October 6 notes

Visualizing microbes

1. Optics concepts
a. Resolving power (R.P)- Minimum distance that 2 objects can be separated from each other and still be distinguished as 2 objects.
i. RP of microscope in class= .2 micrometer
ii. The smaller the RP=stronger. Microscope can see more detail.

iii. How we see stuff: Stuff bounces off surfaces; we interpret patterns of bounced stuff.
b. Wavelength:
i. Different wavelengths of light give it its “color”
ii. Smaller wavelength of light used, the more detailed we can see (R.P. smaller)
iii. You cant see difference between objects if difference between them is smaller then wavelength of light used.
c. Refractive index- how fast light move through a substance
i. Light moves at different speeds through different substances
ii. Light goes off glass at angle so you don’t see as much as thought with light microscope.
iii. Refractive index for immersion oil is almost the same as that of glass (oil immersion too down R.P.)
2. Bright field microscopy
a. R.P. of best light microscopes=0.2 micrometers or 200 nm
b. Oil vs. air refractive index compared to that of glass
c. Why stain? B/C bacteria are so small we wont be able to see them at all otherwise.
d. Advantages
i. Differential staining techniques (gram stain)
ii. Can judge shapes and sizes
• Hard to see very slender structured organism
• Technique kills organisim, cant see motion

3. Dark field microscopy
a. Light bounces off live microbes , making them grow. Can see shape and how they are moving through the liquid.
b. Advantage: Can watch movement of live microbes. Can see very slender structures that you can barely see with right field microscopy.
i. Disadvantages: Loss of resolution cant see as much detail.

Virus cant be seen on microscopy because they are too small compared to resolving power of microscope.

4. Electron Microscopy
a. Why is the R.P. of an electron microscope so much smaller then that of a light microscope?  R.P. of light microscope is 0.002 nm
i. Electrons smaller than smallest light wavelengths
ii. Electron microscope shoots electrons at a sample=electrons bounce off, bounce pattern=image
b. Advantages and disadvantages of electron microscopy
i. Advantage: Allow to see very small items: viruses, cell membrane layer, can see the shape of ribosome’s.
ii. Disadvantage: very labor-intensive, specimens must be dead, is very expensive
How it works: shoot a beam of electrons at something and then it bounces off. You must remove the water from sample and treat sample with metal.

c. Scanning electron microscopy
i. 3-D surface image
d. Transmission EM
i. Thin cross section image
ii. Cam see inside of cell

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