Micr Biology: Observing Microorganisms Through A Microscope

TORTORA • FUNKE • CASE
Microbiology
AN INTRODUCTION
EIGHTH EDITION
B.E Pruitt & Jane J. Stein
Observing Microorganisms Through a

Microscope
Units of Measurement
Know the prefixes and be able to
convert from one to another!
• 1 µm = 10-6 m = 10-3 mm
• 1 nm = 10-9 m = 10-6 mm
• 1000 nm = 1 µm
• 0.001 µm = 1 nm
Metric Units
Microscopy: The Instruments
• Types of microscopes:
• Light -simple or
compound
• Simple microscope
has only one lens.
• Brightfield
• Darkfield
• Phase contrast
• Electron
• Scanning
• Transmission
• other Figure 1.2b
• In a compound
microscope the image
from the objective lens
is magnified again by
the ocular lens.
• Total magnification =
objective lens  ocular
lens
Figure 3.1b
• Resolution is the ability of

the lenses to distinguish two
points.
• A microscope with a
resolving power of 0.4 nm
can distinguish between two
points ≥ 0.4 nm.
• Shorter wavelengths of light
provide greater resolution.
• What color has shortest
wavelength?
• What is the resolution of
our microscopes?
• Refractive index is
the light-bending
ability of a
medium.
• The light may bend
in air so much that
it misses the small
high-magnification
lens.
• Immersion oil is
used to keep light
from bending.
The index of refraction of some common materials are given below.material To determine the index of
n material n refraction (n) you use the
Vacuum 1 Crown Glass 1.52 equation n = c/v, where “c” is
Air 1.0003 Salt 1.54 equal to the speed of light in a
Water 1.33 Asphalt 1.635 vacuum and “v” is the speed of
Ethyl Alcohol 1.36 Heavy Flint Glass 1.65
light in a medium.
Fused Quartz 1.4585 Diamond 2.42
Whale Oil 1.460 Lead 2.6

Figure 3.3
Values of n come from the CRC Handbook of Chemistry and Physics
Brightfield Illumination
• Dark objects are

visible against a
bright background.
• Light reflected off the
specimen does not
enter the objective
lens.
Figure 3.4a, b
Darkfield Illumination
• Light objects are

visible against a
dark background.
• Light reflected off
the specimen
enters the
objective lens.
Figure 3.4a, b
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
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
• Uses electrons instead

of light.
• The shorter wavelength
of electrons gives
greater resolution.
Transmission Electron Microscopy (TEM)
• Ultra thin 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
Scanning-Probe Microscopy
• Atomic force
microscopy uses a
metal and diamond
probe inserted into
the specimen.
• Produces 3-D
images.
Figure 3.9b
Preparation of Specimens for
Light Microscopy
• Tools:
• Loops and needles - compare uses
• Stains
• basic (positive ion like crystal violet)
• Acidic (negative ions like India ink)
• Hanging drop
• Used for motility determination
• All other preparations require:
• 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.
Staining
• Coloring microorganisms for better visibility

• Steps for most preparations (except capsule and
hanging drop:
• Smear
• Air dry
• Fix
• Stain
• Rinse
Preparing Smears for Staining
• Live or unstained
cells have little
contrast with the
surrounding
medium.
However,
researchers do
make discoveries
about cell
behavior looking
at live
specimens.
Preparing Smears for Staining
• Stains consist of a positive or negative ion.

• In a basic dye, the chromophore is a cation.
• In an acidic dye, the chromophore is an anion.
• Staining the background instead of the cell is called
negative staining.
Simple Stains
• Use of a single basic dye is called a simple stain.

• A mordant may be used to hold the stain or coat the
specimen to enlarge it.
Differential Stains: Gram Stain
• Differential stains are used to differentiate between

types of bacteria
• Gram
• Acid fast
• 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.
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 Pink
Safranin
Figure 3.10b
Gram Stain
Differential Stains: 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 basic stain when rinsed
with acid-alcohol, and are usually counterstained (with
a different color basic stain) to see them.
Figure 3.11
Special Stains
• Negative staining is
useful for capsules.
• 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

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TORTORA • FUNKE • CASE

B.E Pruitt & Jane J. Stein

Observing Microorganisms Through a

• Resolution is the ability of

Whale Oil 1.460 Lead 2.6

• Dark objects are

• Light objects are

• Uses electrons instead

• 10,000-100,000; resolution 2.5 nm

• Coloring microorganisms for better visibility

• Stains consist of a positive or negative ion.

• Use of a single basic dye is called a simple stain.

• Differential stains are used to differentiate between

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