Microbiology

Chapter 3
Observing Microorganisms through a Microscope
Units of Measurement

Review Table 3.1

• 1 µm = ______ m = ______ mm
• 1 nm = ______ m = ______ mm
• 1000 nm = ______ µm
• 0.001 µm = ______ nm
 Sizes Among Microorganisms
• Protozoa: 100 µm
• Yeasts: 8 µm
• Bacteria: 1 - 5 µm (some much longer than wide)
• Rickettsia: 0.4 µm = _________ nm
• Chlamydia and Mycoplasma: 0.25 µm
• Viruses: 20 – 250 nm
 Principles of the Compound Light Microscope
• In a compound microscope the image from the
objective lens is magnified again by the ocular lens.
• Magnification: Ocular and objective lenses of
compound microscope
• Total magnification =objective lens  ocular lens
• Resolution: Ability of lens to . . .
Maximum resolving power ___ m
• Contrast: Stains change refractive index  contrast
between bacteria and surrounding medium
Fig 3.1
Shorter wavelengths of light
provide greater resolution.
Refractive Index

• Measures light-bending ability of a

medium
• Light may bend in air so much that
it misses the small high-
magnification lens.
• Immersion oil is used to keep light
from bending.

Fig 3.3
Microscopy: The Instruments
Brightfield Microscopy
• Simplest of all the optical
microscopy illumination.
techniques
• Dark objects are visible
against a bright background.
Darkfield Microscopy
• Light objects visible against
dark background.
• used to enhance the contrast
in unstained samples.
• Instrument of choice for
spirochetes
Spirochetes (Treponema pallidum) viewed with darkfield microscope
Phase-Contrast Microscopy
• Accentuates diffraction of
the light that passes
through a specimen.

Figure 3.4c
 Differential Interference Contrast
Microscopy
• Accentuates
diffraction of the
light that passes
through a specimen;
uses two beams of
light.

Figure 3.5
 Fluorescence Microscopy

• Uses UV light.
• Fluorescent substances
absorb UV light and emit
visible light.
• Cells may be stained with
fluorescent dyes
(fluorochromes).
Figure 3.6b
Fig 3.6a
Principle of
Immunofluorescence
 Confocal Microscopy

• Uses fluorochromes and a

laser light.
• The laser illuminates each
plane in a specimen to
produce a 3-D image.

Figure 3.7
Electron Microscopy: Detailed Images of Cell
Parts
Uses electrons, electromagnetic lenses, and
fluorescent screens
Electron wavelength ~ 100,000 x smaller than visible
light wavelength
Specimens may be stained with heavy metal salts
Two types of EMs:?
Transmission Electron Microscopy (TEM)
• Ultrathin sections of specimens.
• Light passes through specimen, then an electromagnetic
lens, to a screen or film.
• Specimens may be stained with heavy metal salts.

Figure 3.8a
Transmission Electron Microscopy (TEM)
• 10,000-100,000; resolution 2.5 nm

Figure 3.8a
Scanning Electron Microscopy (SEM)
• An electron gun produces a beam of electrons that scans
the surface of a whole specimen.
• Secondary electrons emitted from the specimen produce
the image.

Figure 3.8b
Scanning Electron Microscopy (SEM)

• 1000-10,000; resolution 20 nm

Figure 3.8b
 Scanning-Probe Microscopy
• Scanning tunneling microscopy uses a
metal probe to scan a specimen.
• Resolution 1/100 of an atom.
Figure 3.9a

• Atomic force microscopy uses a

metal and diamond probe
inserted into the specimen.
Figure 3.9b
• Produces 3-D images.
Preparation of Specimens for Light Microscopy
• Staining Techniques Provide Contrast
• Smear  air-dry  heat-fix
• A thin film of a solution of microbes on a slide is a smear.
• A smear is usually fixed to attach the microbes to the slide and to
kill the microbes.
• Basic dyes: cationic chromophore
• Acidic dyes: anionic chromophore  negative staining
(Staining the background instead of the cell is called negative
staining.)
• Three types of staining techniques: Simple, differential, and special
 Simple Stains Differential Stains
• Use a single basic dye. React differently with
• A mordant may be used different bacteria
to hold the stain or coat • Gram stain
the specimen to enlarge
it. • Acid fast stain
 Differential Stains: Gram Stain
• The Gram stain classifies bacteria into gram-positive and
gram-negative.
• Gram-positive bacteria tend to be killed by penicillin and
detergents.
• Gram-negative bacteria are more resistant to antibiotics.
Review of different staining techniques
Important Staining Reactions in Microbiology

For Gram stain

technique compare
to Fig 3-12
Differential Stains: Gram Stain
Color of Color of
Gram + cells Gram – cells
Primary stain: Purple Purple
Crystal violet
Mordant: Purple Purple
Iodine
Decolorizing agent: Purple Colorless
Alcohol-acetone
Counterstain: Purple Red
Safranin
Gram Stain

crystal violet

safranin
 Acid Fast Stain
• Cells that retain a basic stain in the presence of acid-alcohol
are called acid-fast.
• Non–acid-fast cells lose the primary stain when rinsed with
acid-alcohol, and are counterstained with a different color
basic stain.
 Special Stains See Fig 3.14

• Endospore stain: Heat is required to

drive a stain into the endospore.
• Flagella staining: requires a mordant
to make the flagella wide enough to
see.
• Capsule stain uses basic stain and
negative stain