phase) / (Total cells).

2. Multiply this proportion by the total length of the cell cycle.