RP2- microscopes Flashcards
How to prepare squashes of cells from plant root tips?
-Cut a thin slice of root tip using scalpel and mount onto a slide.
-Soak root tip into HCl then rinse.
-Stain for DNA- with toluidine blue.
-Lower coverslip using a mounted at 45 without trapping air bubbles.
-Squash by firmly pressing down on glass slip but do not push sideways.
Why root tips are used?
-Where dividing cells are found/ mitosis occurs.
Why a stain is used?
-To distinguish chromosomes.
-Chromosomes not visible without stain.
Why cover slip needs to be squashed/ pressed down?
-Create a single layer of cells.
-So light passes through to make chromosomes visible.
Why the cover slip should not be pushed sideways?
-Avoids rolling cells together/ breaking chromosomes.
Why the roots should be soaked in acid?
-Separate cells/ cell walls.
-To allow stain to diffuse into cells.
-To allow cells to be more easily squashed.
-To stop mitosis.
How to set-up and use an optical microscope?
-Clip slide onto stage and turn on light.
-Select lowest power objective lens (usually x4).
-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.
-Adjust fine focusing dial to get clear image.
-Swap to higher power objective lens and refocus.
Mitotic index
-Proportion of cells undergoing mitosis- with visible chromosomes.
-Number of cells undergoing mitosis/ total number of cells in sample.
How to determine a reliable MI from observed squashes?
-Count cells in mitosis in field of view.
-Count only whole cells/ only cells on top and right edges- standardise counting.
-Divide this by total number of cells in field of view.
-Repeat with many/ at least 5 fields of view selected randomly- representative sample.
-Calculate a reliable mean.
How to calculate the time cells are in a certain phase of mitosis?
-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.
