topic 2

• Eukaryotic cells


  Cytoplasm containing membrane-bound organelles, DNA enclosed in a nucleus

  
• Prokaryotic cells


  Cytoplasm lacking membrane-bound organelles, genetic material not enclosed in a nucleus

  
• Examples of prokaryotic organisms

  • Bacteria
  • Archaea
• Viruses


  Acellular (not made of cells), non-living (no metabolism, cannot independently move/respire/replicate/excrete)

  
• Structure of a virus particle

  • Nucleic acids surrounded by a capsid (protein coat), attachment proteins allow attachment to specific host cells, no cytoplasm/ribosomes/cell wall/cell-surface membrane, some also surrounded by a lipid envelope

  
• Calculating magnification, real size & image size


  Note formula/rearrange if necessary, convert units if necessary, calculate answer and check units required or if standard form etc. is required
• Unit conversions


  Centimetre (cm) = 0.01 m, Millimetre (mm) = 0.001 m, Micrometre (μm) = 0.000001 m, Nanometre (nm) = 0.000000001 m

  
• Measuring size of object viewed with optical microscope

  Line up eyepiece graticule with stage micrometer, calibrate eyepiece graticule, take micrometer away and use graticule to measure object, calculate size of object, recalibrate eyepiece graticule at different magnifications

  
• Cell fractionation and ultracentrifugation

  Homogenise tissue/use blender, place in cold, isotonic, buffered solution, filter homogenate, ultracentrifugation - separates organelles in order of density/mass
• Eyepiece graticule division


  100 µm/4 = 25 µm
• Describe and explain the principles of cell fractionation and ultracentrifugation as used to separate cell components
• Cell fractionation

  1. Homogenise tissue / use a blender

  2. Place in a cold, isotonic, buffered solution

  3. Filter homogenate

  4. Ultracentrifugation - separates organelles in order of density / mass
• Homogenisation


  • Disrupts cell membrane, breaking open cells and releasing contents / organelles
• Cold, isotonic, buffered solution


  • Cold to reduce enzyme activity → so organelles not broken down / damaged
  • Isotonic so water doesn't move in or out of organelles by osmosis → so they don't burst
  • Buffered to keep pH constant → so enzymes don't denature
• Ultracentrifugation


  • Centrifuge homogenate in a tube at a high speed
  • Remove pellet of heaviest organelle and respin supernatant at a higher speed
  • Repeat at increasing speeds until separated out, each time pellet made of lighter organelles (nuclei → chloroplasts / mitochondria → lysosomes → ER → ribosomes)
• Common mistakes in describing electron microscopes and their use
• Eyepiece graticule


  Spans the full field of view, requires calibration at different magnifications using a micrometre on the stage

  
• Stage micrometer


  Has units, used to calibrate eyepiece graticule

  
• Cell cycle in eukaryotic cells

  1. Interphase (S phase, G1/G2)

  2. Mitosis

  3. Cytokinesis

  
• Mitosis


  1. Prophase

  2. Metaphase

  3. Anaphase

  4. Telophase
• Interphase


  • DNA replicates semi-conservatively, leading to 2 chromatids (identical copies) joined at a centromere
  • Number of organelles & volume of cytoplasm increases, protein synthesis
• Mitosis


  • Nucleus divides to produce 2 nuclei with identical copies of DNA produced by parent cell
• Cytokinesis


  • Cytoplasm and cell membrane (normally) divide to form 2 new genetically identical daughter cells
• Some eukaryotic cells do not undergo the cell cycle
• Mitosis

  • Growth of multicellular organisms by increasing cell number
  • Replacing cells to repair damaged tissues
  • Asexual reproduction
• Tumours and cancers


  Formed by uncontrolled cell division due to mutations in DNA / genes controlling mitosis
• Cancer treatments


  • Disrupt spindle fibre activity / formation
  • Prevent DNA replication during interphase
• Prokaryotic cell replication (binary fission)

  1. Replication of circular DNA

  2. Replication of plasmids

  3. Division of cytoplasm to produce 2 daughter cells
• Virus replication


  1. Attachment to host cell

  2. Injection of viral nucleic acid into host cell

  3. Infected host cell replicates virus particles (nucleic acid replication, viral protein / capsid / enzyme production, virus assembly and release)

  
• Viruses do not undergo cell division as they are non-living
• Preparation of stained squashes of cells from plant root tips

  1. Cut a thin slice of root tip and mount onto a slide

  2. Soak root tip in hydrochloric acid then rinse

  3. Stain for DNA eg. with toluidine blue

  4. Lower coverslip using a mounted needle at 45o without trapping air bubbles

  5. Squash by firmly pressing down on glass slip but do not push sideways
• Why root tips are used

  Where dividing cells are found / mitosis occurs
• Why stain is used

  • To distinguish chromosomes
  • Chromosomes not visible without stain
• Why squash / press down on cover slip


  • (Spreads out cells) to create a single layer of cells
  • So light passes through to make chromosomes visible
• Why not push cover slip sideways

  Avoid rolling cells together / breaking chromosomes
• Why soak roots in acid

  • Separate cells / cell walls
  • To allow stain to diffuse into cells
  • To allow cells to be more easily squashed
  • To stop mitosis
• Setting up and using an optical microscope

  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, then turn to move stage away until image comes into focus

  4. Adjust fine focusing dial to get clear image

  5. Swap to higher power objective lens, then refocus
• Rules of scientific drawing

  • Look similar to specimen / image
  • No sketching / shading - only clear, continuous lines
  • Include a magnification scale
  • Label with straight, uncrossed lines
• Identifying stages of mitosis


  In interphase, chromosomes aren't visible but nuclei are. In mitosis, chromosomes are visible.

  
• Mitotic index


  Proportion of cells undergoing mitosis (with visible chromosomes)