Microscopy

Types of microscope

  • Light microscope

  • Laser scanning confocal microscope

  • Electron microscope

    • Scanning electron microscope (SEM)

    • Transmission electron microscope (TEM)

History of microscopy

microscopy timeline`

Light microscopes

Labelled light microscope

Compound microscope

  • 2 lenses

  • objective lens magnifies the image

  • eyepiece lens magnifies further

  • allows higher magnification vs simple light microscope

  • less chromatic aberration

  • some opaque specimen can be viewed from above

  • compound light microscope

Magnification vs resolution

  • magnification

    • How many times larger the image is compared to the object

  • resolution

    • the ability to distinguish separate objects

    • higher resolution = more detail

Making permanent slides

  • Fixing

    • chemicals like formaldehyde used to preserve specimen in as close to natural state as possible

  • Sectioning

    • specimen dehydrated with alcohol then mounted in wax to form hard block

    • microtome (special sharp knife) used to cut very thin sections

  • Staining

    • specimen often treated with multiple stains to highlight different structures

  • Mounting

    • specimen secured onto a microscope slide and a coverslip placed on top

Sample preparation methods

  • dry mount

    • object to be viewed is placed on a slide and covered with a cover slip

  • wet mount

    • specimen to be viewed is placed on a slide and a drop of liquid added

    • cover slip lowered carefully to avoid air bubbles

  • smear slide

    • sample added to one end of the slide and a second slide pulled back until it reaches the sample

    • slide is then pushed across the surface of the lower side to create a thin smear spreading out sample in a thin layer

  • squash slide

    • wet mount slide prepared

    • lens tissue placed over the coverslip and gently pressed

Methods of staining

  • Positive staining

    • crystal violet or methylene blue

    • positively charged dyes so attracted to negatively charged cell structures

    • leads to staining of cell parts so they are more easily distinguished from cytosol (cell cytoplasm)

    • positive staining

  • negative staining

    • congo red or nigrosin

    • negatively charged so repelled by cell structures

    • leave cells unstained but colour the background so cells stand out

    • negative staining

Differential staining can distinguish between different organisms or different organelles in the same organism

Gram staining

  • used to separate bacteria into two groups

    • gram positive

    • gram negative

    • gram positive vs gram negative bacteria

  • method

    • add crystal violet stain

    • add iodine (fixes dye)

    • wash with alcohol

    • counterstain with safranin

  • gram positive bacteria retain the crystal violet stain and appear blue

  • gram negative bacteria lose the crystal violet stain when washed with alcohol and so appear red due to counterstain

Acid fast

  • used to identify mycobacterium species from other bacteria

    • mycobacteria are red

    • other bacteria species are blue

  • acid fast method