Microscopy

• The confocal microscope uses laser scanning to create a three-dimensional image of a specimen.
• Resolution is the minimum distance between two objects where they can still be seen as two separate objects.
• Dry mount - thin slices of tissue are placed on a glass slide and then covered with a cover slip
• Wet mount - specimens suspended in a liquid such as water or immersion oil, then a cover slip is placed on at an angle.
• Squash slides - A wet mount is first prepared, and then the cover slip is pushed down using a lens tissue
• Smear slides - the edge of a slide is used to smear the sample, creating a thin, even coating and then a cover slip is applied.
• Gram stain technique is used to differentiate between gram-positive and gram-negative bacteria. Crystal violet is applied and then iodine to fix it, then washed with alcohol.
• Gram-positive bacteria - retain the crystal violet stain and will appear blue or purple under a microscope.

  Gram-negative bacteria - lose the stain and are then dyed with safranin dye which makes them appear red.
• Calculation for magnification - Magnification = Image Size / Actual Size.
• 1mm= 1000um(micrometers) Times by 1000 to get to um

  1um =1000nm(nanometers) Times by 1000 to got to nm
• Electron microscopy - uses electrons instead of light to form an image of a specimen

  Produce images with up to x500,000

  Disadvantages -

  • Expensive
  • inside a carefully controlled environment + dedicated space
  • specimens can be damaged by the electron beam and may result in artefacts.
• Two types of electron microscope -

  Transmission electron microscope (TEM) - Beam of electrons sent through the specimen and focused to produce an image. Best resolution of 0.5nm.

  Scanning electron microscope (SEM) - Beam of electrons sent across the surface and reflected electrons are collected. Resolution = 3-10nm. Provide 3D images.
• Light microscope -

  • inexpensive
  • small and portable
  • sample preparation does not normally lead to distortion
  • vacuum not required
  • natural colour of sample is seen
  • up to x2000 magnification
  • resolving power is 200nm
  • specimens can be living or dead
• Electron microscope -

  • expensive
  • large and needs to be installed
  • sample preparation often distorts material
  • vacuum required
  • black and white images produced
  • over x500,000 magnification
  • resolving power - TEM = 0.5nm and SEM = 3-10nm
  • specimens are dead