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Chapter 3:
Observing Microorganisms
Through a Microscope
Units of Measurement
Microorganisms
Appendix D, “Exponential Notation” section
Microscopy: The Instruments
• Light microscopy: use of
any microscope that uses
visible light to view a
specimen
Magnifies specimen
• Compound microscope:
series of lenses
Magnifies specimen
image
image
Concentrates light onto
specimen
MagTotal = Magobjective x Magocular
Figure 3.1b
Microscopy: The Instruments
• Resolution: the ability of the lenses to distinguish two
points as separate (i.e. see fine detail)
─ Expressed as limit of resolution (or resolving power)
◦ Limit of resolution (D): inversely proportional to
resolution
− Smaller D = better resolution
◦ i.e. A microscope with a limit of resolution of 50 nm can
distinguish between two points at least 50 nm apart
─ Shorter wavelengths of light provide greater resolution
Light Microscopy
• Refractive index: the
light-bending ability
─ Staining changes the
refractive index of a
specimen to increase
its contrast with the
background
• Immersion oil acts as
an extension of the
lensmore light is
trapped
Figure 3.3
Light Microscopy:
Brightfield Illumination
• Dark objects are visible
against a bright
background
• Staining is necessary to
increase a specimen’s
refractive index relative
to the background’s
─ Organisms are killed
Figure 3.4a, b
Electron Microscopy
• Uses electron beam instead of lightbeam
─103-105 factor increase in resolution compared
to visible light microscopy
Transmission Electron Microscopy
(TEM)
• Electron beam: 0.006 nm wavelength
─ Vis light: 760-400 nm
• Electrons pass through the specimen, then an
electromagnetic lens, to a screen or film
• Specimens may be stained with heavy metal salts
(enhance electron absorption by specimen)
Figure 3.8a
Transmission Electron Microscopy
(TEM)
• 10,000-100,000X magnification
• Limit of resolution 2.5 nm
• Micrographs are black and white, but false color can be
added electronically
Figure 3.8a
Scanning Electron Microscopy
(SEM)
• Beam of electrons scan the surface of a whole
specimen
• Secondary electrons emitted from the specimen
produce the image
Figure 3.8b
Scanning Electron Microscopy
(SEM)
• 1,000-10,000X magnification
• Limit of resolution 20 nm
Figure 3.8b
Brightfield
illumination
(light microscopy)
Electron
microscopy
Preparation of Specimens for
Light Microscopy
• Smear: a thin film of a solution of microbes on
a slide
• A smear is usually heat-fixed to
─ Attach the microbes to the slide
─ Kill the microbes
Preparing Smears for Staining
• Stains are salts that consist of a positive and negative
ion
─ Acidic dye: the chromophore is an anion
─ Basic dye: the chromophore is a cation
Crystal violet:
• A mordant may be used to intensify staining in one of
several ways (help retain the dye, coat a structure to
enlarge it, etc.)
Simple Staining Techniques
Simple stain:
• Use of a single basic dye
─ Bacteria are negatively charged at
neutral pH
www.bact.wisc.edu
Negative stain:
• Use of acidic dye to stain the
background instead of the cells
microbiology.scu.edu.tw
Differential Stains: Gram Stain
• Differential stains react differently with different
types of bacteria
─ All bacteria are not stained the same color in the end
• The Gram stain classifies bacteria into gram-positive
and gram-negative groups
─ Gram-positive bacteria tend to be killed by penicillin
and detergents
─ Gram-negative bacteria tend to be more resistant to
some antibiotics, but more susceptible to physical
stress
Differential Stains: Gram Stain
Figure 3.10b
Differential Stains: Acid-Fast Stain
• Cells that retain a basic stain in the presence of acidalcohol are called acid-fast
Figure 3.11
Special Stains
Special stains are used to stain specific cell structures
• Heat is required to drive a stain into endospores
• Flagella staining requires a mordant to make the
flagella wide enough to see
Figure 3.12a-c