REQUIRED PRACTICAL 2 - SETUP AND USE OF A MICROSCOPE

to identify the stages of mitosis in the stained squashes and calculation of a mitotic index

• plant cells undergo mitosis at the SHOOT and ROOT TIPS in areas called the MERISTEMS • cells in the MERISTEMS are TOTIPOTENT and retain the ability to DIFFERENTIATE

• the ratio of cells undergoing mitosis to the total number of cells in a sample

cells from the meristem must be viewed under a optical microscope

• optical microscope • microscope slides • cover slips • HCL acid • toluidine blue O stain • distilled water • scalpel • forceps • 100 ml beaker • root tip

• heat 1 mol/dm3 at 60 degrees C in a water bath • cut a small sample of the root tip using a scalpel • transfer the root tip to HCL and incubate for 5 mins • remove the root tip from HCL and wash sample in cold distilled water and remove the tip using a scalpel • place the tip on a microscope slide and add a few drops of stain, toluidine blue O = makes chromosomes visible and will therefore show which cells are undergoing mitosis • lower the cover slip down carefully onto the slide • ensure there are no air bubbles in the slide as this may distort the image • ensure the coverslip doesn’t slide sideways which could damage the chromosomes • place the coverslip under a microscope and set the objective lens on the LOWEST magnification • use the coarse adjustment knob to move the lens down to just above the slide • use the fine adjustment knob to carefully re-adjust the focus until the image is clear • calculate the mitotic index

the number of cells with visible chromosomes/total number of cells in the sample

• HCL acid • toluidine blue O stain • scalpel • broken glass

may cause harm/irritation to eyes or in cuts

• wear eye protection • avoid contact with the skin • tie up long hair

may cause harm/irritation to eyes or in cuts

• wear eye protection • avoid contact with skin

cuts from sharp objects

• cut away from fingers • use forceps to hold sample whilst cutting • keep away from the edge of the desk

cuts from sharp objects

• take care when handling slides and coverslips • keep glassware away from the edge of the desk

as this is where dividing cells are found and mitosis occurs

• to distinguish chromosomes • chromosomes are not visible without a stain

• it spreads out cells to create a single layer of cells • allows light to pass through to make chromosomes visible

to avid rolling cells together/ breaking chromosomes

• separate cells/cell walls • to allow the stain to diffuse into the cells • to allow the cells to be more easily squashed • to stop mitosis

• clip slide onto stage and turn on the light • select the lowest power objective, usually x4 • use the coarse functioning dial to move stage close to lens • OR turn the coarse functioning dial to move the stage away from the lens until the image comes into focus • adjust the fine focusing dial to get a clearer image • swap to a higher power objective lens, then refocus

• interphase is not mitosis • chromosomes aren’t visible BUT nuclei are • in mitosis, chromosomes are visible

• chromosomes are visible/ distinct due to CONDENSING • chromosomes are randomly arranges, as they are not attached to their spindle fibre