BSCI 3234 Lecture Notes - Lecture 3: Antonie Van Leeuwenhoek, Electron Microscope, Nanometre
29 Apr 2018
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Introduction to Microbiology-Lecture 3
Tools of the Microbiologist and Introduction to Prokaryotic Cells
What are some of the most important “tools” of a microbiologist?
⚫ Microscopes
⚫ Centrifuges
⚫ Fluorescent antibodies
⚫ Ability to isolate single
bacteria
⚫ Ability to mutate bacteria
⚫ Ability to culture cells
How many different kinds of microscopes can you name?
⚫ Compound microscope
⚫ Dissecting microscope
⚫ Electron microscope
⚫ Scanning electron
microscope
⚫ Phase-contrast microscope
and more
Table 3.1. Metric Units of Measure
kilometer (km)
kilo = 1000
1000 m = 103
meters
3280 ft or 0.62 miles
meter (m)
Standard unit
3.28 ft
centimeter
(cm)
centi = 1/100
0.01 m = 10-2
meters
0.394 inches
millimeter
(mm)
milli =1/1000
0.001 m = 10-3 m
micrometer
(µm)
micro = 1/1,000,000
0.000001 m = 10-6
m
nanometer
(nm)
10-9 m
Angstrom (Å)
Named for Swedish physicist, Anders
Jonas Ångström
10-10 m = 0.1 nm
Early History of Microscopy
• 1665-Robert Hooke of England man built a compound microscope (light passes
through two lenses and magnification is multiplied by each lens).
• 1683 Antoni van Leeuwenhoek describes bacteria and protozoa using a simple single
lens magnifier
• 1826 Joseph Jackson Lister (father of the surgeon Joseph Lister) developed an effective
compound microscope that became the model of modern microscopes.
Design of a Compound Microscope. Fig. 3.1
• illuminator--source of light
• condenser--directs the light and converges it to pass through the specimen
• objective lens--closest to the specimen, provides partial magnification (usually 10X,
40X, and 100X)
• ocular lens or eye piece--lens closest to the eye, provides final magnification. Eye
pieces are usually a standard 10X or 20X
2
Final magnification = magnification of objective X magnification of the ocular lens. Example:
40X objective x 10X ocular = 400X magnification
What is the resolving power (also termed resolution) of a microscope?
• Ability to distinguish between two points at a specified distance apart.
For light microscopes, the resolution or resolving power is 0.2 m.
• The shorter the wavelength, the better the resolution.
• For electron microscopes (EM), the resolving power is 2.5 nm.
• EM uses shorter wavelengths—beam of focused electrons
What is the refractive index of a material?
• The ability of a material to bend light.
Examples: Air refracts (bends) light more than oil, and much of the light is lost. Immersion oil
has a refractive index similar to glass and bends the light less than air and preserves the
direction of the light (Fig. 3.3. Demonstration of use of oil with oil immersion lenses)
Light Microscopy. Uses visible light to observe microscopic specimens
Brightfield Microscopy--Fig. 3.4 (p. 61) Illumination used in most compound microscopes
Staining for light microscopy
• Ability to visualize an object depends on contrast due to differential staining.
• Without staining with dyes, there is little contrast of specimen.
• Without staining, we need special modified microscopes.
Darkfield Microscopy--Fig. 3.4 (p. 61)
• Condenser lens has an opaque disc that blocks light in the center of the condenser
• The only light to reach the objective lens is reflected off the specimen rather than
through the specimen
• Specimen appears light against a dark background
• One group of human pathogens that can be visualized by darkfield microscopy is the
spirochetes.
• What important human diseases are caused by spirochetes?
Phase Contrast Microscopy--Fig. 3.4 (p. 61)
• Light passes through an annular (ring-shaped) diaphragm
• Internal structures are more sharply contrasted
• One set of light rays pass to the eye directly from the light source. A second set of
light rays is reflected by the specimen.
• The light waves either interact to reinforce (bright areas) or interact to interfere (dark
areas) depending whether the two sources are in phase or out of phase. This
produces sharp contrast in living material
Differential Interference Contrast (DIC) Microscopy (Nomarski)--Fig. 3.5
Uses two light beams. Prisms split the beams to generate greater refractive differences
and better resolution. Image appears to be almost 3-dimesional
Fluorescence Microscopy
