REQUIRED PRACTICAL 2 - optical microscope to identify mitosis stages and calculating mitotic index

Question 1

How to prepare squashes of cells from plant root tips

Answer 1

• Cut thin slice of root tip (5mm) from end - using scalpel and mount onto slide
• Soak root tip in HCL and rinse
• Stain for DNA using toluidine blue stain
• Lower coverslip with a mounted needle at an angle without trapping air bubbles
• Firmly press down on glass slip to squash but don’t push sideways

Question 2

Why are root tips used

Answer 2

Where dividing cells are found/mitosis occurs

Question 2

Why is a stain used

Answer 2

Distinguish chromosomes as they aren’t visible without stain

Question 3

Why does coverslip need to be pressed down

Answer 3

Spreads cells to create a single layer to allow light to pass through to make chromosomes visible

Question 4

Why shouldn’t the cover slip be pushed sideways

Answer 4

Avoid rolling cells together and breaking chromosomes

Question 4

Why do the roots need to be soaked in acid

Answer 4

• Separate cells/cell walls
• Allow stain to diffuse into cells
• Stop mitosis

Question 5

What is mitotic index

Answer 5

• Proportion of cells undergoing mitosis (with visible chromosomes)
• Number of cells undergoing mitosis / total number of cells in sample

Question 5

Rules for scientific drawing

Answer 5

• No sketching, use clear continuous lines, no shading
• Include magnification scale
• Label with straight uncrossed lines

Question 6

How to set up and use an optical microscope

Answer 6

• Clip slide onto stage and turn on light
• Select lowest power objective lens
• Coarse focusing dial to move stage closer to lens
• Coarse focusing dial to move stage away from lens until image is focused
• Adjust fine focusing dial to get a clear image
• Swap to higher power objective lens and refocus

Question 7

How to determine reliable MI from observed squashes

Answer 7

• Count cells undergoing mitosis in field of view
• Count only whole cells -> standardise counting
• Divide by total number of cells in view
• Repeat with many fields of view selected randomly -> representative sample
• Calculate reliable mean

Question 8

How to calculate time cells are in a certain mitosis stage

Answer 8

• Identify proportion of cells in named phase at any one time
  -> number of cells in that phase / total number of cells observed
• Multiply by length of cell cycle

Question 9

How to identify stages of mitosis

Answer 9

• Prophase
  -> chromosomes visible/distinct - because condensing
  -> randomly arranged - no spindle fibre activity/not attached to spindle fibre
• Metaphase
  -> chromosomes lined up on equator - attaching to spindle
• Anaphase
  -> chromatids at poles of spindle
  -> chromatids V shaped - being pulled apart at their centromeres by spindle fibres
• Telophase
  -> chromosomes in 2 sets, one at each pole