z

Chapter 2: Microbial The Microscope

Equipment and
Techniques

A. Equipment § The microscope

z is the
1. Light Microscopy
2. Electron Microscopy microbiologists’s
most important
tool.

The Microscope
Magnification
• The amount the microscope makes the object
bigger. For ex. 10X magnification, makes the object
2 aspects of Microscopy bigger 10X

Magnification Resolving Power or Resolution

• The ability to distinguish fine detail;
• The ability to distinguish 2 objects as separate and distinct.
Resolving Power

WHAT CAUSES MAGNIFICATION? REFRACTION OF LIGHT THROUGH A CONVERGING LENS

Concave Convex

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 Resolving Power: dependent on 2
quantities

Numerical aperture of the lens:

measure of how much light enters
the microscope lens

Wavelength of light used Numerical aperture of the lens: measure of how much light
enters the microscope lens

z
Types of Microscopy

(As the resolving power increases, the distance of the peaks decreases.)
2 General Types
§ Light Microscopy – uses light
as the source of illumination
§ Electron Microscopy- uses a
beam of electrons as source
of illumination
RESOLUTION INCREASES AS THE WAVELENGTH OF LIGHT DECREASES

z z
Types of Light Microscopy Microtome

§ 1.Bright Field - uses a direct light source

-bright light must be focused onto the specimen by

lenses in the condenser
-specimens must be carefully prepared to allow light
to pass through
-appropriate set of lenses (objective and eyepiece)
must be arranged to focus an image of the
specimen in the eye.

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§ 2. Dark Field: dark background

against which dark objects are
illuminated.
-uses a special kind of condenser
that transmit a hollow cone of light
from the source of illumination.

Comparison of Dark Field Microscopy to Optical Enhancement

Dark Field and Rheinberg

•The condenser in a bright field microscope illuminates the

specimen with a solid cone of light.

•Dark field: By inserting an opaque disk (called a "stop") in

the light path so that only a hollow cone of light illuminates
the subject, very fine details can be resolved.
green alga Micrasterias

3. Phase Contrast Microscopy:

• Introduced in the 1930s by a

Dutch physicist, Frits
Zernike
• Improves contrast in
unstained biological
specimens without
significant loss in resolution.
• Based on the principle that
cells differ in refractive
Rheinberg Microscopy: If the central stop is
made of a colored translucent material, the index (a factor by which
image background becomes the color of that light is slowed as it passes
material. through a material)

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Phase Contrast Microscope
§ Device in the
objective lens of the
phase contrast
microscope called
phase ring/phase
plate resulting in a
dark image on a light
microscope

4. Differential Interference
Phase Contrast Contrast (DIC) Microscopy
•Phase contrast greatly
§ Employ a polarizer to
increases the apparent
produce polarized light
contrast between cell
organelles. As seen in the § Uses a polarizer and an
prostate cancer cell (right) analyzer
the phase contrast
background is a mid-line § The polarized light then
gray, phase-dense passes through a prism that
organelles appear dark, and generates 2 distinct beams
there is a bright halo
around the cell.

Differential Interference Contrast (DIC)

• One beauty of DIC is that images have a three-

dimensional appearance with which we are most
familiar in the real world.
• The technique allows the addition of "pseudo-color"
to further improve visual contrast between organelles. Fibroblast cell in culture: A. Bright field B. Phase contrast C. DIC D. Dark field

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 z
4.Fluorescence Microscopy

§ Cells fluoresce either because they contain

naturally fluorescent substances such as chlorophyll
or other fluorescing components

§ Cells are stained with fluorescent dyes and then

illuminated with blue light. The blue light is
absorbed and green light is emitted by the dye.

§ It uses 2 sets of filters.

§ Dyed objects or cells show up in bright color on a
dark background.

Fluorescence Microscopy

One elegant method of improving the contrast between

organelles is by fluorescence microscopy. Fluorescence
is the property of a molecule illuminated with one
wavelength (color) of light to emit a second, longer
wavelength of lower energy. An example is fluorescent
paint that glows when illuminated by a "black light".
Multiple-Fluorescent Probe Microscopy: A cell in mitosis , spindle microtubules
(green) centromeres (red) and the DNA of the condensed chromosome (blue)

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 z
A. Immunofluorescence Microscopy
Uses of Fluorescent microscopes:

§ Antibodies can be used to detect specific molecules

An endothelial cell (above, right) from the bovine pulmonary artery

has been labeled with three different fluorescent stains. The nucleus
fluoresces blue, the microtubules that form the cytoskeleton
fluoresce green, and mitochondria are red.

z Tissue sectioning using z

Immunofluorescence Microscopy B. Confocal Fluorescence

Figure. Multiple labelling Microscopy
immunofluorescence within the
olfactory epithelium showing the
stratified distribution of the mature
(green) and immature (red) olfactory
§A laser is used to
receptor neurons, within the olfactory
mucosa. Their processes combine to illuminate a small
form nerve fibre bundles (red and green
co-localising to orange) that traverse the
lamina propria, to directly innervate the pinhole whose image
brain. Bacterial infection of the brain via
the olfactory pathway is under
investigation using high resolution
is focused at a single
imaging [34] Scale = 50μm. Source:
Source:
point in the specimen
https://www.researchgate.net/publication/2593993
70_Tissue_sectioning_for_epifluorescence_micro
scopy/figures?lo=1 (Nguyen et al., 2013)

Gastrula stage of a Drosophila embryo that has been stained with

a fluorescent probe for actin filaments A. traditional image B. the
confocal image

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 ELECTRON MICROSCOPY
z

§ Uses a beam of
electrons
controlled by a
system of
magnetic fields
§ Dependence of
resolving power
on wavelength
GFP-tagged proteins: A. the upper surface of the leaves of Arabidopsis plant are
covered with huge branched single celled hairs (trichomes) B. Confocal
microscopy reveal the entire actin cytoskeleton of the trichome

z z Transmission Electron Microscope

(TEM)

- Produces its images by

transmitting electrons thru
the specimen; specimen
should be sectioned in
extremely thin slices.
-staining with salts of
uranium or lead

TEM

Two chemical fixatives used in electron microscopy: form cross

link complexes with many organic compounds.

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z
TEM Images:

TEM

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 Transmission Electron Microscope z Scanning Electron Microscope
(SEM)

- Does not transmit electrons

on thin sections, it bombards
the surface of whole, metal
coated specimen with
electrons, while scanning
back and forth over it.

-it involves coating the

specimen with heavy metal

z
Scanning Electron Microscope

SEM

SEM Images

A. SEM of the stereocilia projecting from a hair cell in the inner ear of a bullfrog.
B. DIC C. thin section TEM

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 SEM: developing wheat flower

Scanning Electron Micrograph

This colored electron micrograph shows a T4
bacteriophage, a virus that infects only bacteria.

VIROIDS: Electron Micrograph

TEM of Prions responsible for the “mad cow” disease, or Bovine

Spongiform Encephalopathy disease

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 z
Difference between a SEM and TEM Images Other Types of Microscopy

§ 1. Atomic Force
Microscopy (AFP) or
Scanning Probe
Microscopy (SPM)
§ Gives the ability to
measure intermolecular
forces with the order of
resolution of nanometers

ATOMIC FORCE
MICROSCOPE: provides a 3D
image of biological structures;
tiny stylus is positioned
extremely closed to the
specimen such that weak
repulsive forces are established
between the probe & the atoms

-it has an advantage that

the specimen need not be
treated with fixatives or
coatings; thus allows living
specimens to be viewed,
something that is not
possible with EMs

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2. Scanning Tunneling Microscopy
(STM)

§ is an instrument for
imaging surfaces at the
atomic level.

§ By bringing the tip very

close to the surface,
and by applying an
electrical voltage to the
tip or sample, we can
image the surface at an
extremely small scale –
down to resolving
individual atoms.
High resolution AFM topographs of native membrane proteins.

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STM

Atoms Made Visible

Individual atoms, forming a regular array on the surface
of a crystal of the element germanium, can be seen in
this image made with a scanning tunneling microscope
(STM).

Polysterene particles forming spherical clusters

X-ray Diffraction z
X-RAY DIFFRACTION
Microscope

§ Uses X-RAYS to probe the positions of atoms and

their structural configurations in molecules.

§ X-rays are a form of electromagnetic radiation of

very short wavelength.

§ Many proteins can be crystallized and their three-

dimensional structure can be determined by X-ray
diffraction.

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 The crystal structure of ZnTe(en)0:5 (Zinc Tellerium), determined
by single-crystal X-ray diffraction. Two-monolayerthick ZnTe slabs
are interconnected by ethylenediamine (C2N2H8) molecules
bonded to zinc atoms. Zn-Green, Te-Red, N-Blue,and C-Gray.
Hydrogen atoms are omitted for clarity

Source: www. matternews.com

Reading Assignment
What is the principle behind using oil
immersion in microscopy?

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