rp2 prep stained squash of cell from plant root tip Flashcards
how to prep squashes of cells from plant root tip
cut thin slice of root tip using scalpel
mount onto slide
soak root tip in hcl rinse
stain for dna
lower coverslip using mounted needle at 45 without trapping air bubbles
squash by firmly pressing down on glass slip but dont push sideways
why are root tips used
where dividing cells found and where mitosis occurs
why is stain used
to distinguish chromosomes
why press down on coverslip
spread cells = single layer of cells
so light passes through to make chromosomes visible
why not push coverslip sideways
avoids rolling cells together and breaking chromosomes
why soak roots in acid
separates cells
allows stain to diffuse into cell
allow cells to be squashed more easily
stops mitosis
how to set up and use microscope
clip slide onto stage and turn on light
select lowest power objective lense
use coarse focusing dial to move stage close to lens
turn coarse focusing dial to move stage away from lens till image comes into focus
just fine focusing dial to get clear image
swap to higher power objective lens and refocus
appearance of prophase
chrom visible bc condensing
randomly arranged as not attached to SF
metaphase appearance
chrome line up at equator
bc attaching to spindle
anaphase appearance
chrom at poles of spindle
chrom v shaped bc being pulled apart at their centromeres by SF
telophase
chrom in two sets
one at each pole
mitotic index
prop of cells undergoing mitosis divide by total cells in sample
how to determine reliable MI
count cells in mitosis
count only whole cells
divided by total no of cells
repeat w 5 fields of view selected randomly
calc mean
