Chapter 3

Observing
Microorganisms
Through a
Microscope

© 2013 Pearson Education, Inc. Lectures prepared by Christine L. Case

Copyright © 2013 Pearson Education, Inc.

Lectures prepared by Christine L. Case
Figure 3.2 Microscopes and Magnification.

Unaided eye
≥ 200 m

Light microscope
200 nm – 10 mm Tick
Scanning Actual size
electron
microscope
10 nm – 1 mm
Red blood cells

Transmission
electron
microscope
10 pm – 100  m
E. coli bacteria

T-even bacteriophages
(viruses)
Atomic force
microscope
0.1 nm – 10nm

DNA double helix

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Units of Measurement

▪ 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

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 Microscopy: The Instruments
Figure 1.2b Anton van Leeuwenhoek’s microscopic observations.

Lens
Location of specimen
▪ A simple microscope on pin
has only one lens
Specimen-positioning
screw
Focusing control

Stage-positioning screw

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Microscope replica
 Robert Hooke’s
Microscope

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Light Microscopy

▪ The use of any kind of microscope that uses visible

light to observe specimens
▪ Types of light microscopy
▪ Compound light microscopy
▪ Darkfield microscopy
▪ Phase-contrast microscopy
▪ Differential interference contrast microscopy
▪ Fluorescence microscopy
▪ Confocal microscopy

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© 2013 Pearson Education, Inc.
Figure 3.1a The compound light microscope.

Ocular lens
(eyepiece)
Remagnifies the Fine focusing knob
image formed by
the objective lens
Coarse focusing knob
Body tube
Transmits the
image from the
objective lens to
the ocular lens
Arm

Objective lenses
Primary lenses that
magnify the
specimen
Stage Holds the
microscope slide
in position
Condenser Focuses
light through specimen

Diaphragm Controls the

amount of light entering the
condenser
Illuminator Light source
Principal parts and
functions
Base

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Compound Light Microscopy
▪ In a compound microscope, the image from the
objective lens is magnified again by the ocular lens
▪ Total magnification = objective lens  ocular lens
Ocular lens
Line of
vision Path of light
Figure 3.1b The compound light
microscope.
Prism

Body tube

Objective lenses

Specimen
Condenser lenses

Illuminator
Base with source of illumination

The path of
light (bottom
to top)
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Compound Light Microscopy

▪ 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

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Compound Light Microscopy

▪ The refractive index is a measure of 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

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Figure 3.3 Refraction in the compound microscope using an oil immersion objective lens.

Unrefracted Oil immersion

light objective lens

Without immersion oil

most light is refracted
and lost
Immersion oil

Air
Glass slide

Condenser
lenses

Condenser

Iris diaphragm
Light source
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Brightfield Illumination

▪ Dark objects are visible against a bright background

▪ Light rays are directed through the specimen.
▪ Light reflected off the specimen does not enter the
objective lens

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Figure 3.4a Brightfield, darkfield, and phase-contrast microscopy.

Eye Eye Eye

Ocular lens
Ocular lens Diffraction plates
Only light reflected Undiffracted light
by the specimen is (unaltered by specimen)
captured by the Objective lens
Objective lens objective lens Refracted or diffracted
light (altered by
Specimen specimen)
Unreflected light Specimen
Condenser lens Condenser lens
Opaque disk Annular diaphragm

Light Light Light

-used with stained specimens

-able to see internal structures and outline of
external structures

Brightfield
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Darkfield Illumination

▪ Light objects are visible against a dark background

▪ Light reflected off the specimen enters the objective
lens

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Figure 3.4b Brightfield, darkfield, and phase-contrast microscopy.

Eye Eye Eye

Ocular lens
Ocular lens Diffraction plates
Only light reflected Undiffracted light
by the specimen is (unaltered by specimen)
captured by the Objective lens
Objective lens objective lens Refracted or diffracted
light (altered by
Specimen specimen)
Unreflected light Specimen
Condenser lens Condenser lens
Opaque disk Annular diaphragm

Light Light Light

-see outline of cells

-see some external and
internal features
-used for living organisms
-used when organism
can’t be seen with
brightfield or when
Darkfield staining distorts/destroys
specimen
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Phase-Contrast Microscopy

▪ Accentuates diffraction of the light that passes

through a specimen

▪ accentuates = makes more prominent, more noticeable

▪ diffraction = bending, scattering of light rays

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Figure 3.4c Brightfield, darkfield, and phase-contrast microscopy.

Eye Eye Eye

Ocular lens
Ocular lens Diffraction plates
Only light reflected Undiffracted light
by the specimen is (unaltered by specimen)
captured by the Objective lens
Objective lens objective lens Refracted or diffracted
light (altered by
Specimen specimen)
Unreflected light Specimen
Condenser lens Condenser lens
Opaque disk Annular diaphragm

Light Light Light

-direct and diffracted light are brought together to

produce the image
-in phase, bright
-out of phase, dark

Phase-contrast
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Differential Interference Contrast
Microscopy
▪ Accentuates diffraction of the light that passes
through a specimen; uses two beams of light
Figure 3.5 Differential interference contrast (DIC) microscopy.

-uses a prism to split the light

-do not need stains
-contrasting colors in 3D

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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 The principle of
immunofluorescence.

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 Fluorescence Microscopy

© 2013 Pearson Education, Inc. Figure 3.6a

Confocal Microscopy
▪ Cells are stained with fluorochrome dyes
▪ Short-wavelength (blue) light is used to excite the dyes
▪ The light illuminates each plane in a specimen to produce a
three-dimensional image
▪ Up to 100 µm deep

Figure 3.7 Confocal Nucleus

microscopy.

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Two-Photon Microscopy
▪ Cells are stained with fluorochrome dyes
▪ Two photons of long-wavelength (red) light are used to excite
the dyes
▪ Used to study cells attached to a surface
▪ Up to 1 mm deep
Food vacuoles
Figure 3.8 Two-photon
microscopy (TPM).

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Scanning Acoustic Microscopy (SAM)
▪ Measures sound waves that are reflected back from an object
▪ Used to study cells attached to a surface; can study living cells
▪ Resolution 1 µm
Figure 3.9 Scanning
acoustic microscopy
(SAM) of a bacterial
biofilm on glass.

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Electron Microscopy

▪ Uses electrons instead of light

▪ The shorter wavelength of electrons gives greater
resolution

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Transmission Electron Microscopy
(TEM)

▪ Ultrathin sections of specimens

▪ A beam of electrons passes through specimen. An
electromagnetic lens is used to focus the beam of
electrons much like the condenser in a light
microscope
▪ Specimens may be stained with heavy-metal salts
▪ Lead, osmium, tungsten, or uranium
▪ Absorb electrons creating image with dark areas in stained region

▪ 10,000–100,000; resolution 2.5 nm

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Figure 3.10a Transmission and scanning electron microscopy.

Electron gun
Primary electron beam
Electron beam
Electromagnetic Electromagnetic lenses
condenser lens Viewing
Specimen Viewing screen
Electromagnetic eyepiece
Electron
objective lens
collector
Electromagnetic
projector lens
Secondary
Fluorescent screen electrons
or photographic
plate Specimen
Amplifier

Transmission
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Scanning Electron Microscopy (SEM)

▪ An electron gun produces a beam of electrons that

scans the surface of a whole specimen
▪ Metal salts (gold, palladium) cover specimen
▪ Secondary electrons emitted from the specimen
produce the image
▪ 1,000–10,000; resolution 20 nm

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Figure 3.10b Transmission and scanning electron microscopy.

Electron gun
Primary electron beam
Electron beam
Electromagnetic Electromagnetic lenses
condenser lens Viewing
Specimen Viewing screen
Electromagnetic eyepiece
Electron
objective lens
collector
Electromagnetic
projector lens
Secondary
Fluorescent screen electrons
or photographic
plate Specimen
Amplifier

Scanning
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Scanned-Probe Microscopy:
Atomic/Molecular Detail
▪ Scanning tunneling microscopy (STM) uses a
metal probe to scan a specimen
▪ Resolution 1/100 of an atom
▪ No special preparation
▪ Uses electrical current

Protein from E.coli

Figure 3.11a Scanned-probe microscopy.

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Scanned-Probe Microscopy:
Atomic/Molecular Detail
▪ Atomic force microscopy (AFM) uses a
metal-and-diamond probe inserted into the
specimen
▪ Produces three-dimensional images

Figure 3.11b Scanned-probe

microscopy.

Toxin from C. perfringens

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Preparing Smears for Staining

▪ Staining: coloring the microbe with a dye that

emphasizes certain structures
▪ Smear: a thin film of a solution of microbes on
a slide
▪ A smear is usually fixed to attach the microbes to
the slide and to kill the microbes

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Preparing Smears for Staining

▪ Live or unstained cells have little contrast with

the surrounding medium. Researchers do make
discoveries about cell behavior by observing live
specimens.

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Preparing Smears for Staining

▪ Stains consist of positive and negative ions

▪ 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

Basic Dyes: Carbolfuchsin, Crystal Violet,

Methylene Blue, Malachite Green, Safranin

Acidic Dyes: Acid Fuchsin, Eosin, Nigrosin

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Simple Stains

▪ Simple stain: use of a single basic dye

▪ A mordant may be used to hold the stain or coat the
specimen to enlarge it

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Differential Stains

▪ Used to distinguish between bacteria

▪ Gram stain
▪ Acid-fast stain

Gram Stain
▪ Classifies bacteria into Gram-positive
or Gram-negative
▪ Gram-positive bacteria tend to be killed by penicillin and
detergents
▪ Gram-negative bacteria are more resistant to antibiotics

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Gram Stain
Color of Color of
Gram-Positive Cells Gram-Negative Cells

Primary Stain:
Purple Purple
Crystal Violet

Mordant:
Purple Purple
Iodine

Decolorizing Agent:
Purple Colorless
Alcohol-Acetone

Counterstain:
Purple Red/Pink
Safranin

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Figure 3.12b Gram staining.

Rod
(gram-negative)

Cocci
(gram-positive)

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Acid-Fast Stain

▪ Stained waxy cell wall is not decolorized by

acid-alcohol
▪ Mycobacterium
▪ Nocardia
Color of Color of
Acid-Fast Non–Acid-Fast
Primary Stain:
Red/Hot Pink Red/Hot Pink
Carbolfuchsin

Decolorizing Agent:
Red/Hot Pink Colorless
Acid-alcohol

Counterstain:
Red/Hot Pink Blue
Methylene Blue

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Figure 3.13 Acid-fast bacteria.

M. bovis

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Special Stains

▪ Used to distinguish parts of cells

▪ Capsule stain
▪ Endospore stain
▪ Flagella stain

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Negative Staining for Capsules
▪ The cells are stained
▪ Negative stain (the background is stained)

Capsules
Figure 3.14a
Special staining.

Negative staining
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Endospore Staining
▪ Primary stain: malachite green, usually with heat
▪ Decolorize cells: water
▪ Counterstain: safranin

Figure 3.14b
Special
staining.

Endospore
Endospore staining

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Flagella Staining
▪ Mordant on flagella
▪ Carbolfuchsin simple stain
Figure 3.14c Special staining.

Flagellum

Flagella staining

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