Introduction to Microbiology & Parasitology

CHAPTER 1
Learning Outcomes

 Define microbiology, pathogen, nonpathogen,

and opportunistic pathogen

 Explain the importance of microbes

 Explain the relationship between microbes

and infectious diseases

 Differentiate between infectious diseases and

microbial intoxications

 Describe the classifications of

microorganisms

What is Microbiology?

 Microbiology is the study of microbes.

 With only rare exceptions, individual
microbes can be observed only with the use
of various types of microscopes.

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 Why Study Microbiology? products or processes is called
biotechnology.
 The microbes that live on and in the human
body are referred to as our indigenous  An antibiotic is a substance produced by a
microbiota. microbe that is effective in killing or
inhibiting the growth of other microbes.
 Indigenous microbiota inhibit the growth of
pathogens in areas of the body where they  In genetic engineering, a gene or genes from
live by occupying space, depleting the food one organism (e.g., from a bacterium, a
supply, and secreting materials. human, an animal, or a plant) is/are inserted
into a bacterial or yeast cell.
 Some microbes that colonize (inhabit) our
bodies are known as opportunistic pathogens
(opportunists) -- the microbes do not cause us
any problems but they have the potential to
cause infections should there be any
opportunity.

 Microbes are essential for life on this planet;

they contribute more oxygen to our
atmosphere than do plants.
 Many microbes are involved in the
decomposition of dead organisms and the
waste products of living organisms –
decomposers / saprophytes

 Decomposition is the process by which

substances are broken down into simpler
forms of matter

 A saprophyte is an organism that lives on

dead or decaying organic matter

 Some microbes are capable of decomposing

industrial wastes (oil spills, for example).

 Bioremediation involves the use of

genetically engineered microbes to clean up
pollutants.
 The study of the relationships between
microbes and the environment is called
microbial ecology

 Microbes serve as important links in food

chains

 The use of living organisms or their

derivatives to make or modify useful

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 First Microorganisms on Earth
 Scientists tell us that Earth was formed about
4.5 billion years ago and, for the first 800
million to 1 billion years of Earth’s existence,
there was no life on this planet.
 Fossils of primitive microbes (as many as 11
different types) found in ancient sandstone
formations in northwestern Australia date back
to about 3.5 billion years ago.
 By comparison, animals and humans are
relative newcomers. Animals made their
appearance on Earth between 900 and 650
million years ago.
 Candidates for the first microbes on Earth are
archaea and cyanobacteria.
 Many people believe that syphilis was carried
to Europe by Native Americans who were
brought to Portugal by Christopher Columbus.
The French called syphilis the Neapolitan
disease; the Italians called it the French or
Spanish disease; and the English called it the
French pox.
 Other names for syphilis were Spanish,
German, Polish, and Turkish pocks. The name
“syphilis” was not given to the disease until
1530.
Pioneers in the Science of Microbiology
 1673-1723, Antoni van Leeuwenhoek
(Dutch) described live microorganisms that he
observed in teeth scrapings, rain water, and
peppercorn infusions.

Earliest Known Infectious Diseases 1861: Louis Pasteur demonstrated that

microorganisms are present in the air.
 The earliest known account of a “pestilence”
occurred in Egypt about 3180 BC.

 This may represent the first recorded epidemic, Results

although words such as pestilence and plague Conditions
were used without definition in early writings.
Nutrient broth placed in flask, Microbial
 Around 1900 BC, near the end of the Trojan heated, not sealed growth
War, the Greek army was decimated by an
epidemic of what is thought to have been
bubonic plague. Nutrient broth placed in flask, No microbial
heated, then sealed growth
 The Ebers papyrus, describing epidemic
fevers, was discovered in a tomb in Thebes, Spontaneous generation or biogenesis?
Egypt; it was written around 1500 BC.

 A disease thought to be smallpox occurred in

China around 1122 BC. Epidemics of plague
occurred in Rome in 790, 710, and 640 BC, and
in Greece around 430 BC.

 There are early accounts of rabies, anthrax,

dysentery, smallpox, botulism, measles,
typhoid fever, typhus fever, diphtheria, and
syphilis. The syphilis story is quite interesting.
It made its first appearance in Europe in 1493.

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  Bacteria that use alcohol and produce acetic
acid spoil wine by turning it to vinegar (acetic
acid).

 Pasteur demonstrated that these spoilage

bacteria could be killed by heat that was not hot
enough to evaporate the alcohol in wine. This
application of a high heat for a short time is
called pasteurization.
 Pasteur’s process involved heating wine to
55°C and holding it at that temperature for
several minutes.
 Today, pasteurization is accomplished by
heating liquids to 63°C to 65°C for 30 minutes
or to 73°C to 75°C for 15 seconds.
 It should be noted that pasteurization does not
Next experiment, Pasteur’s S-shaped flask kept kill all of the microbes in liquids—just the
microbes out but let air in. These experiments form pathogens.
the basis of aseptic technique
The Germ Theory of Disease

 1876: Robert Koch provided proof that a

bacterium causes anthrax and provided the
experimental steps, Koch’s postulates, used to
prove that a specific microbe causes a specific
disease.
 Koch was a physician and Pasteur’s young
rival

 Koch's Postulates are used to prove the cause

of an infectious disease.
 Koch's Postulates are a sequence of
The Golden Age of Microbiology
experimental steps to relate a specific
(1857-1914) microbe to a specific disease.

Beginning with Pasteur’s work, discoveries included

the relationship between microbes and disease,
immunity, and antimicrobial drugs

 Pasteur showed that microbes are responsible

for fermentation.
 Fermentation is the conversation of sugar to
alcohol to make beer and wine.
 Microbial growth is also responsible for
spoilage of food.

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 Classification of Microbes

Taxonomy

 The science of classifying organisms

 Provides universal names for organisms

 Provides a reference for identifying

organisms

 Systematics or phylogeny

- The study of the evolutionary history of

organisms

 All Species Inventory (2001-2025)

- To identify all species of life on Earth

Taxonomic Hierarchy

Domain

Kingdom

Phylum

Class

Order

Family
 1928: Alexander Fleming discovered the first Genus
antibiotic.
 He observed that Penicillium fungus made an Species
antibiotic, penicillin, that killed S. aureus.
 1940s: Penicillin was tested clinically and
mass produced. Mnemonics: Dumb Kings Play Chess On Funny
Green Squares
Modern Developments Binomal

Nomenclature uses the Genus and Species name to

 Bacteriology is the study of bacteria.
identify each creature.
 Mycology is the study of fungi.
- Each name is Latinized
 Parasitology is the study of protozoa and - There is a specific way to write each name.
parasitic worms.
Homo sapiens
 Recent advances in genomics, the study of an
organism’s genes, have provided new tools for  The first word is capitalized
classifying microorganisms
 Name is in italic
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 e.g. Homo sapiens - H. sapiens Classification of Microbes

 Eukaryotic species:

 A group of closely related organisms that

breed among themselves

 Prokaryotic species:

 A population of cells with similar

characteristics

 Clone: Population of cells derived from a

single cell

 Strain: Genetically different cells within

a clone

 Viral species:

 Population of viruses with similar

characteristics that occupies a particular
ecological niche

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Owner: Abby Mauricio
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Introduction to Microbiology & Parasitology
CHAPTER 2
Viewing the Microbial World
Learning Outcomes
 Explain the interrelationships among the
following metric system units of length:
centimeters, millimeters, micrometers, and
nanometers
 State the metric units used to express the sizes of
bacteria, protozoa, and viruses
 Compare and contrast the various types of
microscopes
Sizes of Microbes
 Microbes are very tiny or microscopic
 In microbiology, metric units (primarily
micrometers and nanometers) are used to express
the size of microbes
 It should be noted that the old terms micron (μ) a
and millimicron (mμ) have been replaced by the
terms micrometer (μm) and nanometer (nm),
respectively.
 An angstrom (Å) is 0.1 nm.
 Using this scale, human red blood cells are about
7 μm in diameter.
 In the microbiology laboratory, the sizes of
 Before it can be used to measure objects,
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Units of Measurement
-6 -3
 1 µm = 10 m = 10 mm
-9 -6
 1 nm = 10 m = 10 mm
 1000 nm = 1 µm
 0.001 µm = 1 nm
 The sizes of bacteria and protozoa are usually
expressed in terms of micrometers. For
example, a typical spherical bacterium
(coccus; pl., cocci) is approximately 1μm in
diameter.
 A typical rod-shaped bacterium (bacillus; pl.,
bacilli) is about 1 μm wide x 3 μm long,
although some bacilli are shorter, and some
form very long filaments.
 The sizes of viruses are expressed in terms of
nanometers. Most of the viruses that cause
human disease range in size from about 10 to
300 nm, although some (e.g., Ebola virus, a
cause of hemorrhagic fever) can be as long as
1,000 nm (1 μm).
 Some very large protozoa reach a length of
2,000 μm (2 mm).
cellular microbes are measured using an ocular
micrometer, a tiny ruler within the eyepiece
(ocular) of the compound light microscope.
however, the ocular micrometer must first be
 calibrated, using a measuring device called a
stage micrometer.
 Calibration must be performed for each of the
objective lenses to determine the distance
between the marks on the ocular micrometer.
 The ocular micrometer can then be used to
measure lengths and widths of microbes and
other objects on the specimen slide.
Microscopy: The Instruments
 In a compound microscope the image from the
objective lens is magnified again by the ocular
lens.
 Total magnification = objective lens  ocular lens
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 Resolution is the ability of the lenses to
distinguish two points.
 A microscope with a resolving power of 0.4 nm
can distinguish between two points ≥ 0.4 nm.
 Shorter wavelengths of light provide greater
resolution
Scale
 Refractive index is the light-bending ability of a
medium.
 The light may bend in air so much that it misses
the small high-magnification lens.
 Immersion oil is used to keep light from bending.  Because visible light (from a built-in light bulb)
is used as the source of illumination, the
compound microscope is also referred to as a
compound light microscope.
 It is the wavelength of visible light
(approximately 0.45 μm) that limits the size of
objects that c be seen using the compound light
microscope.
 When using the compound light microscope,
objects cannot be seen if they are smaller than
half of the wavelength of visible light (i.e.,
smaller than about 0.225 µm).

Microscopes

 A microscope is an optical instrument that is used

to observe tiny objects, often objects that cannot
be seen at all with the unaided human eye (the
“naked eye”).
 Each optical instrument has a limit as to what can
be seen using that instrument.
 This limit is referred to as the resolving power
or resolution of the instrument.

Simple Microscopes

 A simple microscope is defined as a microscope

containing only one magnifying lens.
 Actually, a magnifying glass could be considered
a simple microscope.
 Images seen when using a magnifying glass
usually appear about 3 to 20 times larger than the
object’s actual size.
 During the late 1600s, Anton van Leeuwenhoek
used simple microscopes to observe many tiny
objects, including bacteria and protozoa.
 Because of his unique ability to grind glass
lenses, scientists believe that Leeuwenhoek’s
simple microscopes had a maximum magnifying
power of about 3300 (300 times).
Compound Microscopes
 The compound light microscopes used in today’s
laboratories contain two magnifying lens
systems.
 Within the eyepiece or ocular is a lens called the
ocular lens which has a magnifying power of
x10.
 The second magnifying lens system is in the
objective which is immediately above the object
to be viewed.

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 The four objectives used in most laboratory
compound light microscopes are x4, x10, x40,
and x100 objectives.
 The x4 objective is rarely used in microbiology
laboratories.
 Usually, specimens are first observed using the
x10 objective.
 Once the specimen is in focus, the high power or
“high-dry” objective is then swung into position.
This lens can be used to study algae, protozoa,
and other large microorganisms.
 However, the oil-immersion objective
 (total magnification = x 1,000) must be used to
study bacteria,
 because they are so tiny.
 To use the oil immersion objective, a drop of
immersion oil must first be placed between the
specimen and the objective;
 the immersion oil reduces the scattering of light
and ensures that the light will enter the oil-
immersion lens.
 For optimal observation of the specimen, the
light must be properly adjusted and focused.
 The condenser, located beneath the stage,
focuses light onto the specimen, adjusts the
amount of light, and shapes the cone of light
entering the objective.
 Generally, the higher the magnification, the more
light that is needed.
 As magnification is increased, the amount of
light striking the object being examined must
also be increased.
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 There are three correct ways to accomplish this:
 by opening the iris diaphragm in the
condenser,
 by opening the field diaphragm, and
 by increasing the intensity of light being
emitted from the microscope’s light bulb, by
turning the rheostat knob clockwise.
 Turning the knob that raises and lowers the
condenser is an incorrect way to adjust lighting.
 Magnification alone is of little value unless the
enlarged image possesses increased detail and
clarity.
 Image clarity depends on the microscope’s
resolving power (or resolution), which is the
ability of the lens system to distinguish between
two adjacent objects.
 If two objects are moved closer and closer
together, there comes a point when the objects
are so close together that the lens system can no
longer resolve them as two separate objects.
 Knowing the resolving power of an optical
instrument also defines the smallest object that
can be seen with that instrument.
 For example, the resolving power of the unaided
human eye is approximately 0.2 mm.
 The resolving power of the compound light
microscope is approximately 1,000 times better
than the resolving power of the unaided human
eye.
 Using a compound light microscope, we can see
objects down to about 0.2 μm in diameter.

 Because objects are observed against a bright
background (or “bright field”) when using a
compound light microscope, that microscope is
sometimes referred to as a brightfield
microscope.
Brightfield Illumination
 Dark objects are visible against a bright
background.
 Light reflected off the specimen does not enter
the objective lens.
Darkfield Illumination
 If the regularly used condenser is replaced with
what is known as a darkfield condenser,
illuminated objects are seen against a dark
background (or “dark field”), and the microscope
has been converted into a darkfield microscope.
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 Light objects are visible against a dark
background.
 Light reflected off the specimen enters the
objective lens.
 In the clinical microbiology laboratory,
darkfield microscopy is routinely used to
diagnose primary syphilis (the initial stage of
syphilis).
 The etiologic (causative) agent of syphilis—a
spiral-shaped bacterium, named Treponema
pallidum—cannot be seen with a brightfield
microscope because it is thinner than 0.2 μm and,
therefore, is beneath the resolving power of the
compound light microscope.
 T. pallidum can be seen using a darkfield
microscope, however, much in the same way that
you can “see” dust particles in a beam of sunlight.
Darkfield Microscopy
 Other types of compound microscopes include
phase-contrast microscopes and fluorescence
microscopes.
 Phase-contrast microscopes can be used to
observe unstained living microorganisms.
Because the light refracted by living cells is
different from the light refracted by the
surrounding medium, contrast is increased, and
the organisms are more easily seen.
 Fluorescence microscopes contain a built-in
ultraviolet (UV) light source. When UV light
strikes certain dyes and pigments, these
substances emit a longer wavelength light,
causing them to glow against a dark background.
 Fluorescence microscopy is often used in
immunology laboratories to demonstrate that
antibodies stained with a fluorescent dye have
combined with specific antigens; this is a type of
immunodiagnostic procedure.
Phase-Contrast Microscopy
 Accentuates diffraction of the light that passes
through a specimen.
Fluorescence Microscopy
 Uses UV light.
 Fluorescent substances absorb UV light and
emit visible light.
 Cells may be stained with fluorescent dyes
(fluorochromes).
Differential Interference Contrast
 Accentuates diffraction of the light that passes
through a specimen; uses two beams of light.
Confocal Microscopy
 Uses fluorochromes and a laser light.
 The laser illuminates each plane in a specimen to
produce a 3-D image.
Atomic Force Microscopes
 Neither TEMs nor SEMs enable scientists to
observe live microbes because of the required
specimen-processing procedures and subjection
of the specimens to a vacuum.
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 Atomic force microscopy (AFM) enables
scientists to observe living cells at extremely
high magnification and resolution under
physiological conditions.
 Using AFM, it is possible to observe single live
cells in aqueous solutions where dynamic
physiological processes can be observed in real
time.
 Unlike the SEM, which provides a two-
dimensional image of a sample, the AFM
provides a true three-dimensional surface profile.
 Figure 2-14 is a diagrammatic representation of
an AFM. A silicon or silicon nitride cantilever
having a sharp tip (probe) at its end is used to
scan the specimen surface.
 When the tip is brought in proximity to a sample
surface, forces between the tip and the sample
lead to a deflection of the cantilever.
 Typically, the deflection is measured using a laser
spot reflected from the top surface of the
cantilever into an array of photodiodes, creating
an image on a monitor screen.
 Additional information regarding AFM can be
found using an Internet search engine.
Electron Microscopy
 Uses electrons instead of light.
 The shorter wavelength of electrons gives greater
resolution
 Although extremely small infectious agents, such
as rabies and smallpox viruses, were known to
exist, they could not be seen until the electron
microscope was developed.
 It should be noted that electron microscopes
cannot be used to observe living organisms.
 Organisms are killed during the specimen-
processing procedures.
 Even if they were not, they would be unable to
survive in the vacuum created within the electron
microscope.
 Electron microscopes use an electron beam as a
source of illumination and magnets to focus the
beam.
 Because the wavelength of electrons traveling in
a vacuum is much shorter than the wavelength of
visible light— about 100,000 times shorter—
electron microscopes have a much greater
resolving power than compound light
microscopes.
 There are two types of electron microscopes:  Secondary electrons emitted from the specimen
transmission electron microscopes (TEMs) produce the image.
and scanning electron microscopes (SEMs).

Transmission Electron Microscopy (TEM)

 Ultrathin sections of specimens.

 Light passes through specimen, then an
electromagnetic lens, to a screen or film.
 Specimens may be stained with heavy metal
salts.

Electron Microscopes

 A transmission electron microscope (TEM)

has a tall column, at the top of which an electron
gun fires a beam of electrons downward.
 When an extremely thin specimen (less than
1 μm thick) is placed into the electron beam,
some of the electrons are transmitted through the
specimen, and some are blocked.
 An image of the specimen is produced on a
phosphor-coated screen at the bottom of the
microscope’s column.
 The object can be magnified up to approximately
1 million times.
 Thus, using a TEM, a magnification is achieved
that is about 1,000 times greater than the
maximum magnification achieved using a
compound light microscope. Even very tiny
microbes (e.g., viruses) can be observed using a
TEM.
 Because thin sections of cells are examined,
transmission electron microscopy enables
scientists to study the internal structure of cells.
 Special staining procedures are used to increase
contrast between different parts of the cell.
 The first TEMs were developed during the late
1920s and early 1930s, but it was not until the
early 1950s that electron microscopes began to
be used routinely to study cells.
Electron Microscopes
 A scanning electron microscopy (SEM), has a
shorter column, and instead of being placed into
the electron beam, the specimen is placed at the
bottom of the column.
 Electrons that bounce off the surface of the
specimen are captured by detectors, and an image
of the specimen appears on a monitor.
 SEMs are used to observe the outer surfaces of
specimens (i.e., surface detail).
 Although the resolving power of SEMs (about 20
nm) is not quite as good as the resolving power
of TEMs (about 0.2 nm), it is still possible to
observe extremely tiny objects using an SEM.
 SEMs became available during the late 1960s
Scanning Electron Microscopy (SEM)
 1000-10,000; resolution 20 nm

Transmission Electron Microscopy (TEM)

 10,000-100,000; resolution 2.5 n

Scanning Electron Microscopy (SEM)

 An electron gun produces a beam of electrons

that scans the surface of a whole specimen.
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  In a basic dye, the chromophore is a cation.
 In an acidic dye, the chromophore is an anion.
 Staining the background instead of the cell is
called negative staining.

Simple Stains
 Use of a single basic dye is called a simple
stain.
 A mordant may be used to hold the stain or coat
the specimen to enlarge it.

Differential Stains: Gram Stain

 The Gram stain classifies bacteria into gram-

positive and gram-negative.
 Gram-positive bacteria tend to be killed by
Preparation of Specimens for penicillin and detergents.
Light Microscopy  Gram-negative bacteria are more resistant to
antibiotics.
 A thin film of a solution of microbes on a slide is
a smear.
 A smear is usually fixed to attach the microbes to
the slide and to kill the microbes.
Color of Color of
Preparing Smears for Staining Gram + cells Gram – cells
 Live or unstained cells have little contrast with
the surrounding medium.
 However, researchers do make discoveries about Primary stain: Purple Purple
cell behavior looking at live specimens. Crystal violet

Mordant: Purple Purple

Iodine

Decolorizing Purple Colorless

agent:
Alcohol-
acetone

Counterstain: Purple Red

Safranin

 Stains consist of a positive and negative ion.

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Differential Stains: Acid-Fast Stain
 Cells that retain a basic stain in the presence of
acid-alcohol are called acid-fast.
 Non–acid-fast cells lose the basic stain when
rinsed with acid-alcohol, and are usually
counterstained (with a different color basic stain)
to see them.
Special Stains
 Negative staining is useful for capsules.
 Heat is required to drive a stain into endospores.
 Flagella staining requires a mordant to make the
flagella wide enough to see.
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Preparation and Staining of Specimens
 increases visibility of specimen
 accentuates specific morphological features
 preserves specimens
Fixation
 process by which internal and external structures
are preserved and fixed in position
 process by which organism is killed and firmly
attached to microscope slide
 heat fixing
 preserves overall morphology but not
internal structures
 chemical fixing
 protects fine cellular substructure and
morphology of larger, more delicate
organisms
Dyes and Simple Staining
dyes
 make internal and external structures of cell
more visible by increasing contrast with
background
 have two common features
chromophore groups
 chemical groups with conjugated
double bonds
 give dye its color
 ability to bind cells
simple staining
 a single staining agent is used
 basic dyes are frequently used
 dyes with positive charges
 e.g., crystal violet
 Differential Staining

 divides microorganisms into groups based on

their staining properties
 e.g., Gram stain Acid-fast staining
 e.g., acid-fast stain
 particularly useful for staining members of the
Gram staining genus Mycobacterium

 most widely used differential staining procedure e.g., Mycobacterium tuberculosis – causes
 divides Bacteria into two groups based on tuberculosis
differences in cell wall structure
e.g., Mycobacterium leprae – causes leprosy

 high lipid content in cell walls is responsible for

their staining characteristics

primary stain Staining Specific Structures

 Negative staining
mordant  often used to visualize capsules
surrounding bacteria
decolorization  capsules are colorless against a stained
background
 Spore staining
 double staining technique
counterstain  bacterial endospore is one color and
vegetative cell is a different color
 Flagella staining
positive  mordant applied to increase thickness of
negative flagella

METHODS IN LIGHT MICROSCOPY

1. Hanging
 drop preparations
 makes use of a slide with a hollow portion at the
center
 Microorganisms in fluid media
 Size, shape and arrangement of microorganisms
and their motion are observed

Escherichia coli – a gram-negative rod

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 2. Wet Mounts
 Drop of fluid containing cells to be examined is
placed on a slide; covered with cover slip and
observed under the microscope.
 Best observed in diminished light intensity, or if
dark-field or phase contrast microscope is used.
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3. Oil Immersion
 used to increase the resolution of a
microscope by immersing both the objective
lens and the specimen in a transparent oil
 Immersion oils are transparent oils that
have specific optical and viscosity
characteristics necessary for use in
microscopy. An example is cedar wood oil.
 Oil immersion is essential for viewing
individual bacteria or details of the striations
of skeletal muscle.
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