3.2.1.3 Methods of studying cells

• How to calibrate the graticule?

  • requires a special microscope slide called a stage micrometer
  • Stage micrometer also has a scale etched onto it, normally 2mm long with smaller sub-divisions of 0.1mm
  • Stage micrometer will change.It allows you to calculate how many micrometers each division on your graticule represents.
  • Number of divisions on stage micrometer/ number of divisions on gracticule.
• What are eyepiece graticules?

  A glass disc that is placed on the eyepiece of the microscope.The disc has a scale etched onto it.The scale is typically 10mm long, and is divided into 100 sub-divisions.
• What does the resolution and magnification of light microscope say?

  The relatively long wavelength of light rays means that a light microscope can only distinguish between 2 objects if they are 0.2 micrometers or further apart. This means that any 2 objects that are 0.2 micrometers or more apart will be seen more separately.Limits the magnification to x1500
• How do light microscopes work?

  Uses lenses to project an image of an object onto the eye.They have a lower magnification than electron microscopes, due to their resolving power.
• What are light microscopes also known as?

  Optical Microscopes/Light Microscopes
• What is the formula for calculating magnification?

  I (image size) = A (actual size) x M (magnification)
• Describe the transmission electron microscopy.

  • Very thin slices are made and then surrounded by heavy metals.
  • Electrons don't pass through the heavily-striped parts because they are absorbed.
  • Those that pass through are focused by electromagnets onto a fluorescent screen or photographic plate.
  • Allows us to see inside cells and organelles (cross section or 2D only)
• Describe the scanning electron microscopy?

  • Specimens are coated with a thin layer of metal to increase conductivity and contrast.
  • The electrons are reflected from by the surface of the specimen producing a 3D image.
  • This type of microscope is used to view full cells or tissues.
• What does the greater resolution mean?

  Greater resolution means we are able to see smaller structures in much more detail.
• How is the electron different than the light microscope?

  Exposes specimen to electrons rather than light.
• How does the electron microscope be able to create a higher resolution and greater magnification?

  Due to the much shorter wavelengths of electrons, these microscopes are able to create a higher resolution image and greater magnification.
• What is resolution?

  The ability to distinguish between two points.
• What is magnification?

  How many times bigger an image appears.
• When using a microscope what do you have to consider?

  • Magnification 
  • Resolution
• What is step 1 of ultracentrifugation of animal cells?

  The tube of filtrate is placed in the centrifuge and spun at slow speed.
• What is step 2 of ultracentrifugation of animal cells?

  The heaviest organelle in animal cells, the nuclei, are forced to the bottom of the tube where they form a thin sediment or pellet.
• What is step 3 of ultracentrifugation of animal cells?

  The fluid at the top of the tube, the supernatant, is removed, leaving the sediment.
• What is step 4 of ultracentrifugation of animal cells?

  The supernatant is transferred to another tube and is spun at a faster speed.
• What is step 5 of ultracentrifugation of animal cells?

  The next heaviest organelles in animal cells, the mitochondria, are forced to the bottom of the tube.
• What is step 6 of ultracentrifugation of animal cells?

  The process of ultracentrifugation of animal cells is continued in this way, so that with each increase in speed, the next heaviest organelle is sedimented and separated out.
• Describe the process of ultracentrifugation:

  • A centrifuge spins the homogenate at very high speeds to separate substances by their weight.
  • The faster it spins, the greater the centrifugal force.
  • When spinning at slower speeds, larger fragments collect at the bottom of the tube while smaller ones remain suspended in the liquid.
• What is ultracentrifugation?

  The process by which the fragments in the filtered homogenate are separated in a machine called a centrifuge. 
• Why is the homogenate filtered?

  To remove any complete cells or large pieces of debris?
• What is the resultant fluid called in homegenation?

  Homogenate
• What is homogenation?

  Cells are broken by a homogeniser (blender). This releases the organelles from the cell.
• What are the stages of cell fractionation?

  1. Homogenation 
  2. Ultracentrifugation
• What are some conditions that the buffer solution have to have?

  • Cold - reduces enzymes activity that might break down the organelles.
  • Buffered - so that pH does not fluctuate. Changes in pH could alter the structure of organelles.
  • Same water potential - prevent the organelles bursting or shrinking as a result of osmosis.
• What is cell fractionation?

  A process where cells are broken up to release the different organelles they contain and are separated out based on their size/mass using gravity.