MICROSCOPIC OBSERVATION of MICROORGANISM

Infection occurs
when a type of
bacteria called
Helicobacter pylori
(H. pylori) infects
stomach.
Helicobacter pylori
A common cause of
peptic ulcers

Chapter 2
•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 (small) and the
Greek word skopos (to look at).

•The microscope is the instrument most characteristic of the microbiology

laboratory.

•The magnification it provides enables us to see microorganism and structures

otherwise invisible to the naked eye.
Microscopes are used to
magnify small objects.

• Because different
microscopes have
different resolution
ranges, the size
of a specimen determines
which microscopes can
be used to view the
specimen effectively.

• A red icon indicates

that a micrograph has
been artificially colorized.
 Compound Light Microscopy
•A modern compound light microscope - a series of lenses , uses visible
light --- source of illumination.
Illuminator
•compound light microscope, ---- examine very small specimens
and fine detail. Condenser lens

•A series of finely ground lenses forms a clearly focused image Specimen

that is many times larger than the specimen itself.
The compound light microscope consists of three sets of lenses Objective lens
Eye piece lens
•Magnification is achieved -----light rays from an
•illuminator (the light source),------pass through Human eye
a condenser, ----- lenses direct the light rays through the specimen.

•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.

•The total magnification of a specimen by multiplying the objective lens

•Most microscopes used in microbiology
---10x (low power), 40x (high power),
and 100x (oil immersion).
•Most ocular lenses magnify specimens
by a factor of 10.
•The total magnifications would be 100x
for low power, 400x for high power, and
1000x for oil immersion.

•Resolution (also called resolving power, d) is the ability of the lenses to

distinguish fine detail and structure.
•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.

Immersion oil is used to improve the resolution of a light microscope at high

power. It has the same refractive index as glass and is placed between the
high power objective and the glass slide. With no layer of air, more light from
the specimen enters the objective lens instead of being refracted outside of it,
resulting in a sharper image.
The resolution (resolving power, d) of a microscope is determined by the
equation:
d = 0.61λ / n sin ϴ

where λ is the wavelength of the light source,

n is the refractive index* of the air or liquid between the objective lens and the
specimen and
ϴ is the aperture angle (a measure of the light-gathering ability of the lens).

The expression n sinϴ is called the numerical aperture and for high quality
lenses has a value of around 1.4.
The lowest wavelength of light visible to the human eye is approximately 400
nm, so the maximum resolving power for a light microscope is approximately
d = 0.61 × 400 / 1.4
= 0.17 μm

*The refractive index is a measure of the light-bending ability of a medium.

•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.
 Supplementary

The numerical aperture of a microscope objective is a measure of its ability

to gather light and resolve fine specimen detail at a fixed object distance.
Darkfield Microscopy

A darkfield 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.

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.

The specimen appears light against a black background—the dark field

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 , the causative agent of syphilis.
Phase-Contrast Microscopy

•Another way to observe microorganisms is with a phase-contrast microscope

•It permits detailed examination of internal structures in living microorganisms

•Not necessary to fix (attach the microbes to the microscope slide) or stain the specimen

•The principle of phase-contrast microscopy is based on the wave nature of light rays

•light rays can be in phase (their peaks and valleys match) or out of phase.

•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.

•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)

•In phase-contrast microscopy, the internal structures of a cell become more sharply
defined.
Supplementary
Supplementary
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 not ,the specimen 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.
The principal use of fluorescence microscopy is a diagnostic technique called
the fluorescent-antibody (FA) technique, or immunofluorescence.

Fluorescent antibodies for a particular antigen are obtained by injecting an

animal with a specific antigen, such as a bacterium, and the animal then begins
to produce antibodies against that antigen. Over time, the antibodies are
removed from the serum of the animal.

A fluorochrome is chemically combined with the antibodies.

These fluorescent antibodies are then added to a microscope slide containing
an unknown bacterium.

If this unknown bacterium is the same bacterium that was injected into the
animal, the fluorescent antibodies bind to antigens on the surface of the
bacterium, causing it to fluoresce.

This technique can detect bacteria or other pathogenic microorganisms, even

within cells, tissues, or other clinical specimens

Immunofluorescence is especially useful in diagnosing syphilis and

rabies.
The principle of immunofluorescence.
(a) A type of fluorochrome is combined
with antibodies against a specific type
of bacterium. When the preparation is
added to bacterial cells on a microscope
slide, the antibodies attach to the bacterial
cells, and the cells fluoresce when
illuminated with ultraviolet light.
(b) In the fluorescent treponemal
antibody absorption (FTA-ABS) test for
syphilis shown here, Treponema pallidum
shows up as green cells against a darker
background.
 Confocal Microscope
Confocal microscopy is a technique in light microscopy used to reconstruct three-
dimensional images.
Specimens are stained with fluorochromes, and one plane of a small region of a
specimen is illuminated with a short-wavelength (blue) light which passes the returned
light through an aperture aligned with the illuminated
region.

Successive planes and regions are illuminated until the

entire specimen has been scanned. Confocal microscopy
uses a pinhole aperture, it eliminates the blurring
exceptionally clear two-dimensional images can be
obtained,

Most confocal microscopes are used in conjunction

with computers to construct three-dimensional images
of entire cells and cellular components.

In addition, confocal microscopy can be used to

evaluate cellular physiology by monitoring the
distributions and concentrations of substances such as
ATP and calcium ions.
 Electron Microscopy

Objects smaller than about 0.2 μm, 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.

The better resolution of electron microscopes is due to the shorter wavelengths

of electrons; the wavelengths of electrons are 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.
 Supplementary

avian influenza virus

 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.

The beam is focused on a small area of the specimen by an electromagnetic

condenser lens—directing the beam of electrons in a straight line to illuminate
the specimen.

Electron microscopes use electromagnetic lenses to control illumination, focus,

and magnification.
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 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.

The transmission electron microscope can resolve objects as close together as

10 pm, and objects are generally magnified 10,000 to 100,000x
Transmission electron microscopy has high resolution and is extremely valuable for
examining different layers of specimens.

Disadvantages : Because electrons have limited penetrating power, only a very thin
section of a specimen (about 100 nm) can be studied effectively. Thus, the
specimen has no three-dimensional aspect.

In addition, specimens must be fixed, dehydrated, and viewed under a high vacuum
to prevent electron scattering. These treatments not only kill the specimen, but also
cause some shrinkage and distortion.

Scanning Electron Microscopy

A scanning electron microscope (SEM) provides striking three-dimensional views of
specimens.
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 of the specimen, 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
 Staining
Definition:
Staining simply defines coloring the microorganisms with a
dye that emphasizes certain structures.

Fixation
Before the microorganisms can be stained, however, they
must be fixed (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.

Smear:
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.
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 basic dyes is in the positive ion; in acidic dyes, it 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,

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.

Valuable for observing overall cell shapes, sizes, and capsules because the
cells are made highly visible against a contrasting dark background.

Examples of acidic dyes are eosin, acid fuchsin, and nigrosin.

Staining methods
A. Simple staining

A simple stain is an aqueous or alcohol solution of a single basic dye.

The primary purpose of a simple stain is to highlight the entire

microorganism so that cellular shapes and basic structures are visible.

The stain is applied to the fixed smear for a certain length of time and
then washed off, and the slide is dried and examined.

Mordant, a chemical is added to the solution to intensify the staining

Some of the simple stains commonly used in the laboratory are

methylene blue, carbolfuchsin, crystal violet, and safranin.
B. Differential staining
Differential stains react differently with different kinds of bacteria

Differences between microbial cells or parts of cell can be seen with

differential staining techniques.
•These involve more than one dye solutions.
•These dyes must be added one after another
Two main differential staining methods:
1. Gram Stain
2. Acid Fast stain
(1) Gram staining
•One of the most important and widely used differential staining
methods
•first described in 1884 by Christian Gram of Denmark.
•classifies bacteria into two large groups, gram-positive and gram-
negative
•The purple dye and the iodine combine in the cytoplasm of each bacterium and
color it dark violet or purple.

•Bacteria that retain this color after decolorization are classified as gram-positive;

•Bacteria that lose the dark violet or purple color after decolorization are classified as
gram-negative.

Gram-negative bacteria are colorless after the alcohol wash, no longer visible.
basic dye safranin is applied; it turns the gram-negative bacteria pink.

•Stains such as safranin that have a contrasting color to the primary stain are called
counterstains.

•Gram-positive bacteria have a thicker peptidoglycan (disaccharides and amino acids)

cell wall than gram-negative bacteria.
•Gram-negative bacteria contain a layer of lipopolysaccharide (lipids and
polysaccharides) as part of their cell wall.

•Inside the cells, the crystal violet and iodine combine to form CV–I. Gram-positive
cells retain the dye and remain purple. Gram-negative cells do not retain the dye;
they are colorless until counterstained with a red dye.
Uses of Gram stain

① Gram stain is very useful for classification of bacteria

② This stain is also particularly useful in the hospital

diagnostic laboratory. For example,

Gram-negative spherical bacteria found in a spinal fluid

specimen strongly suggests meningitis caused by
meningococcus bacterium.

Such information, useful in selecting and antibiotic (or

other treatment) for the patient, is available before results
of culture tests have identified the microorganism.
 Acid-Fast Stain
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,
two important pathogens Mycobacterium tuberculosis, the causative
agent of tuberculosis, and
Mycobacterium leprae , the causative agent of leprosy.

This stain is also used to identify the pathogenic strains of the genus
Nocardia.

Bacteria in the genera Mycobacterium and Nocardia are acid-fast.

1. 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.

2. Then the slide is cooled and washed with water.

3. 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 pink or red color because the carbolfuchsin is more soluble in the cell
wall lipids than in the acid-alcohol .

4. In non–acid-fast bacteria, whose cell walls lack the lipid components, the
carbolfuchsin is rapidly removed during decolorization, leaving the cells
colorless.

5.The smear is then stained with a methylene

blue counterstain.

6. Non–acid-fast cells appear blue after the

counterstain is applied.
 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.

a) Negative Staining for Capsules

Many microorganisms contain a gelatinous covering called a capsule
(determining the organism’s virulence)

b) Endospore (Spore) Staining

An endospore is a special resistant, dormant structure formed
within a cell that protects a bacterium from adverse environmental
conditions.

c) Flagella Staining
Bacterial flagella (singular: flagellum) are structures of
locomotion too small to be seen with a light microscope without
staining.
 C. Special Staining Methods
Stains specific parts of microorganisms