Required Practical 2 Flashcards

Preparation of stained squashes of cells from plant root tips; set-up and use of an opitcal microscope to identify the stages of mitosis in these stained squashes and calculation of a mitotic index.

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1
Q

Describe how to prepare squashes of cells from plant root tips.

A
  1. Cut a thin slice of root tip using scalpel and mount onto a slide.
  2. Soak root tip in hydrochloric acid.
  3. Stain for DNA (e.g. with toluidine blue).
  4. Lower coverslip using a mounted needle at 45° without trapping air bubbles.
  5. Squash by firmly pressing down on glass slip but do not push sideways.
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2
Q

Why is a stain used?

A

To distinguish chromosomes which are not visible without a stain.

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3
Q

Why are root tips used?

A

This is where dividing cells are found/mitosis occurs here.

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4
Q

Why squash/press down on coverslip?

A

To create a single layer of cells so light passes through to make chromosomes visible.

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5
Q

Why not push cover slip sideways?

A

Avoid rolling cells together/breaking chromosomes.

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6
Q

Why soak roots in acid?

A
  • Separate cells/cell walls.
  • To allow stain to diffuse into cells.
  • To allow cells to be more easily squashed.
  • To stop mitosis.
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7
Q

Describe how to set up and use an optical microscope.

A
  1. Clip slide onto stage and turn on light.
  2. Select lowest power objective lens.
  3. Use coarse focusing dial to move stage close to lens. Turn coarse focusing dial to move stage away from lens until image comes into focus.
  4. Adjust fine focusing dial to get clear image.
  5. Swap to higher power objective lens, then refocus.
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8
Q

What are the rules of scientific drawing?

A
  • Look similar to specimen/image - draw parts to the same scale / relative size.
  • No sketching - only clear, continuous lines.
  • No shading / hatching.
  • Include a magnification scale.
  • Label with straight, uncrossed lines.
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9
Q

What stage of mitosis is shown in the diagram below? Explain how you know.

A

PROPHASE

  • Chromosomes visible/distinct because they’re condensing.
  • Randomly arranged because no spindle activity/not attached to spindle fibres.
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10
Q

What stage of mitosis is shown in the diagram below? Explain how you know.

A

METAPHASE

  • Chromosomes lined up on equator because attaching to spindle.
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11
Q

What stage of mitosis is shown in the diagram below? Explain how you know.

A

ANAPHASE

  • Chromatids at poles of spindle.
  • Chromatids V-shaped because being pulled apart at their centromeres by spindle fibres.
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12
Q

What stage of mitosis is shown in the diagram below? Explain how you know.

A

TELOPHASE

  • Chromosomes in two sets, one at each pole.
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13
Q

What is a mitotic index?

A
  • Proportion of cells undergoing mitosis with visible chromosomes
  • Mitotic index = number of cells undergoing mitosis / total number of cells in sample
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14
Q

Explain how to determine a reliable mitotic index from observed squashes.

A
  1. Count cells in mitosis in field of view.
  2. Count only whole cells - standardise counting.
  3. Divide this by total number of cells in field of view.
  4. Repeat with at least 5 fields of view selected randomly - representative sample.
  5. Calculate a reliable mean of mitotic index.
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15
Q

Suggest how to calculate the time cells are in a certain phase of mitosis.

A
  1. Identify proportion of cells in named phase at any one time.
    • Number of cells in that phase / total number of cells observed.
  2. Multiply by length of cell cycle.
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16
Q

How do you calculate actual size?

A

Actual size = size of image / magnification