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2. MICROSCOPY TECHNIQUES : THE
INSTRUMENT & DYES AND STAINING
Summary of Microscopic Instruments and their Characteristics
INSTRUMENT MECHANISM
Light Microscopes visible light passes through a
series of lenses to produce a
magnified image.
a. Bright Field Illuminates the field of view
evenly
b. Phase Contrast Illuminates the filed of view
evenly
c. Interference Two light beams pass through
the specimen and then
recombine
d. Dark-Field Light is directed toward the
specimen and then recombine
e. Fluorescence Projects ultraviolet light, causing
fluorescent molecules in the
specimen to emit longer
wavelength light
f. Confocal Mirrors scan a laser beam across
successive regions and planes of
a specimen. From that data, a
computer constructs an image
ELECTRON MICROSCOPES Use electrons beams in place of
visible to produce the magnified
image
a. Transmission Transmits a bean of electrons
through a specimen
b. Scanning A beam of electrons scans back
and forth over the surface of a
specimen
SCANNING PROBE Make it possible to view images
MICROSCOPES on an atomic scale
a. Scanning tunnelling A sharp metallic probe causes
electrons to tunnel between the
probe and a conductive surface
b. Atomic Force A tip bends in response to even the
slightest force between it and the
sample
USES / COMMENT
Relatively easy to use.
Considerably less expensive than
confocal and electron
microscopes.
Most common type of
microscope
Make unstained cells more
readily visible
Causes the specimen to appear
as 3D image
Make unstained cells more
readily visible ; organisms stand
out as bright objects against a
dark background
Used to observe cells that have
been stained or tagged with a
fluorescent dye
Used to construct a 3D
structure ; provides detailed
sectional views of intact cells
Can clearly magnify images
100,000x
Elaborate specimen preparation,
which may introduce artifacts, is
required.
Used for observing surface
details; produces 3D effect
Produce a map showing the
bumps and valleys of the atoms
on that surface
First scanning probe microscope
developed.
Can operate in air and in liquids.
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MICROSCOPIC TECHNIQUES : Dyes and Staining

STAIN CHARACTERISTICS
Simple Stains  Employ a basic dye to impart a color to a cell. Easy way to
increase the contrast between otherwise colorless cells and a
transparent background.

(See steps)

 Spread thin film specimen over slide

 Allow to dry
 Pass slide through flame to fix specimen
 Flood with stain. Rinse and dry
 Examine with microscope

Differential Stains Are used to distinguish one group of bacteria from another. They take
advantage of the fact that certain bacteria have distinctly different
chemical structures in some of their components.

Most frequently used differential staining techniques are the GRAM

STAIN and ACID-FAST STAIN.
(1 )GRAM STAIN Used to separate bacteria into tow major groups, GRAM-Positive and
Gram-Negative.

The staining characteristics of these groups reflect a fundamental

difference in the chemical structure of their cell walls. This is by far the
most widely used staining procedure.

See Steps

Step 1 : Crystal Violet

(Primary Stain)

Step 2 : Iodine (Mordant)

Step 3 : Alcohol (decolorizer)

Step 4 : Safranin (Counterstain)

(2) ACID-FAST STAIN Used to detect members of the genus Mycobacterium in a specimen.
Due to the lipid composition of their cell walls, these organisms do not
readily take up stains.
Harsh methods are needed to stain these microorganisms.
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Ex. Tuberculosis, Hansen’s Disease (Leprosy)

Special Stains Stain specific structures inside or outside of a cell.
(1) Capsule Stain Because the viscous capsule does not readily take up stains, it stands out
against a stained background. This is an example of a negative stain.
(2) Endospore Stain Stains endospores, a type of dormant cell that does not readily take up
stains These are produced by the genera Bacillus and Clostridium
(3) Flagella Stain The staining agent adheres to and coats the otherwise thin flagella,
enabling them to be seen with the light microscope
Fluorescent Dyes and Tags Fluorescent dyes and tags absorb ultraviolet light and then emit light of
a longer wavelength. They are used in conjunction with a fluorescent
microscope.
(1) Fluorescent Dyes Some fluorescent dyes bind to compounds found in all cells ; others bind
to compounds specific to only certain types of cells
(2) Fluorescent Tags Antibodies to which a fluorescent molecule has been attached are used
to tag specific molecules

SUMMARY OF PROKARYOTIC CELLS

Structure Characteristics

Extra Cellular
Filamentous Appendages Composed of subunits of proteins that form a
helical chain
(1) Flagella Provides the most common mechanism of motility
(2) Pili Different types of pili have different functions. The
common types, often called fimbrae, enable cells
to ahdere to surfaces. A few types mediate
twitching or gliding motility. Sex pili are involved in
a mechanism of DNA transfer
Surface Layers

1. Glycocalyx Layer outside the cell wall, usually made of

polysaccharide.
2. Capsule Distinct and gelatinous. Enables bacteria to adhere
to specific surfaces; allow some organisms to
thwart innate defense systems and thus cause
disease.
3. Slime Layer Diffuse and irregular. Enables bacteria to adhere to
specific surfaces.
CELL WALL Peptidoglycan is the molecule common to all
bacterial cell walls. Provides rigidity to prevent the
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cell from lysing.

(1) Gram Positive
(2) Gram Negative
CELL BOUNDARY
Thick layer of peptidoglycan that contains teichoic
acids and lipoteichoic acids.
Thin layer of peptidoglycan surrounded by an
outer membrane. The outer leaflet of the outer
membrane is lipopolysaccharide.

1. Cytoplasmic Membrane Phospholipid bilayer embedded with proteins. A

barrier between the cytoplasm and the outside
environment. Also functions as a discriminating
conduit between the cell and its surroundings.
INTRACELLULAR
(1) DNA Contains the genetic information of the cell
Chromosomal Carries the genetic information that is essential to
a cell. Typically a single, circular, double-stranded
DNA molecule.
Plasmid Carries genetic information that my be
advantageous to a cell in certain situations
(2) Endospore A type of dormant cell that is extraordinary
resistant to damaging conditions including heat,
desiccation, ultraviolet light, and toxic chemicals.
(3) Gas vesicles Small, rigid structures that provides buoyancy to a
cell.
(4) Granules Accumulations of high molecular weight polymers,
which are synthesized from a nutrient that a cell
has in relative excess
(5) Ribosomes Intimately involved in protein synthesis. Two
subunits, 30S and 50S, join to form the 70S
ribosome, which serves as the structure that
facilitates the joining of amino acids.

A SUMMARY OF EUKARYOTIC CELLS STRUCTURES

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PLASMA MEMBRANE Asymmetric lipid bilayer embedded with proteins.

Selective permeability, conduit to external
environment.
INTERNAL PROTEIN STRUCTURES
(1) Cilia Appear to project out of a cell. Beat in a synchrony
to provide movement.
(2) Cytoskeleton Dynamic filamentous network that provides
structure to the cell.
(3) Flagella Appear to project out of a cell. Propel or push the
cell with a whiplike or thrashing motion.
(4) Ribosomes Two subunits, 60S and 40S, join to fomr the 80S
ribosome.
MEMBRANE BOUND ORGANELLES
Chloroplasts Harvest the energy of sunlight to generate ATP.
The ATP is then used to fuel synthesis of organic
compounds.
Endoplasmic Reticulum Site of synthesis of macromolecules destined for
other organelles or the external environment.
a. Rough Attached ribosomes extrude the proteins they are
synthesizing through pores that lead into the
lumen of the organelle.
b. Smooth Site of lipid synthesis and degradation, and Ca2+
storage.
Golgi Apparatus Site where macromolecules that are synthesized in
the endoplasmic reticulum are modified before
they are transported in vesicles to other
destinations.
Lysosome Digestion of foodstuffs
Mitochondria Harvests the energy released during the
degradation of organic compounds to generate
ATP. Within the highly folded inner membrane are
the components of the electron transport chain
and the proteins that use proton motive force to
synthezie ATP
Nucleus Contains the DNA

Peroxisome Oxidation of lipids and toxic chemicals occurs.