2

Microscopy

Copyright McGraw-Hill Global Education Holdings, LLC. Permission required for reproduction or display.

Microscopy
Microorganisms range in size from the
smallest viruses which are measured in
nanometers (nm), to the largest protists and
bacteria, which can be about 200
micrometers (m).

Lenses and the Bending of

Light
Light is refracted (bent) when
passing from one medium to
another
Refractive index
a measure of how greatly a
substance slows the velocity of
light

Direction and magnitude of

bending is determined by the
refractive indices of the two media
forming the interface

Lenses
Focus light rays at a
specific place called
the focal point
Distance between
center of lens and
focal point is the
focal length
Strength of lens
related to focal
length
short focal length
more
magnification

The Light Microscope

Many varieties
bright-field microscope
dark-field microscope
phase-contrast microscope
fluorescence microscope
confocal microscope

Compound microscopes
image formed by action of 2 lenses

The Bright-Field Microscope

Produces a dark image
against a brighter
background
Has several objective
lenses
parfocal microscopes
remain in focus when
objectives are changed

Total magnification
product of the
magnifications of the
ocular lenses and the
objective lenses

Microscope Resolution
Ability of a lens to separate or distinguish
small objects that are close together
Wavelength of light used is major factor in
resolution
shorter wavelength greater resolution

Working distance
distance

between the front surface of lens and

surface of cover glass or specimen when it is in
sharp focus

If air is replaced with immersion oil, many light rays

that did not enter the objective due to reflection and
refraction at the surfaces of the objective lens and
slide will now do so. This results in an increase in
resolution and numerical aperture.

10

The Dark-Field Microscope

Image is formed by light
reflected or refracted by
specimen
Produces a bright image of
the object against a dark
background
Used to observe living,
unstained preparations
used to observe internal
structures in eukaryotic
microorganisms
used to identify bacteria
such as Treponema
pallidum, the causative
agent of syphilis

11

The Phase-Contrast Microscope

Converts differences in
refractive index/cell density
into detected variations in
light intensity
Some light rays from hollow
cone of light passing through
unstained cell slowed/out of
phase (dark against bright
background)
Excellent way to observe
living cells

12

13

The Differential Interference

Contrast Microscope (DIC)
Creates image by detecting
differences in refractive indices
and thickness of different parts
of specimen
Excellent way to observe living
cells
live, unstained cells appear
brightly colored and threedimensional
cell walls, endospores, granules,
vacuoles, and nuclei are clearly
visible
14

The Fluorescence Microscope

Exposes specimen to ultraviolet, violet, or blue light
Specimens usually stained with fluorochromes
Shows a bright image of the object resulting from
the fluorescent light emitted by the specimen
Has applications in medical microbiology and
microbial ecology studies

15

Fluorescence Microscopy
Essential tool in microbiology
fluorochrome-labeled probes, such as
antibodies, or fluorochrome dyes tag specific
cell constituents for identification of unknown
pathogens
localization of specific proteins in cells

16

Confocal Microscopy
Confocal scanning
laser microscopy
(CLSM) creates
sharp, composite 3D
image of specimens
by using laser beam,
aperture to eliminate
stray light, and
computer interface
Numerous
applications including
study of biofilms
17

Preparation and Staining of

Specimens
Increases visibility of specimen
Accentuates specific morphological features
Preserves specimens

18

Fixation
Preserves internal and external structures and fixes
them in position
Organisms usually killed and firmly attached to
microscope slide
heat fixation routinely used with bacteria and
archaea
preserves overall morphology but not internal structures

chemical fixation used with larger, more delicate

organisms
protects fine cellular substructure and morphology

19

Dyes and Simple Staining

Dyes
make internal and external structures of cell
more visible by increasing contrast with
background

20

Dyes and Simple Staining

Dyes
Ionizable dyes have charged
groups
basic dyes have positive charges
acid dyes have negative charges

Simple stains
a single stain is used
use can determine size, shape,
and arrangement of bacteria

21

Differential Staining
Divides microorganisms into groups based on
their staining properties
e.g., Gram stain
e.g., acid-fast stain

Differential stain used to detect presence or

absence of structures
endospores, flagella, capsules

22

Gram Staining
Most widely used
differential staining
procedure
Divides bacteria into
two groups, Grampositive and Gramnegative, based on
differences in cell
wall structure

23

Acid-Fast Staining
Particularly useful for staining members of the
genus Mycobacterium
e.g., Mycobacterium tuberculosis causes
tuberculosis
e.g., Mycobacterium leprae causes leprosy
high lipid content in cell walls (mycolic acid) is
responsible for their staining characteristics

24

Staining Specific Structures

Endospore staining
heated, double-staining technique
bacterial endospore is one color and vegetative
cell is a different color

Capsule stain used to visualize polysaccharide

capsules surrounding bacteria
negative stain - capsules may be colorless against
a stained background

Flagella staining
mordant applied to increase thickness of flagella
25

Electron Microscopy
Electrons replace light as the illuminating
beam
Wavelength of electron beam is much
shorter than light, resulting in much higher
resolution
Allows for study of microbial morphology in
great detail

26

27

The Transmission Electron

Microscope (TEM)
Electrons scatter when
they pass through thin
sections of a specimen
Transmitted electrons
are under vacuum which
reduces scatter and are
used to produce clear
image
Denser regions in
specimen scatter more
electrons and appear
darker

28

29

30

Specimen Preparation
Analogous to procedures used for light
microscopy
For transmission electron microscopy,
specimens must be cut very thin
Specimens are chemically fixed and stained
with electron dense materials, such as heavy
metals, that differentially scatter electrons

31

Other Preparation Methods

Negative stain
heavy metals do not
penetrate the specimen but
render dark background
used for study of viruses,
bacterial gas vacuoles

Shadowing
coating specimen with a thin
film of a heavy metal on only
one side
useful for viral morphology,
flagella, DNA

32

Other Preparation Methods

Freeze-etching
freeze specimen then
fracture along lines of
greatest weakness (e.g.,
membranes)
allows for 3-D observation
of shapes of intracellular
structures
reduces artifacts

33

The Scanning Electron Microscope

Uses electrons excited from
the surface of a specimen
to create detailed image
Produces a realistic 3D
image of specimens
surface features
Can determine actual in situ
location of microorganisms
in ecological niches
34