Microscope and Microscopy: Brightfield microscopy,

Darkfield microscopy, Phase contrast microscopy,

Fluorescence microscopy, Electron microscopy, Preparation
and staining of specimens for microscopy
Microscope and Microscopy
• Microorganisms are much too small to be seen with the unaided eye;
they must be observed with a microscope.
• The word microscope is derived from the Latin word micro, which
means small, and the Greek word skopos, to look at.
• Modern microbiologists use microscopes that produce with great
clarity, magnifications that range from ten to thousands of times
greater than those of van Leeuwenhoek's single
• Some microbes are more readily visible than others because of their
larger size or more easily observable features.
• Many microbes, however. must undergo several staining procedures
before their cell walls, capsules, and other structures lose their
colorless natural state.
• The standard unit of length in the metric system is the meter (m). A
major advantage of the metric system is that the units are related to
each other by factors of 10.
• Thus, I m equals 10 decimeters (dm) or 100 centimeters (cm) or 1000
millimeters (mm).
• Units in the U.S. system of measure do not have the advantage of easy
conversion by a single factor of 10. For example, we use 3 feet or 36
inches to equal I yard.
• Microorganisms and their structural components are measured in even
smaller units, such as micrometers and nanometers.
• A micrometer (j-lm) is equal to 0.000001 m (10--6 m).
• The prefix micro indicates that the unit following it should be divided
by I million, or 106 (see the "Exponential Notation" section in
Appendix B).
• A nanometer (nm) is equal to 0.000000001 m (10--9 m). Angstrom (A)
was previously used for 10-10 m, or 0.1 nm.
• The simple microscope used by van Leeuwenhoek in the seventeenth
century had only one lens and was similar to a magnifying glass.
• However, van Leeuwenhoek was the best lens grinder in the world in his
day.
• His lenses were ground with such precision that a single lens could magnify
a microbe 300X.
• His simple microscopes enabled him to be the first person to see bacteria
• Contemporaries of van Leeuwenhoek, such as Robert Hooke, built
compound microscopes, which have multiple lenses.
• In fact, a Dutch spectacle maker, Zaccharias Janssen, is credited with
making the first compound microscope around 1600.
• However, these early compound microscopes were of poor
quality and could not be used to see bacteria.
• It was not until about 1830 that a significantly better microscope
was developed by Joseph Jackson Lister (the father of Joseph
Lister).
• Various improvements to Lister's microscope resulted in the
development of the modern compound microscope, the kind used
in microbiology laboratories today.
• Microscopic studies of live specimens have revealed dramatic
interactions between microbes
Light Microscopy:
Compound light Microscopy
• A modern compo und light microscope has a series of lenses and uses
visible light as its source of illumination (Figure 3.1a).
• With a compound light microscope, we can examine very small
specimens as well as some of their fine detail.
• A series of finely ground lenses (Figure 3.1b) forms a clearly focused
image that is many times larger than the specimen itself.
• This magnification is achieved when light rays from an illuminator,
the light source, pass through a condenser, which has lenses that direct
the light rays through the specimen.
• From here, light rays pass into the objective lenses, the lenses closest
to the specimen.
• The image of the specimen is magnified again by the ocular lens, or
eyepiece.
• We can calculate the total magnification of a specimen by multiplying
the objective lens magnification (power) by the ocular lens
magnification(power)
• Most microscopes used in microbiology have several objective lenses,
including 10X (low power).
• 40X (high power), and 100X (oil immersion, which is described
shortly). Most ocular lenses magnify specimens by a factor of 10.
• Multiplying the magnification of a specific objective lens with that of
the ocular, we see that the total magnifications would be 100X for low
power, 400X for high power, and 1000X for oil immersion.
• Some compound light microscopes can achieve a magnification of
2000X with the oil immersion lens.
• Resolution (also called resolving power) is the ability of the lenses to
distinguish fine detail and structure.
• Specifically, it refers to the ability of the lenses to distinguish two points a
specified distance apart.
• For example, if a microscope has a resolving power of 0.4 nm, it can
distinguish two points if they are at least 0.4 nm apart.
• A general principle of microscopy is that the shorter the wavelength of light
used in the instrument, the greater the resolution.
• The white light used in a compound light microscope has a relatively long
wavelength and cannot resolve structures smaller than about 0.2 µm.
• Considerations limit the magnification achieved by even the best compound
light microscopes to about 2000X.
• By comparison, van Leeuwenhoek's microscopes had a resolution of I µm.
• To obtain a clear, finely detailed image under a compound light
microscope, specimens must be made to contrast sharply with their
medium (substance in which they are suspended).
• To attain such contrast, we must change the refractive index of specimens
from that of their medium.
• The refractive index is a measure of the light-bending ability of a
medium.
• We change the refractive index of specimens by staining them, a
procedure we will discuss shortly.
• Light rays move in a straight line through a single medium.
• After the specimen is stained, when light rays pass through the two
materials (the specimen and its medium) with different refractive
indexes, the rays change direction (refract) from a straight path by
bending or changing angle at the boundary between the materials and
increase the image's contrast between the specimen and the medium.
• As the light rays travel away from the specimen, they spread out and
enter the objective lens, and the image is thereby magnified.
• The immersion oil has the same refractive index as glass, so the oil
becomes part of the optics of the glass of the microscope.
• Unless immersion oil is used, light rays arc refracted as they enter the
air from the slide, and the objective lens would have to be increased in
diameter to capture most of them.
• The oil has the same effect as increasing the objective lens diameter;
therefore, it improves the resolving power of the lenses.
• If oil is not used with an oil immersion objective lens, the image
becomes fuzzy, with poor resolution.
• Under usual operating conditions, the field of vision in a compound
light microscope is brightly illuminated.
• By focusing the light, the condenser produces a brightfield
illumination
Dark field Microscopy:
• A dark field microscope is used to examine live microorganisms that
either are invisible in the ordinary light microscope, cannot be stained
by standard methods, or are so distorted by staining that their
characteristics then cannot be identified.
• Instead of the normal condenser, a darkfield microscope uses a
darkfield condenser that contains an opaque disk.
• The disk blocks light that would enter the objective lens directly
• Only light that is reflected off (turned away from) the specimen enters
the objective lens.
• Because there is no direct background light, the specimen appears
light against a black background-the dark field (Figure 3.4b).
• This technique is frequently used to examine unstained
microorganisms suspended in liquid.
• One use for darkfield microscopy is the examination of very thin
spirochetes, such as Treponema pallidum (tre-po- ne'ma pal'li-d um),
the causative agent of syphilis.
Phase-Contrast Microscopy:
• Phase-contrast microscopy is especially useful because it
permits detailed examination of internal structures in living
microorganisms.
• It is not necessary to fix (attach the microbes to the
microscope slide) or stain the specimen procedures that could
distort or kill the microorganisms.
• The principle of phase-contrast microscopy is based on the wave
nature of light rays and the fact that light rays can be in phase (their
peaks and valleys match) or out of phase.
• If the wave peak of light rays from one source coincides with the
wave peak of light rays from another source, the rays interact to
produce reinforcement (relative brightness).
• However, if the wave peak from one light source coincides with the
wave trough from another light source, the rays interact to produce
interference (relative darkness).
• In a phase-contrast microscope, one set of light rays comes directly
from the light source.
• The other set comes from light that is reflected or diffracted from a
particular structure in the specimen.
• Diffraction is the scattering of light rays as they "touch" a specimen's
edge.
• The diffracted rays are bent away from the parallel light rays that pass
farther from the specimen.
• When the two sets of light rays- direct rays and reflected or diffracted
rays- are brought together, they form an image of the specimen on the
ocular lens, containing areas that are relatively light (in phase),
through shades of gray, to black (out of phase; Figure 3.4c).
• In phase-contrast microscopy, the internal structures of a cell become
more sharply defined
Fluorescence Microscopy:
• Fluorescence microscopy takes advantage of fluorescence, the ability
of substances to absorb short wavelengths of light (ultraviolet) and
give off light at a longer wavelength (visible).
• Some organisms fluoresce naturally under ultraviolet light; if the
specimen to be viewed does not naturally fluoresce, it is stained with
one of a group of fluorescent dyes called Fluorochromes.
• When microorganisms stained with a fluorochrome are examined
under a fluorescence microscope with an ultraviolet or near ultraviolet
light source, they appear as luminescent, bright objects against a dark
background.
• Fluorochromes have special attractions for different microorganisms.
• For example, the fluorochrome auramine O, which glows yellow when
exposed to ultraviolet light, is strongly absorbed by Mycobacterium
tuberculosis, the bacterium that causes tuberculosis.
• When the dye is applied to a sample of material suspected of
containing the bacterium, the bacterium can be detected by the
appearance of bright yellow organisms against a dark background.
• Bacilllls anthracis, the causative agent of anthrax, appears apple green
when stained with another fluorochrome, fluorescein isothiocyanate
(FITC).
• The principal use of fluorescence microscopy is a diagnostic technique
called the fluorescent-antibody (FA) technique, or
immunofluorescence.
• Antibodies are natural defense molecules that are produced by humans
and many animals in reaction to a foreign substance, or antigen.
• Fluorescent antibodies for a particular antigen are obtained as
follows: an animal is injected with a specific antigen, such as a
bacterium, and the animal then begins to produce antibodies against
that antigen.
• After a sufficient time, the antibodies are removed from the serum of
the animal.
• Shown in Figure 3.6a, a fluorochrome is chemically combined with
the antibodies
Electron Microscopy :
• Objects smaller than about 0.2 µ.m, such as viruses or the internal
structures of cells, must be examined with an electron microscope.
• In electron microscopy, a beam of electrons is used instead of light.
Like light, free electrons travel in waves.
• The resolving power of the electron microscope is far greater than that
of the other microscopes described here so far.
• The better resolution of electron microscopes is due to the shorter
wavelengths of electrons; the wavelengths of electrons arc about
100,000 times smaller than the wavelengths of visible light.
• Thus, electron microscopes are used to examine structures too small to
be resolved with light microscopes.
• Images produced by electron microscopes are always black and white,
but they may be colored artificially to accentuate certain details.
• Instead of using glass lenses, an electron microscope uses
electromagnetic lenses to focus a beam of electrons onto a specimen.
• There are two types of electron microscopes: the transmission electron
microscope and the scanning electron microscope
Transmission Electron:
• Microscopy In the transmission electron microscope (TEM), a finely
focused beam of electrons from an electron gun passes through a
specially prepared, ultrathin section of the specimen (Figure 3.108).
• The beam is focused on a small area of the specimen by an
electromagnetic condenser lens that performs roughly the same
function as the condenser of a light microscope-directing the beam of
electrons in a straight line to illuminate the specimen.
• Electron microscopes use electromagnetic lenses to control illumination,
focus, and magnification.
• Instead of being placed on a glass slide, as in light microscopes, the
specimen is usually placed on a copper mesh grid.
• The beam of electrons passes through the specimen and then through an
electromagnetic objective lens, which magnifies the image.
• Finally, the electrons are focused by an electromagnetic projector lens
(rather than by an ocular lens as in a light microscope) onto a fluorescent
screen or photographic plate.
• The final image. called a transmission electron micrograph. appears as
many light and dark areas, depending on the number of electrons absorbed
by different areas of the specimen.
Scanning Electron Microscopy:
• The scanning electron microscope (SEM) overcomes the problem of
sectioning associated with a transmission electron microscope.
• A scanning electron microscope provides striking three -dimensional
views of specimens (Figure 3.1 Db).
• In scanning electron microscopy, an electron gun produces a finely
focused beam of electrons called the primary electron beam.
• These electrons pass through electromagnetic lenses and are directed
over the surface of the specimen.
• The primary electron beam knocks electrons out of the surface and the
secondary electrons thus produced are transmitted to an electron
collector, amplified, and used to produce an image on a viewing screen
or photographic plate.
• The image is called a scanning electron micrograph.
• This microscope is especially useful in studying the surface structures
of intact cells and viruses.
• In practice, it can resolve objects as dose together as 10 nm, and
objects are generally magnified 1000 to 1O,000X.
Preparation and staining of specimens for microscopy
• Preparing Smears for Staining Most initial observations of
microorganisms are made with stained preparations.
• Staining simply means coloring the microorganisms with a dye that
emphasizes certain structures.
• Before the microorganisms can be stained, however, they must be
ftxed (attached) to the microscope slide.
• Fixing simultaneously kills the microorganisms and fixes them to the
slide.
• It also preserves various parts of microbes in their natural state with
only minimal distortion .
• When a specimen is to be fixed, a thin film of material containing the
microorganisms is spread over the surface of the slide.
• This film, called a smear, is allowed to air dry. In most staining
procedures the slide is then fixed by passing it through the flame of a
• Bunsen burner several times, smear side up, or by covering the slide with
methyl alcohol for I minute.
• Stain is applied and then washed off with water; then the slide is blotted
with absorbent paper.
• Without fixing, the stain might wash the microbes off the slide.
• The stained microorganisms are now ready for microscopic examination.
• Stains are salts composed of a positive and a negative ion,
one of which is colored and is known as the chromophore.
• The color of so-called basic dyes is in the positive ion; in
acidic dyes, is in the negative ion.
• Bacteria are slightly negatively charged at pH 7.
• Thus, the colored positive ion in a basic dye is attracted to the
negatively charged bacterial cell.
• Basic dyes, which include crystal violet, methylene blue,
malachite green, and safranin, are more commonly used than
acidic dyes.
• Acidic dyes are not attracted to most types of bacteria because the dye's
negative ions are repelled by the negatively charged bacterial surface, so
the stain colors the background instead.
• Preparing colorless bacteria against a colored background is called
negative staining.
• It is valuable for observing overall cell shapes, sizes, and capsules because
the cells are made highly visible against a contrasting dark background
• Distortions of cell size and shape are minimized because fixing is not
necessary and the cells do not pick up the stain.
• Examples of acidic dyes are eosin, acid fuchsin, and nigrosin.
To apply acidic or basic dyes, microbiologists use three
kinds of staining techniques: simple, differential, and
special.
Simple Stains/ Staining:
• A simple stain is an aqueous or alcohol solution of a single basic dye.
• Although different dyes bind specifically to different parts of cells, the
primary purpose of a simple stain is to highlight the entire
microorganism
• Cellular shapes and basic structures are visible.
• The stain is applied 10 the fixed smear for a certain length of lime and
then washed off, and the slide is dried and examined.
Simple Staining
Differential Stains/Staining:

• Unlike simple stains, differential stains react differently

with different kinds of bacteria and thus can be used to
distinguish them.
• The differential stains most frequently used for bacteria
are the Gram stain and the acid-fast stain.
Gram Stain / Staining:
• The Gram stain was developed in 1884 by the Danish bacteriologist
Hans Christian Gram.
• It is one of the most useful staining procedures because it classifies
bacteria into two large groups: gram-positive and gram-negative.
• In this procedure (Figure 3.12a),
- A heat-fixed smear is covered with a basic purple dye, usually crystal
violet.
- Because the purple stain imparts its color to all cells, it is referred to as
a primary stain.
- After a short time, the purple dye is washed off, and the smear is
covered with iodine, a mordant.
• When the iodine is washed off, both gram- positive and gram-
negative bacteria appear dark violet or purple.
• Next, the slide is washed with alcohol or an alcohol-acetone
solution.
• This solution is a decoloruing agent, which removes the purple from
the cells of some species but not from others.
• The alcohol is rinsed off, and the slide is then stained with safranin,
a basic red dye.
• The smear is washed again, blotted dry, and examined
microscopically.
Acid-Fast Stain/ Staining:
• Another important differential stain (one that differentiates bacteria into
distinctive groups) is the acid-fast stain, which binds strongly only to
bacteria that have a waxy material in their cell walls.
• Microbiologists use this stain to identify all bacteria in the genus
Mycobacterium, including the two important pathogens
• Mycobacterium tuberculosis, the causative agent of tuberculosis, and
Mycobactcrium leprae (lep' rn), the causative agent of leprosy.
• This stain is also used to identify the pathogenic strains of the genus
Nocardia (no-kar'de-a).
• Bacteria the genera Mycobacterium and Nocardia are acid-fast
• In the acid-fast staining procedure, the red dye carbolfuchsin
is applied to a fixed smear, and the slide is gently heated for
several minutes.
• Heating enhances penetration and retention of the dye.
• Then the slide is cooled and washed with water.
• The smear is next treated with acid-alcohol, a decolorizer,
which removes the red stain from bacteria that are not acid -
fast.
• The acid-fast microorganisms retain the red color because
the carbolfuchsin is more soluble in the cell wall lipids than
in the acid-alcohol (Figure 3.13).
• In non acid fast bacteria whose cell walls lack the lipid
components, the carbolfuchsin is rapidly removed during
decolorization, leaving the cells colorless.
• The smear is then stained with a methylene blue counterstain.
• Non-acid-fast cells appear blue after application of the
counterstain.
Special Stains:
• Special stains are used to color and isolate specific parts of
microorganisms, such as endospores and flagella, and to
reveal the presence of capsules.
Negative Staining for Capsules
• Many microorganisms contain a gelatinous covering called a capsule.
which we will discuss in our examination of the prokaryotic cell
• In medical microbiology, demonstrating the presence of a capsule is a
means of determining the organism's virulence, the degree to which a
pathogen can cause disease.
• Capsule staining is more difficult than other types of staining
procedures because capsular materials are soluble in water and may be
dislodged or removed during rigorous washing.
•
• To demonstrate the presence of capsules, a microbiologist
can mix the bacteria in a solution containing a fine colloidal
suspension of colored particles (usually India ink or nigrosin)
to provide a contrasting background and then stain the
bacteria with a simple stain, such as safranin
• Because of their chemical composition, capsules do not
accept most biological dyes, such as safranin, and thus appear
as halos surrounding each stained bacterial cell.
Endospore (Spore) Staining:
• An endospore is a special resistant, dormant structure formed
within a cell that protects a bacterium from adverse
environmental conditions.
• Although endospores arc relatively uncommon in bacterial
cells, they can be formed by a few genera of bacteria.
• Endospores cannot be stained by ordinary methods, such as
simple staining and Gram staining, because the dyes do not
penetrate the wall of the endospore
• The most commonly used endospore stain is the Schaeffer
Fulton endospore stain (Figure 3.14b).
• Malachite green, the primary stain, is applied to a heat-fixed
smear and heated to steaming for about 5 minutes.
• The heat helps the stain penetrate the endospore wall.
• Then the preparation is washed for about 30 seconds with
water to remove the malachite green from all of the cells'
parts except the endospores.
• Next, safranin, a counterstain, is applied to the smear to stain
portions of the cell other than endospores.
• In a properly prepared smear, the endospores appear green
within red or pink cells.
• Because endospores are highly refractive, they can be
detected under the light microscope when unstained, but they
cannot be differentiated from inclusions of stored material
without a special stain .
Flagella Staining:
• Bacterial flagella (singular: flagellum) are structures of loco -
motion too small to be seen with a light microscope without
staining.
• A tedious and delicate staining procedure uses a mordant and
the stain carbolfuchsin to build up the diameters of the
flagella until they become visible under the light micro -
scope (Figure 3.14c).
• Microbiologists use the number and arrangement of flagella
as diagnostic aids.