MBIO 1010 Lecture 4

• Principles of light microscopy

  • Compound light microscopes uses visible light to illuminate cells
  • many different types of light microscopy
  • Bright-field
  • Phase-contrast
  • Dark-field
  • Flueorescence
• Bright-field

  Specimens are visualized because of differences in contrast between specimen and surroundings

  Two sets of lenses form the image - Objective lens (usually 10x-100x mag) and ocular lens (usually 10x-20x mag)

  Maximum magnification is ~2,000x
• Total magnification = objective mag x ocular mag
• magnification light path

  visualized image 100x, 400x, 1000x -> eye -> ocular lens 10x -> objective lens 10x, 40x or 100x (oil) -> specimen -> light source
• condenser just condenses the light, doesn't magnify
• Magnification


  The ability to make an object larger - think of magnifying glass
• Resolution


  The ability to distinguish two adjacent object as separate and distinct - the zoom, you can tell two things apart

  limit of resolution for light microscope is about 0.2 micrometer

  Two points can be distinguished if they are at least 0.2 micrometers apart

  light must pass between two points for them to be viewed as separate objects
• Resolution is the limiting factor
• 0.2 micrometers is the same as 200 nm
• As wavelength decreases resolution improves - the shorter the wavelength the better the resolution
• Past 1,000x doesn't improve our microscope
• Improving contrast results in a better final image
• Staining improves contrast

  • dyes are organic compounds that bind to specific cellular materials
  • common stains are
  • methylene blue
  • safranin
  • crystal violet
• Light microscope is the physical microscope, light microscopy is the action of doing it
• Electron microscopy have better resolution than light microscopy
• Types of staining 


  • Simple staining
  • Differential stains
• Simple staining

  One dye is used to color specimen - chromophore is the coloured portion of a dye (2 types of chromophores)
• Types of chromophores

  • Basic dye - positively charged molecules on cell surface
  • Acidic dye - negatively charged chromophore
• Basic dye (positively charged chromophore)

  • binds to negatively charged molecules on cell surface
• Acidic dye (negatively charged chromophore)

  • repelled by cell surface
  • used to stain background
  • negative stain
• Preparing samples for staining

  1. preparing a smear: spread culture in thin film over slide
  2. heat fixing and staining: pass slide through flame to heat fix - flood slide with stain; rinse and pat dry
  3. microscopy
• Negative stains are useful for capsules and looking for shape
• Immersion oil - attaches the slide to the lens for no air
• Negative stain (acidic) - have all the same steps in preparing for a stain except for no heat fixing and no rinsing of the stain
• Gram positive/negative depends on cell wall
• Differential stains

  The gram stain - separates bacteria into 2 groups based on cell wall structure
• Gram positive

  • cells that retain a primary stain (will look purple)
  • cytoplasmic membrane (equiv to inner membrane) and a peptidoglycan
• Gram negative

  • cells that loose the primary stain and take the color of the counterstain (red or pink)
  • inner membrane and outer membrane and peptidoglycan
• Iodine is a morden - attaches itself to crystal violet
• the decolorizer is alcohol
• decolorizer and gram negative - makes big holes in outer membrane and because of that the iodine crystal complex makes it colorless

  decolorizer and gram positive - peptidoglycan is so thick that it can't make holes and so will remain purple
• Counterstain - second stain (safranin) red or pink
• Because of the difference in their cell wall we are able to use a differential stain to differentiate them between gram positive and gram negative
• Acid Fast stain

  • Are neither positive or negative gram stain - its own thing
  • Detects mycolic acid in the cell wall of the genus mycobacerium
  • mycobacterium retains primary stain - fuchsia (pink)
  • anything else on slide - color of counterstain (blue)
• Endospore stain

  • another differential stain is an endospore stain
  • endospores retain primary color green, cells counterstained are pink (ex. bacillus anthracis spores)
  • endospores are internal structures that are super resistant which helps with survival - when environments get really harsh they are able to sustain it
  • the one with the little green dots will have endospores and if you heat it up the ones with the little green dots will survive
• Phase-contrast microscopy

  • Phase ring amplifies differences in the refractive index of cells and surroundings
  • improves the contrast of a sample without the use of a stain
  • allows for the visualization of live samples
  • resulting image is dark cells on a light background
  • because stains need heat fixing and due to this cells are dead and we can't assess motility (movement) and it can also distort some features phase-contrast microscopy helps with this as it amplifies differences without the use of a stain and we can view their motility
• Dark field microscopy

  • specimen is illuminated with a hollow cone of light
  • only refracted light enters the objective
  • specimen appears as a bright object on a dark background
  • used to observe bacteria that don't stain well (ex. treponema pallidum - the causative agent of syphilis)
  • challenging technique and not many labs have them - less common
• Micrographs = resulting image
• Fluorescence microscopy

  • used to visualize specimens that fluoresce
  • emit light of one color when illuminated with another color of light
  • cells may fluoresce naturally
  • ex. photosynthetic cyanobacteria have chlorophyll
  • absorbs light at 430nm (blue-violet)
  • emits at 670 nm (red)
  • Or after staining with fluorescent dye
  • DAPI specifically binds to DNA
• Differential interference contrast (DIC) microscopy

  • uses a polarizer to create two distinct beams of polarized light
  • gives structures such as endospores, vacuoles, and granules a 3D appearance
  • another way to play with contrast - helps with 3D view
  • structures not visible by bright-field microscopy are sometimes visible by DIC