Chapter 2 - Basic Components of Living Systems

Light microscope

• two lenses

  • objective and eyepiece

  • allows for higher magnification

• portable

• simple preparation

• natural colour is seen (or stains)

• x2000 magnification

• resolving power = 200nm

• specimens can be living or dead

Dry mount

• solid specimens sliced thinly (sectioning)

• cover slip placed over sample

Wet mount

• specimens suspended in water/immersion oil

• cover slip placed at angle

• aquatic samples/living organisms

Squash slides

• wet mount prepared

• lens tissue used to press down cover slip

• soft samples

Smear slides

• edge of a slide at 45 degrees used to smear sample

• cover slip placed on top

Positively charged stains

• crystal violet

• methylene blue

• attracted to negatively charged materials in cytoplasm

Negatively charged stains

• nigrosin

• congo red

• repelled by negative cytosol

• cells stay unstained but stand out against the background

Gram stain

• separates bacteria into Gram-positive and Gram-negative

• crystal violet is applied to the slide

• then iodine applied (fixes dye)

• washed with alcohol

• Gram-positive bacteria retain the stain

• Gram-negative bacteria lose the stain

• these are stained with safranin dye (counterstain)

Acid-fast

• differentiates species of mycobacterium

• lipid carries carbolfuchsin dye into cells

• cells washed with acid-alcohol solution

• mycobacterium retain the stain

Stages in pre-prepared slides

• fixing

  • chemicals e.g. formaldehydes used to preserve specimens

• sectioning

  • specimens dehydrated with alcohols and place in a mould

  • thinly sliced

• staining

  • treated with stains

• mounting

  • placed on a slide with a cover slip

Magnification

• how many times larger the image is than the actual size of the object

Resolution

• the ability to distinguish between different objects

Magnification calculation

image size / actual size

Microscope calibration

1. put stage micrometer in place and the eyepiece graticule in the eyepiece

2. focus the scale on the micrometer slide

3. line up the micrometer scale with the eyepiece scale

4. xEPG = y SM

5. 100 SM =1mm

6. each devision = 1mm/100 = 0.01mm

7. 0.01mm x y = 0.01ymm = 10yμm

8. 1EPG = number of micrometers / number of graticule divisions

9. 1EPG = 10y/x

10. magnification factor = 10y/x

11. graticule divisions x magnification factor = measurement

Electron microscopy

• beam of electrons (wavelength < 1nm)

• more detail of ultrastructure

• electrons have a smaller wavelength than light waves

• magnification up to 500,000

• expensive

• very large

• complex sample preparation

• material often distorted

• vacuum required

• black & white images produced (coloured digitally)

• specimens are dead

Transmission electron microscope

• beam of electrons transmitted through a specimen

• best resolution - 0.5nm

• 2D images

Scanning electron microscope

• beam of electrons is sent across the surface of a specimen

• reflected electrons are collected

• resolving power = 3-10nm

• produces 3D images

Artefact

• a visible structural detail caused by processing the specimen

Examples of artefacts

• trapped bubbles

• dust