Studying Cells

• What is the purpose of cell fractionation?


  To separate organelles from the cell
• Why does the solution in cell fractionation have to be ice cold?

  To reduce enzyme activity that would break down the organelle.
• Why does the solution in cell fractionation have to be isotonic and what does this mean?


  To prevent damage to the organelles through osmosis

  isotonic: having the same water potential so there is no osmotic effect
• Why does the solution in cell fractionation have to be a buffer?


  To maintain pH so that proteins won't denature.
• What is the function of ultracentrifugation?


  To separate organelles using density
• What is the order of organelle density (most dense -> least dense) ?

  Nuclei, chloroplasts, mitochondria, lysosomes, endoplasmic reticulum, ribosomes
• Describe the procedure of cell fractionation.


  . Collect a sample of tissue and place in an ice cold, isotonic, buffer solution

  . Put the sample and solution into a homogeniser to break open the cell membranes, releasing the organelles

  . Take the homogenate and filter it to remove any tissue debris

  . (after this is ultracentrifugation)
• Describe the procedure of ultracentrifugation.


  . Take the filtrate and spin in a centrifuge, on a low speed at first

  . The densest organelle will settle (sediment out) to form a pellet at the bottom of the tube (this is the nuclei)

  . Pour off the supernatant and spin this again at a higher speed

  . The next densest organelle will form the next pellet

  . Repeat the process at increasing speeds to gain pellets of the other organelles (the last pellet to form is the least dense organelle - ribosomes)
• What is magnification?


  The ability to enlarge an object.
• What is resolution?


  The ability to distinguish between two points so that they can be seen as two separate items.
• What is the resolving power of a microscope?


  The minimum distance apart that two objects can be in order for them to appear as two separate items.
• What is the primary function of lenses in an optical microscope?


  A beam of light is focused with lenses
• Why must specimens be thin in an optical microscope?


  So that light can pass through them
• Why do specimens need staining in optical microscopy?


  Because lots of biological material is colorless
• What types of specimens can be viewed with an optical microscope?


  Both living and non-living specimens
• What is a limitation of optical microscopes regarding organelles?


  They can only see large organelles and not detailed structures within them
• What is the resolving power of an optical microscope?


  2 µm
• How does the resolving power of optical microscopes compare to electron microscopes?


  It is lower than that of electron microscopes, so detail seen is limited
• What is the wavelength of the beam used in optical microscopy?


  The wavelength is relatively long
• What type of image is produced by an optical microscope?


  The image produced is flat and 2D
• Why do any colors seen in optical microscopy occur?


  Due to staining or natural pigments
• What is generally true about the colour of images produced by optical microscopes?


  The image does not have colour
• What is the primary method of electron beam direction in a SEM?


  A beam of electrons is directed from above
• How does the pattern of electron scattering in a SEM relate to the specimen?


  The pattern of scattering depends on the contours of the specimen surface
• Why does a specimen not need to be very thin in a SEM?


  Because the electrons do not penetrate the specimen
• What is a challenge associated with staining specimens for SEM?


  Staining is complicated
• What is a potential issue with images produced by SEM regarding artefacts?


  The image can contain artefacts that may not represent the specimen accurately
• Why can't living specimens be viewed in a SEM?


  The specimen has to be placed in a vacuum
• What is the resolving power of a SEM?


  20 nm
• How does the wavelength of the electron beam in a SEM compare to that of light?


  The wavelength of the beam is short
• What type of image is produced by a SEM?


  A 3D image is produced using computer analysis of the pattern of scattered electrons
• What is the color format of images produced by a SEM?


  The image is black and white, with colors added using a computer
• What does SEM stand for?


  Scanning electron microscope
• What does TEM stand for?


  Transmission electron microscope
• What type of beam does a TEM use to produce an image?


  A beam of electrons
• How does the density of a specimen affect the appearance in a TEM image?


  Dense areas absorb more electrons and appear darker
• Why must the specimen be extremely thin in a TEM?


  So that electrons can pass through it
• What is a complication associated with staining in TEM?


  Staining is complicated
• What are artefacts in the context of TEM imaging?


  Artefacts are things not part of the specimen resulting from preparation
• Why can't living specimens be viewed in a TEM?


  Because the specimen needs to be in a vacuum