RPQ 2

• safety goggles, lab coat, gloves when using stain
• 1. add some 1 M HCl to a boiling tube. there should be just enough acid to cover the root tip - so the acid should only be a few mm deep - put in a water bath that has been allowed to reach 60 degrees
• 2. use a scalpel to cut 1cm from the tip from a growing root (e.g. of an onion). It needs to be the tip bc that is where growth occurs and so that it where mitosis takes place
• 3. carefully transfer the root tip into the boiling tube containing the acid. Incubate for about 5 minutes
• 4. use tweezers to remove the root tip from the tube and use a pipette to rinse it well with cold water - leave the tip to dry on a paper towel
• 5. place the root tip on a microscope slide and cut 2mm from the very tip of it
• 6. use a mounted needle to break the tip open and spread the cells out thinly
• 7. add a few drops of stain and leave it for a few minutes - the stain will make the chromosomes easier to see under a microscope.
• there are many different stains:

  • toluidine blue O
  • ethano-orcein
  • feulgen stain - needs an extra rinse
• 8. place a cover slip over the cells and put a folded piece of paper on top. push down firmly to squash the tissue. Squashing the tissue will make the tissue thinner and allow light to pass through it. don't smear the overslip sideways as this will damage the chromosomes
• 9. now can look at all stages of mitosis
• Using an optical microscope

  1. Clip the slide onto the stage

  2. Select the lowest powered objective lens

  3. Use the coarse adjustment knob to bring the stage up to just below the objective lens

  4. Look down the eyepiece and use the coarse adjustment knob to move the stage downwards until the image is roughly in focus

  5. Adjust the focus with the fine adjustment knob until you get a clear image
• Higher-powered objective lens


  Provides greater magnification
• Increasing magnification


  1. Swap to a higher-powered objective lens

  2. Refocus
• if asked to draw cells undergoing mitosis under the microscope, make sure the relative sizes of objects in your drawing are accurate and that you write down the magnification the specimen was viewed under - need to label and give a title
• the mitotic index is the proportion of cells in a tissue sample that are undergoing mitosis - lets you work out how quickly the tissue is growing and if there's anything weird going on
• mitotic index = number of cells with visible chromosomes / total number of cells observed
• a plant root tip is constantly growing so you would expect a high mitotic index i.e. lots of cells in mitosis
• in other samples, a high mitotic index could mean that tissue repair is taking place or that there is cancerous growth in the tissue
• need to be able to calculate the actual size of cells looking at - where the eyepiece graticule and stage micrometer come in
• an eyepiece graticule is fitted onto the eyepiece - like a transparent ruler with numbers but no units
• the stage micrometer is placed on the stage - it is a microscope slide with an accurate scale (it has units) and is used to work out the value of the divisions on the eyepiece graticule at a particular magnification - means that when you take the stage micrometer away and replace it with the slide containing your tissue sample - will be able to measure the size of the cell
• • line up the eyepiece graticule and the stage micrometer
  • each division on the stage micrometer is 0.1 mm long
  • work out how many divisions on the eye piece graticule fit into one division on the stage micrometer
  • divide the 0.1 (stage micrometer) by 4.5 (e.g. eyepiece graticule) = 0.022mm
  • if a cell is 4 eyepiece divisions long at a certain mag - 4 x 0.022 = 0.09 mm
• if given an image of cells under microscope can calc actual size using: actual size = size of image / magnification
• root tips are used in this investigation because they are a growing part of the plant and therefore mitosis will be occurring in cells allowing it to be observed
• we use several roots as this increases the likelihood that we will be able to view some stage of mitosis and get results as the process damages the cells (HCl)
• the root tips are placed in 1M HCl acid at 60 degrees for 5 min as this hysdrolyses the lamellae which holds the cell together - separates the layers of the cell and allows the stain to enter the nucleus
• the root tips are placed in the toluidine blue O stain for 2 min as it binds to chromatin and DNA, staining it blue and making the chromosomes visible
• the root tips are gently squashed in order to produce a layer that is one cell thick as this will make it easier to view the different phases of mitosis, but gently to avoid rupturing the cell and to avoid breaking the coverslip
• to improve the definition of the chromosomes within the cells you could increase the time spent in the HCl or increase the time spent in the stain
• a mitotic index provides information about how fast and how much cell division is taking place - if high - suggests rapid cell division - could indicate the presence of a tumour/ how fast a tumour is developing
• HCl hydrolyses the lamellae - cells held together by pectin - separates this to allow the stain to enter the nucleus