RP1 - MICROSCOPY

• Optical microscope


  Used to look at cells on a prepared microscope slide
• Optical microscope


  • Has a stage to place the microscope slide
  • Has a light source (lamp or mirror) to illuminate the slide
  • Has objective lenses with different magnifications (4x, 10x, 40x)
  • Has an eyepiece lens with 10x magnification
  • Has coarse and fine focusing dials
• Using an optical microscope to view a prepared slide


  1. Place slide on stage and secure with clips

  2. Select lowest power (4x) objective lens and slowly lower it until it almost touches the slide

  3. Look through eyepiece and use coarse focus to bring cells into view

  4. Use fine focus to clearly see the cells

  5. Calculate total magnification by multiplying eyepiece (10x) and objective (4x, 10x, 40x) magnifications
• What can be seen in animal cells

  • Nucleus, cytoplasm, cell membrane, possible mitochondria
• What can be seen in plant cells

  • Cell wall, cytoplasm, nucleus, possible vacuole and chloroplasts
• An optical microscope can only show limited detail, it cannot see organelles like ribosomes
• A magnification scale should be included on drawings made from an optical microscope
• step 1


  1. Add a drop of water to a clean slide.
  2. Carefully extract the cells of interest and place them on the slide, in the water – common choices are human cheek cells (animal) and onion epidermal cells (plant).
  3. Highlight the cells using an appropriate stain (Iodine for onion cells and methylene blue for cheek cells).
  4. Finally, place a cover slip over the top of the specimen.

  
• step 2 - view under light microscope

  1. Carefully place the slide onto the stage and clip it in place.
  2. Select the objective lens with the lowest power and therefore lowest magnification.
  3. While looking down the eyepiece, move the stage up and down using the coarse adjustment knob until the image becomes more focussed.
  4. Use the fine-adjustment knob to further focus the image until it is clear. 
  5. Switch to a higher powered objective lens and refocus if greater magnification is required.

  
• step 3 - sketch


  Biological drawings should be scientific so draw what you see down the microscope with clear unbroken lines and in the correct proportions.

  1. Label the different cell structures with clear straight lines, add a title and state the magnification that the cell was observed under.

  
• Prepare a microscope slide

  1. Plant eg. onion cells: peel off thin layer of tissue using forceps, place on water drop on slide

  2. Animal eg. cheek cells: swab inside cheek with a cotton bud, smear onto centre of slide

  3. Add a drop of stain with a pipette

  4. Lower coverslip at an angle without trapping air bubbles
• Use a microscope to view cells

  1. Clip slide onto stage and turn on light

  2. Select lowest power objective lens (usually x 4)

  3. Use coarse focusing dial to move stage close to lens

  4. Turn coarse focusing dial to move stage away from lens until image comes into focus

  5. Adjust fine focusing dial to get clear image

  6. Swap to higher power objective lens, then refocus
• Total magnification of a microscope

  Magnification of eyepiece lens x magnification of objective lens
• Estimate the sizes of cells using a microscope

  1. Measure diameter of field of view (by using microscope to observe a transparent ruler)

  2. Divide this by number of cells that span field of view
• Rules of scientific drawing

  • No sketching or shading - only use clear, continuous lines (with a sharp pencil)
  • Include a magnification scale (eg. x 400)
  • Label important features eg. nucleus
• Why use a stain?

  To make sub-cellular structures visible
• Why is a thin layer of onion tissue used?

  To let light pass through
• Why is the cover slip lowered at an angle?

  To avoid trapping air bubbles
• Why is it easier to view cells using the low power objective lens first?

  • Biggest/widest field of view
  • Easier to focus
• How could a student observe structures within a cell in greater detail?

  Use a higher power objective lens (or use an electron microscope but a student wouldn't have access)
• Equipment


  • Light microscope
  • Microscope slide
  • Cover slip
  • Onion
  • Forceps
  • Iodine solution
  • White tile
  • Scalpel
  • Any other prepared plant and animal cell slides
• Method


  1. Peel off a thin layer of the onion using forceps

  2. Place onto the microscope slide with a drop of water using a pipette, making sure the Layer of onion lies flat

  3. Add 2 drops of iodine solution to stain the cells

  4. Place the cover slip at an angle. This strategy ensures no air bubbles are trapped

  5. Remove any excess stain by soaking it with paper towels

  6. Place the slide on the stage of the microscope

  7. Turn the objective lens to select the lowest power

  8. Set up the microscope-don't look into the eyepiece yet. Instead, use the course adjustment knob to raise the stage - the cover slip should be close to the objective lens

  9. Now look into the eyepiece and turn the course adjustment knob to move the stage away until the image comes into focus (doing this helps avoid you breaking the slide)

  10. Turn the objective lens to a higher magnification power

  11. Repeat the same process as above and then look into the eyepiece and tum the fine adjustment knob until the image comes into focus
• Make a labelled drawing of a few of the cells you can see, including any features eg. cell wall, nucleus. Write down the magnification.
• Safety precautions


  • Wear safety goggles when handling the iodine solution