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Chapter 1
The History and Scope of Microbiology
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What is microbiology?
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Biology is the study of living organisms
Micro = very small
Microbiology is the study of microbes, which are
extremely small (microscopic) living organisms and
certain non-living entities
these organisms are relatively simple in their construction
and lack highly differentiated cells and distinct tissues
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The Importance of Microorganisms
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most populous group of organisms
are found everywhere on the planet and are essential for life
play a major role in recycling essential elements
Microorganisms are involved in the decomposition of dead
organisms and waste products (fertilizers as returning inorganic
nutrients to the soil, Nitrates, Phosphates etc)
Saprophytes or decomposers are organisms that live on dead
and/or decaying organic matter,
source of nutrients and some carry out photosynthesis,
Photosynthetic algae and bacteria (such as cyanobacteria)
produce much of the oxygen in our atmosphere (more than the
plants)
The use of microbes to clean up toxic wastes and other industrial
waste (oils) products is known as bioremediation
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benefit society by their production of food, beverages,
antibiotics and vitamins
Algae and bacteria serve as food for tiny animals; they are
important links in food chains
Microbes that live in the intestinal tracts of animals aid in the
digestion of food and produce beneficial substances E. coli, vit
K, B1
For many years, microorganisms have been used as “cell
models”; the more that scientists learned about microbial cells,
the more they learned about cells in general
In genetic engineering, a gene or genes from one organism
is/are inserted into a bacterial or yeast cell; the cell that
receives the new gene(s) is then capable of producing the gene
product(s) coded for by the new gene(s)
The use of living organisms or their derivatives to make or
modify useful products or processes is call biotechnology
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Members of the microbial world
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procaryotic cells lack a true membrane-delimited nucleus
eucaryotic cells have a membrane-enclosed nucleus, are more complex
morphologically and are usually larger than procaryotic cells
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Living microbes are known as cellular microbes or
microorganisms; examples include bacteria, archaea
‫العتيقة‬, some algae, protozoa ‫األوليات‬, and some fungi
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Non-living microbes are known as acellular microbes
or infectious particles; examples include viroids,
prions, and viruses
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Microorganisms are ubiquitous (they are found
virtually everywhere)
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Classification schemes
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five kingdom scheme includes Monera
‫األصليات‬, Protista ‫الطالئعيات‬, Fungi,
Animalia and Plantae with microbes
placed in the first three kingdoms
Six kingdom classification (bacteria)
? viruses
three domain alternative, based on a
comparison of ribosomal RNA, divides
microorganisms into Bacteria (true
bacteria), Archaea and Eucarya
(eucaryotes)
The small ribosomal subunit is
composed of only one rRNA molecule,
which is coded for by a gene called the
16S rRNA gene in procaryotes and the
18S rRNA gene in eucaryotes
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Domain Bacteria – all procaryotic
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most are single-celled
most have peptidoglycan in cell wall
can survive broad range of environments
most are non-pathogenic and play major role in nutrient recycling
cyanobacteria produce oxygen as a result of photosynthesis
Domain Archaea – all procaryotic
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procaryotic
distinguished from Bacteria by unique ribosomal RNA sequences
lack peptidoglycan in cell wall
many found in extreme environments
no pathogenic species known
Unusual metabolic characteristics, methanogenes
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Domain Eucarya – all eucaryotic
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animals, plants and eucaryotic microorganisms
 Microorganisms include protists (unicellular algae, protozoa,
slime molds and water molds) and fungi
 Most are larger than procaryotic cells
Viruses (animals and bacteria), viroids (plants), virusoids
(plants, hepaitis), prions(proteins)
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acellular
smallest of all microbes 10000 smaller than bact
cause a range of diseases including some cancers
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Discovery of Microorganisms
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Antony van Leeuwenhoek (16321723)
 first person to observe and
describe microorganisms
accurately
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The Conflict over Spontaneous Generation
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spontaneous generation
 living organisms can develop from nonliving or decomposing
matter
Francesco Redi (1626-1697)
 disproved spontaneous generation for large animals
 showed that maggots on decaying meat came from fly eggs
But could spontaneous generation be true for
microorganisms?
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John Needham (1713-1781)
 his experiment:
mutton broth in flasks  boiled sealed
 results: broth became cloudy and contained microorganisms
Lazzaro Spallanzani (1729-1799)
 his experiment:
broth in flasks sealed  boiled
 results: no growth of microorganisms
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Louis Pasteur (1822-1895)
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his experiments
 placed nutrient solution in flasks
 created flasks with long, curved necks
 boiled the solutions
 left flasks exposed to air
results: no growth of microorganisms
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Final blow to theory of spontaneous generation
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John Tyndall (1820-1893)
 demonstrated that dust carries microorganisms
 showed that if dust was absent, nutrient broths remained sterile, even if
directly exposed to air
 also provided evidence for the existence of exceptionally heat-resistant
forms of bacteria (endospores)
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The Role of Microorganisms in Disease
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was not immediately obvious
establishing connection depended on development of techniques for
studying microbes
once established, led to study of host defenses - immunology
The golden age of microbiology (1857-1914)
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Many disease producing organisms discovered
Microbial metabolism studies undertaken
Microbiological techniques refined
A better understanding of the role of immunity and ways to
control and prevent infection by microbes
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Recognition of the Relationship between Microorganisms
and Disease
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Agostini Bassi (1773-1856)
 showed that a disease of silkworms was caused by a fungus
More evidence…
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M. J. Berkeley (ca. 1845)
 demonstrated that the great Potato Blight of Ireland was caused by
a water mold
Heinrich de Bary (1853)
 showed that smut and rust fungi caused cereal crop diseases
Louis Pasteur
 showed that the pébrine disease of silkworms was caused by a
protozoan
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Other evidence…
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Joseph Lister
 provided indirect evidence that microorganisms were the causal agents of
disease
 developed a system of surgery designed to prevent microorganisms from
entering wounds as well as methods for treating instruments and surgical
dressings
 his patients had fewer postoperative infections
Final proof…
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Robert Koch (1843-1910)
 established the relationship between Bacillus anthracis and anthrax
 used criteria developed by his teacher Jacob Henle (1809-1895)
 these criteria now known as Koch’s postulates
 still used today to establish the link between a particular
microorganism and a particular disease
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Koch’s postulates
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The microorganism must be present in every case of the disease but
absent from healthy individuals.
The suspected microorganism must be isolated and grown in a pure
culture.
The same disease must result when the isolated microorganism is
inoculated into a healthy host.
The same microorganism must be isolated again from the diseased
host.
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The Development of Techniques for Studying Microbial
Pathogens
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Koch’s work led to discovery or development of:
 agar
 petri dish
 nutrient broth and nutrient agar
 methods for isolating microorganisms
Other developments…
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Charles Chamberland (1851-1908)
 developed porcelain bacterial filters used by Ivanoski and
Beijerinck to study tobacco mosaic disease
 determined that extracts from diseased plants had
infectious agents present which were smaller than
bacteria and passed through the filters
 Infectious agents were eventually shown to be viruses
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Immunological Studies
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Edward Jenner (ca. 1798)
 used a vaccination procedure to protect individuals from smallpox
NOTE: this preceded the work establishing the role of microorganisms
in disease
Other developments…
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Pasteur and Roux
 discovered that incubation of cultures for long intervals
between transfers caused pathogens to lose their ability to
cause disease
Pasteur and his coworkers
 developed vaccines for chicken cholera, anthrax, and rabies
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More developments…
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Emil von Behring (1854-1917) and Shibasaburo Kitasato (1852-1931)
 developed antitoxins for diphtheria and tetanus
 evidence for humoral immunity
Elie Metchnikoff (1845-1916)
 discovered bacteria-engulfing, phagocytic cells in the blood
 evidence for cellular immunity
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The Development of Industrial Microbiology and
Microbial Ecology
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Louis Pasteur
 demonstrated that alcohol fermentations and other fermentations
were the result of microbial activity
 developed the process of pasteurization to preserve wine during
storage
Additional Developments…
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Sergei Winogradsky (1856-1953) and Martinus Beijerinck (1851-1931)
 studied soil microorganisms and discovered numerous interesting
metabolic processes (e.g., nitrogen fixation)
 pioneered the use of enrichment cultures and selective media
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First Microorganisms on Earth
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Fossils of primitive microorganisms date back about 3.5
billion years ago.
Candidates for the first microorganisms on Earth are
archaea and cyanobacteria LUCA
Infectious diseases of humans and animals have existed for
as long as humans and animals have inhabited the planet.
Earliest known account of pestilence occurred in Egypt in
about 3180 BC.
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The Scope and Relevance of Microbiology
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importance of microorganisms
 first living organisms on planet
 live everywhere life is possible
 more numerous than any other kind of organisms
 global ecosystem depends on their activities ecology
 influence human society in many ways diseases, benefits, life
Microbiology has basic and applied aspects
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Basic aspects are concerned with individual groups of microbes,
microbial physiology, genetics, molecular biology and taxonomy
Applied aspects are concerned with practical problems –
disease, water, food and industrial microbiology
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Microbiology actually represents many fields of study
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Examples
 medical microbiology is concerned with diseases of humans
and animals
 immunology is concerned with how the immune system protects
a host from pathogens
 microbial ecology is concerned with the relationship of
organisms with their environment
 microbial genetics and molecular biology are concerned with
the understanding of how genetic information functions and
regulates the development of cells and organisms
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The Future of Microbiology:
Challenges and opportunities for future microbiologists
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infectious disease
new and improved industrial processes
microbial diversity and microbial ecology
 less than 1% of earth’s microbial population has been cultured
More challenges and opportunities…
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biofilms
genome analysis
microbes as model systems
assessment of implications of new discoveries and technologies
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Chapter 2
The Study of Microbial Structure: Microscopy
and Specimen Preparation
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Lenses and the Bending of Light
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light is refracted (bent) when passing from one medium to another
refractive index
 a measure of how greatly a substance slows the velocity of light
direction and magnitude of bending is determined by the refractive indices
of the two media forming the interface
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Lenses
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focus light rays at a specific place called the focal point
distance between center of lens and focal point is the focal length
strength of lens related to focal length
 short focal length more magnification
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The Light Microscope
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bright-field microscope
dark-field microscope
phase-contrast microscope
fluorescence microscope
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The Bright-Field Microscope
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produces a dark image against a brighter background
has several objective lenses
 parfocal microscopes remain in focus when objectives are changed
total magnification
 product of the magnifications of the ocular lenses and the objective lenses
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Microscope Resolution
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ability of a lens to separate or distinguish small objects that are close
together
wavelength of light used is major factor in resolution
shorter wavelength  greater resolution
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S. aureus
Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins
S. aureus and red blood cells as seen by light microscopy (photomicrograph)
The Dark-Field Microscope
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Image is formed by light reflected or refracted by specimen
produces a bright image of the object against a dark background
used to observe living, unstained preparations
 For eucaryotes has been used to observe internal structures
 For procaryotes has been used to identify bacteria such as
Treponema pallidum, the causative agent of syphilis
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Darkfield Microscopy of
Treponema pallidum (the bacterium that
causes syphilis)
The Phase-Contrast Microscope
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enhances the contrast between intracellular
structures having slight differences in
refractive index
excellent way to observe living cells
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Especially useful for detecting bacterial
components such as endospores and
inclusion bodies that have refractive
indices different from that of water
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The Differential Interference Contrast Microscope (DIC)
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creates image by detecting differences in refractive indices and
thickness of different parts of specimen
excellent way to observe living cells
 Live, unstained cells appear brightly colored and three-dimensional
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The Fluorescence Microscope
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exposes specimen to ultraviolet, violet, or blue light
specimens usually stained with fluorochromes
shows a bright image of the object resulting from the fluorescent light
emitted by the specimen
Has applications in medical microbiology and microbial ecology studies
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Preparation and Staining of Specimens
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increases visibility of specimen
accentuates specific morphological features
preserves specimens
Fixation
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preserves internal and external structures and fixes them in position
organisms usually killed and firmly attached to microscope slide
 heat fixation – routine use with procaryotes
 preserves overall morphology but not internal structures
 chemical fixation – used with larger, more delicate organisms
 protects fine cellular substructure and morphology
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Dyes and Simple Staining
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dyes
 Ionizable dyes have charged groups
 basic dyes have positive charges
 acid dyes have negative charges
 chromophore groups
 chemical groups with conjugated double bonds
simple stains
 a single stain is used
 use can determine size, shape and arrangement of bacteria
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Differential Staining
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divides microorganisms into groups based on their staining properties
Gram staining
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divides bacteria into two groups based on differences in cell wall
structure
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Acid-fast staining
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particularly useful for staining members of the genus
Mycobacterium
e.g., Mycobacterium tuberculosis – causes
tuberculosis
e.g., Mycobacterium leprae – causes leprosy
 high lipid content in cell walls is responsible for
their staining characteristics
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Staining Specific Structures
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negative staining
 e.g., capsule stain used to visualize capsules surrounding bacteria
 capsules are colorless against a stained background
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endospore staining
 double staining technique
 bacterial endospore is one color and vegetative cell is a
different color
flagella staining
 mordant applied to increase thickness of flagella
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Electron Microscopy
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beams of electrons are used to
produce images
wavelength of electron beam is much
shorter than light, resulting in much
higher resolution
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The Transmission Electron Microscope (TEM)
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electrons scatter when they pass through thin sections of a specimen
transmitted electrons (those that do not scatter) are used to produce image
denser regions in specimen, scatter more electrons and appear darker
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Specimen Preparation
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analogous to procedures used for light microscopy
for transmission electron microscopy, specimens must be cut
very thin
specimens are chemically fixed and stained with electron dense
material
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Other preparation methods
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shadowing
 coating specimen with a thin film of a heavy metal
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•freeze-etching
freeze specimen then fracture along lines of greatest weakness
(e.g., membranes)
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The Scanning Electron Microscope
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uses electrons reflected from the surface of a specimen to create image
produces a 3-dimensional image of specimen’s surface features
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Newer Techniques in Microscopy
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confocal scanning laser (CLSM) microscopy and scanning probe
microscopy
have extremely high resolution
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Confocal Microscopy
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laser beam used to illuminate a variety of planes in the specimen
computer compiles images created from each point to generate a 3dimensional image
used extensively to observe biofilms
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Scanning Probe Microscopy
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scanning tunneling microscope
 steady current (tunneling current) maintained between microscope
probe and specimen
 up and down movement of probe as it maintains current is detected and
used to create image of surface of specimen
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Scanning Probe Microscopy
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atomic force microscope
 sharp probe moves over surface of specimen at constant distance
 up and down movement of probe as it maintains constant distance is
detected and used to create image
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