21BTC202T - MICROBIOLOGY

Unit 1: Microscopy and Structure of Prokaryotes

Unit 1: Microscopy and Structure of Prokaryotes

• Introduction to Microbiology.

• Characterization, Classification, and Identification of microbes.

• Microscopy - Light, Electron, and Advanced Microscopy.

• Structure of prokaryotes - Bacteria.

• Structure of prokaryotes - Mycoplasma.

• Actinomycetes - Morphology, Structure, Cultivation, Reproduction, and Pathogenicity

 Introduction to Microbiology
• Microbiology is the study of microorganisms, a large and diverse group of microscopic organisms that exist as
single cells or cell clusters; it also includes viruses, which are microscopic but not cellular.

• The science of microbiology revolves around two interconnected themes:

(1) understanding the nature and functioning of the microbial world, and

(2) applying our understanding of the microbial world for the benefit of humankind and planet Earth.

• As a basic biological science, microbiology uses microbial cells to probe the fundamental processes of life.

• Microbiologists have developed a sophisticated understanding of the chemical and physical basis of life and
have learned that all cells share much in common.

• As an applied biological science, microbiology is at the forefront of many important breakthroughs in human
and veterinary medicine, agriculture, and industry.

• From infectious diseases to soil fertility to the fuel you put in your automobile, microorganisms affect the
everyday lives of humans in both beneficial and detrimental ways.
• Microorganisms are defined as those organisms
too small to be seen clearly by the unaided eye.
• They are generally 1 millimeter or less in diameter.
• Microbiology is the study of all living organisms
that are too small to be visible with the naked eye.
• It is derived from Greek words:
Mikros – which means small
Bios – which means life
Logos – which means study of
• This includes bacteria, archaea, viruses, fungi,
prions, protozoa and algae, collectively known as
'microbes'.
• These microbes play key roles in nutrient cycling,
biodegradation/biodeterioration, climate change,
food spoilage, the cause and control of disease,
and biotechnology.
 Prokaryotes and Eukaryotes

• Examination of the internal structure of cells reveals two

patterns, called prokaryote and eukaryote.
• Prokaryotes include the Bacteria and the Archaea and
consist of small and structurally rather simple cells.
• Eukaryotes are typically much larger than prokaryotes and
contain an assortment of membrane enclosed cytoplasmic
structures called organelles.
• These include, most prominently, the DNA-containing
nucleus but also mitochondria and chloroplasts, organelles
that specialize in supplying the cell with energy, and
various other organelles.
• Eukaryotic microorganisms include algae, protozoa and
other protists, and the fungi.
• The cells of plants and animals are also eukaryotic.
Classification of living organisms

SYSTEM GIVEN BY BASIS

2 kingdom Linneaus Cellwall

Ernst Cellularity
3 kingdom
Haeckel level

Compartmen
talization of
4 kingdom Copeland
cell
organelles

Cell type,
RH wall, mode
5 kingdom
Whittaker of nutrition,
motility
 5 kingdom classification

• proposewd by RH Whittaker in 1969.

• Criteria for classification

• Complexity of cell structure – Prokaryote, Eukaryote

• Complexity of organism – Unicellular, Multicellular

• Mode of nutrition – plant (autotroph) Fungi (heterotroph and

saprobic absorption) and animals (heterotroph and ingestion)
• Lifestyle – producers (plant), consumers (animals) decomposers
(fungi)
• Phylogenetic relationship – prokaryote to eukaryote, unicellular to
multicellular
Major groups of microbes
 DISCOVERY ERA
“Spontaneous generation”

 Aristotle (384-322) and others believed that living

organisms could develop from non-living materials.
 In 13th century, Rogen Bacon described that the
disease caused by a minute “seed” or “germ”.

Antony Van Leeuwenhoek (1632 – 1723)

 Descriptions of Protozoa, basic types of bacteria,
yeasts and algae.
 Father of Bacteriology and protozoology.
 In 1676, he observed and described microorganisms
such as bacteria and protozoa as “Animalcules”.
 The term microbe is used by Sedillot in 1878.
TRANSITION ERA
Francesco Redi (1626 - 1697)
 He showed that maggots would not arise from
decaying meat, when it is covered.
John Needham (1713 – 1781)
 Supporter of the spontaneous generation theory.
 He proposed that tiny organism(animalcules)
arose spontaneously on the mutton gravy. He
covered the flasks with cork as done by Redi, Still
the microbes appeared on mutton broth.

Lazzaro spallanzai (1729 – 1799)

 He demonstrated that air carried germs to the
culture medium.
 He showed that boiled broth would not give rise
to microscopic forms of life.
 GOLDEN ERA
Louis Pasteur
 He is the father of Medical Microbiology.
 He pointed that no growth took place in
swan neck shaped tubes because dust and
germs had been trapped on the walls of the
curved necks but if the necks were broken off
so that dust fell directly down into the flask,
microbial growth commenced immediately.

 Pasteur in 1897 suggested that mild heating

at 62.8°C (145°F) for 30 minutes rather than
boiling was enough to destroy the undesirable
organisms without ruining the taste of the
product, the process was called Pasteurization.
 He invented the processes of pasteurization,
fermentation and the development of effective
vaccines ( rabies and anthrax).
 Pasteur demonstrated diseases of silkworm was
due to a protozoan parasite.
Contributions of Loius pasteur
 He coined the term “microbiology”, aerobic,
anaerobic.
 He disproved the theory of spontaneous
germination.
 He demonstrated that anthrax was caused by
bacteria and also produced the vaccine for the
disease.
 He developed live attenuated vaccine for the
disease.
John Tyndall (1820 - 1893)
 He discovered highly resistant bacterial
structure, later known as endospore.
 Prolonged boiling or intermittent heating was
necessary to kill these spores, to make the
infusion completely sterilized, a process known as
Tyndallisation.

Lord Joseph Lister (1827-1912)

 He is the father of antiseptic surgery.
 Lister concluded that wound infections too
were due to microorganisms.
 He also devised a method to destroy
microorganisms in the operation theatre by
spraying a fine mist of carbolic acid into the air.
Robert Koch (1893-1910)
 He demonstrated the role of bacteria in
causing disease.
 He perfected the technique of isolating
bacteria in pure culture.
 Robert Koch used gelatin to prepare solid
media but it was not an ideal because
(i) Since gelatin is a protein, it is
digested by many bacteria capable of
producing a proteolytic exoenzyme gelatinase
that hydrolyses the protein to amino acids.
(ii) It melts when the temperature
rises above 25°C.
Koch’s Postulates
Fanne Eilshemius Hesse (1850 - 1934)
 One of Koch's assistant first proposed the use
of agar in culture media.
 It was not attacked by most bacteria.
 Agar is better than gelatin because of its
higher melting pointing (96°c) and solidifying
(40 – 45°c)points.

Richard Petri (1887)

 He developed the Petri dish (plate), a
container used for solid culture media.
Edward Jenner (1749-1823)
 First to prevent small pox.
 He discovered the technique of
vaccination.
Alexander Flemming
 He discovered the penicillin from
Penicillium notatum that destroy several
pathogenic bacteria.
Paul Erlich (1920)
 He discovered the treatment of syphilis by
using arsenic.
 He Studied toxins and antitoxins in
quantitative terms & laid foundation of
biological standardization.
 IMPORTANT DISCOVERIES

Bacteria
 Hansen (1874) – Leprosy bacillus
 Neisser (1879) – Gonococcus
 Ogston (1881) – Staphylococcus
 Loeffler (1884) – Diphtheria bacillus
 Roux and Yersin – Diphtheria toxin
Viruses
 Beijerinck (1898) - Coined the term Virus for filterable infectious agents.
 Pasteur developed Rabies vaccine.
 Charles Chamberland, one of Pasteur’s associates constructed a porcelain
bacterial filter.
 Twort and d’Herelle - Bacteriophages.
 Edward Jenner - Vaccination for Smallpox.
 MODERN ERA
Nobel Laureates
Year Nobel laureates Contribution

1901 Von behring Dipth antitox

1902 Ronald Ross Malaria

1905 Robert Koch TB
1908 Metchnikoff Phagocytosis
1945 Flemming Penicillin
1962 Watson, Crick Structure DNA
1968 Holley, Khorana Genetic code
1997 Pruisner Prions
2002 Brenner, Hervitz Genetic regulation of organ
development &cell death
 Characterization, Classification and Identification of microorganisms

• Many different approaches are used to classify and identify microorganisms that have been isolated and grown
in pure culture.

• For clarity, we divide these approaches into two groups:

• classical and molecular.

• The most durable identifications are those that are based on a combination of approaches.

Classical Characteristics

• Classical approaches to taxonomy make use of morphological, physiological, biochemical, and ecological
characteristics.

• These characteristics have been employed in microbial taxonomy for many years and form the basis for
phenetic (phenotypic) classification.

• When used in combination, they are quite useful in routine identification of well-characterized microbes.
Morphological Characteristics

• Morphological features are important in

microbial taxonomy for many reasons.

• Morphology is easy to study and analyze,

particularly in eukaryotic microorganisms and
more complex bacteria and archaea.

• In addition, morphological comparisons are

valuable because structural features depend on
the expression of many genes and are usually
genetically stable.

• Thus morphological similarity often is a good

indication of phylogenetic relatedness.
Physiological and Metabolic Characteristics

• Physiological and metabolic characteristics are useful

because they are directly related to the nature and activity
of microbial proteins.

• For instance, the detection of specific end products of

fermentation in a newly discovered microorganism reveals
the presence of specific catabolic enzymes and the genes
that encode them.

• Therefore analysis of characteristics, such as energy

metabolism and nutrient transport, provides an indirect
comparison of microbial genomes.
 Biochemical Characteristics
• Among the more useful biochemical characteristics used in
microbial taxonomy are bacterial fatty acids, which can be analyzed
using a technique called fatty acid methyl ester (FAME) analysis.
• A fatty acid profile reveals differences in chain length, degree of
saturation, branched chains, and hydroxyl groups.
• Microbes of the same species will have identical fatty acid profiles,
provided they are grown under the same conditions; this limits
FAME analysis to only those microbes that can be grown in pure
culture.
• Finally, because the identification of a species is done by comparing
the results of the unknown microbe in question with the FAME
profile of other, known microbes, identification is only possible if
the species in question has been previously analyzed.
• Nonetheless, FAME analysis is particularly important in public
health, food, and water microbiology.
• In these applications, microbiologists seek to identify specific
pathogens.
 Mass spectrometry(MS)
• Advances in mass spectrometry (MS) have resulted in the fast and accurate
identification of bacteria based on the presence of specific, highly abundant proteins.
• The specific type of MS used is called matrix-assisted laser desorption/ionization-time
of flight (MALDI-ToF).
• MALDI-ToF enables the analysis of complex biomolecules that could not previously
be studied by MS.
• The material to be analyzed is dried on a sample holder (called a target) and then
mixed with a molecular film called a matrix.
• When a UV laser beam strikes the sample target, the matrix helps stimulate release of
the sample from the surface; this is matrix-assisted desorption.
• Once the matrix-sample is released (desorbed), the matrix transfers protons to the
sample, which then becomes ionized; this is matrix-assisted ionization.
• Once ionized, biomolecules are “flown” across a space within the instrument; the
time taken for a molecule to fly from one side to the other is used to determine the
mass of each molecule; this is time of flight.
• In its simplest form, MALDI-ToF identification of bacteria involves the transfer of
whole cells from a single colony grown under specific conditions to a sample target.
• Once dried, a matrix is deposited and the bacterial samples are analyzed. MALDI-ToF
yields the masses of many highly abundant bacterial proteins.
• Like FAME analysis, each experimentally derived protein profile must be matched to
a protein profile from a known bacterium.
• MALDI-ToF is becoming increasingly important in medical microbiology laboratories
where the same strains of organisms are regularly encountered.
• Like FAME, microbes must be grown under very specific conditions and MALDI-ToF
cannot be used to identify newly discovered microbes.
 Ecological Characteristics

• The ability of a microorganism to colonize a specific environment is of taxonomic value.

• Some microbes may be very similar in many other respects but inhabit different ecological niches,
suggesting they may not be as closely related as first suspected.

• Some examples of taxonomically important ecological properties are life cycle patterns; the nature of
symbiotic relationships; the ability to cause disease in a particular host; and habitat preferences such as
requirements for temperature, pH, oxygen, and osmotic concentration.

• Many growth requirements are considered physiological characteristics as well.

Molecular Characteristics

• Molecular analysis is the feasible means of collecting a large and accurate data set that explores microbial
evolution.
• Phylogenetic inferences based on molecular approaches provide the most robust analysis of microbial
evolution.
• Microbial genomes can be directly compared and taxonomic similarity can be estimated in many ways.
• DNA sequence can be determined for one or a few genes, or for an entire genome, depending on the degree
of identification required.
• The recent rapid drop in cost and time to generate a genome sequence has made this approach viable for
many taxonomic applications.
• Molecular techniques developed in the late twentieth century are now in transition to techniques based on
whole-genome sequencing (WGS).
• In many instances, the original nomenclature remains, although the technique is performed In silico rather
than In vitro.
• As with phenetic approaches, comparison to standards and type strains forms the basis for identification and
phylogenetic placement.
• Many classical properties used in strain identification are now inferred from genome sequences.
 Whole-Genome Comparison

• As the field of taxonomy shifts to whole-genome sequencing

(WGS) for strain identification and taxonomic classification,
new quantitative measures of relatedness are being developed
and evaluated.
• As with any metric, standardization of methods and
interpretations are required.
• Average nucleotide identity (ANI) is now widely considered to
be the standard for species identification.
• This technique uses pairwise alignments between all sequences
shared between two genomes and calculates the fraction of
identical nucleotides.
• ANI values for two genomes of the same species should be at
least 95 to 96%.
• ANI is poised to replace a biochemical technique called DNA-
DNA hybridization (DDH).
• DDH is performed by mixing genomic DNA from two strains.
• The mixture is heated until denaturation occurs, and then slowly
cooled to allow renaturation.
• Non complementary regions remain unpaired, and the degree of
renaturation is calculated.
• This is a relatively complex assay and results depend on the quality of
the extracted DNA and other factors.
• By contrast, bioinformatics software quickly calculates digital DDH
values using WGS data, although this technique is less commonly
used than ANI.
GC Content determination
• Another biochemical technique gone digital is determination of G + C
content.
• This metric is a simple percentage of the bases in DNA that are G +
C, and it is readily determined computationally from WGS data.
• Organisms range from around 30% to 80% G + C.
• Despite the wide range of variation, the G + C content of strains
within a species is constant and varies little within a genus
 Species and Subspecies

Species

• collection of bacterial cells which share an overall

similar pattern of traits in contrast to other bacteria
whose pattern differs significantly

Strain or variety

• culture derived from a single parent that differs in

structure or metabolism from other cultures of that
species (biovars, morphovars)
*A biovar is a variant prokaryotic strain that differs
Type
physiologically or biochemically from other strains in
• subspecies that can show differences in antigenic
a particular species.
makeup (serotype or serovar), susceptibility to bacterial
* Morphovars (or morphotypes) are those strains that
viruses (phage type) and in pathogenicity (pathotype)
differ morphologically.
 Microscopy
Better Microscope Resolution Means a Clearer Image
• A microscope is a laboratory instrument used to examine objects that are
too small to be seen by the naked eye.
• The most important part of the microscope is the objective lens, which must
produce a clear image, not just a magnified one.
• Resolution is the ability of a lens to separate or distinguish between small
objects that are close together.
• At best, the resolution of a bright-field microscope is 0.2 µm, which is about
the size of a very small bacterium.
• Resolution is in part dependent on the numerical aperture (n sin θ) of a lens.
• Numerical aperture is defined by two components: n is the refractive index
of the medium in which the lens works (e.g., air = 1) and θ is 1/2 the angle
of the cone of light entering an objective.
• A cone with a narrow angle does not adequately separate the rays of light
emanating from closely packed objects, and the images are not resolved. A
cone of light with a very wide angle is able to separate the rays, and the
closely packed objects appear widely separated and resolved.
• Some objective lenses work in air; since sin θ cannot be greater than 1 (the
maximum θ is 90° and sin 90° is 1.00), no lens working in air can have a
numerical aperture greater than 1.00.
• The only practical way to raise the numerical aperture above 1.00, and
therefore achieve higher resolution, is to increase the refractive index with
• If air is replaced with immersion oil, many light rays that would
otherwise not enter the objective due to reflection and refraction at the
surfaces of the objective lens and slide will now do so.

• This results in an increase in numerical aperture and resolution.

• Resolution is described mathematically by an equation developed in

the 1870s by Ernst Abbé (1840–1905), a German physicist.

• The Abbé equation states that the minimal distance (d) between two
objects that reveals them as separate entities depends on the
wavelength of light (λ) used to illuminate the specimen and on the
numerical aperture of the lens (n sin θ), which is the ability of the lens
to gather light.
• The smaller d is, the better the resolution, and finer detail can be discerned in a specimen; d becomes
smaller as the wavelength of light used decreases and as the numerical aperture increases.

• Thus the greatest resolution is obtained using a lens with the largest possible numerical aperture and light
of the shortest wavelength, at the blue end of the visible spectrum (in the range of 450 to 500 nm).

• Although most condensers have a numerical aperture between 1.2 and 1.4, the numerical aperture will not
exceed about 0.9 unless the top of the condenser is oiled to the bottom of the slide.

• The most accurate calculation of a microscope’s resolving power considers both the numerical aperture of
the objective lens and that of the condenser, as is evident from the following equation, where NA is the
numerical aperture.
 Bright-Field Microscope
Dark Object, Bright Background
• The bright-field microscope is routinely used to examine both stained and unstained specimens.
• It forms a dark image against a lighter background, thus it has a “bright field.”
• The image seen when viewing a specimen with a compound microscope is created by the objective
and ocular lenses working together.
• Light from the illuminated specimen is focused by the objective lens, creating an enlarged image
within the microscope.
• The ocular lens further magnifies this primary image.
• The total magnification is calculated by multiplying the objective and eyepiece magnifications
together.
• For example, if a 45× objective lens is used with a 10× eyepiece, the overall magnification of the
specimen is 450×.
 Visualizing Living, Unstained Microbes
Dark-field microscope, Phase-contrast microscope, and Differential interference
contrast microscopes.

• Bright-field microscopes are probably the most common microscope found in teaching, research,
and clinical laboratories.

• However, many microbes are unpigmented so are not clearly visible because there is little
difference in contrast between the cells, subcellular structures, and water.

• One solution to this problem is to stain cells before observation.

• Staining procedures usually kill cells.

• Three types of light microscopes create detailed, clear images of living specimens: dark-field
microscopes, phase-contrast microscopes, and differential interference contrast microscopes.
 Dark-Field Microscope
Bright Object, Dark Background

• The dark-field microscope produces detailed images of living, unstained

cells and organisms by simply changing the way in which they are
illuminated.
• A hollow cone of light is focused on the specimen in such a way that
unreflected and unrefracted rays do not enter the objective.
• Only light that has been reflected or refracted by the specimen forms an
image.
• The field surrounding a specimen appears black, while the object itself is
brightly illuminated.
• The dark-field microscope can reveal considerable internal structure in
larger eukaryotic microorganisms.
Advantages:
• Ideal for viewing objects that are unstained live organisms, transparent
and absorb little or no light.
• Used for visualization of spirochetes such as Treponema pallidum
(syphilis), Borrelia burgdorferi (lyme borreliosis) and Leptospira
interrogans (leptospirosis) in clinical samples. Spirochetes cannot be
seen by light microscopy because of their thin dimensions.
 Phase-Contrast Microscope

• Phase contrast is a light microscopy technique used to

enhance the contrast of images of transparent and
colourless specimens.

• It enables visualisation of cells and cell components that

would be difficult to see using an ordinary light
microscope.

• As phase contrast microscopy does not require cells to be

killed, fixed or stained, the technique enables living cells,
usually in culture, to be visualised in their natural state.

• This means biological processes can be seen and recorded

at high contrast and specimen detail can be observed.
• Phase-contrast microscopes take advantage of the phenomenon to create differences
in light intensity that provide contrast to allow the viewer to see a clearer, more
detailed image of the specimen.
• They do so by separating the two types of light so that the undeviated light
(primarily from the surroundings) can be manipulated and then recombined with the
deviated light (from the bacterium) to form an image.
• Two components allow this to occur: a condenser annulus and a phase plate.
• The condenser annulus is an opaque disk with a thin transparent ring.
• A ring of light is directed by the condenser annulus to the condenser, which focuses
the light on the specimen.
• Deviated and undeviated light then pass through the objective toward the phase
plate.
• The phase plate has a thin ring through which the undeviated light (i.e., from the
surroundings) is focused.
• In a common type of phase contrast microscopy (positive phase contrast), the ring is
coated with a substance that advances the phase of the undeviated light by ¼
wavelength.
• The deviated light is focused on the rest of the phase plate, which lets the deviated
light pass through unchanged.
• After leaving the phase plate, the deviated and undeviated light are now out
of phase by ½ wavelength.
• When the two rays of light recombine to form an image, they cancel each
other out, a phenomenon called destructive interference.
• Destructive interference is also seen if the crest of a wave of water meets the
trough of another wave—the two cancel each other out and the surface of the
water remains calm at the point where they meet.
• The resulting image consists of a darker bacterium against a lighter
background. Yeast in Bright field microscope

Advantages
• Phase-contrast microscopy is especially useful for studying microbial
motility, determining the shape of living cells, and detecting bacterial
structures such as endospores and inclusions.
• These are clearly visible because they have refractive indices markedly
different from that of water.
• Phase-contrast microscopes also are widely used to study eukaryotic cells. Yeast in Phase contrast microscope
Differential Interference Contrast Microscope

• The differential interference contrast (DIC) microscope is

similar to the phase-contrast microscope in that it creates
an image by detecting differences in refractive indices and
thickness.
• Two beams of plane-polarized light at right angles to each
other are generated by prisms.
• In one design, the object beam passes through the
specimen, while the reference beam passes through a
clear area of the slide.
• After passing through the specimen, the two beams
combine and interfere with each other to form an image.
• A live, unstained specimen appears brightly colored and
seems to pop out from the background, giving the viewer
the sense that a three-dimensional image is being viewed.
• Structures such as cell walls, endospores, granules,
vacuoles, and nuclei are clearly visible
 Saccharomyces cerevisiae

Phase contrast microscope

Differential Interference Contrast Microscope

 Fluorescence Microscope
Use Emitted Light to Create Images
• The light microscopes thus far considered produce an image from light that passes through a specimen.
• An object also can be seen because it emits light. This is the basis of fluorescence microscopy.
• When some molecules absorb radiant energy, they become excited and release much of their trapped energy as light.
• Any light emitted by an excited molecule has a longer wavelength (i.e., has lower energy) than the radiation originally
absorbed.
• Fluorescent light is emitted very quickly by the excited molecule as it gives up its trapped energy and returns to a more stable
state.
• The fluorescence microscope excites a specimen with a specific wavelength of light that triggers the emission of fluorescent
light by the object, which forms the image.
• The most commonly used fluorescence microscopy is epifluorescence microscopy, also called incident light or reflected light
fluorescence microscopy.
• Epifluorescence (Greek epi, upon) microscopes illuminate specimens from above.
• The objective lens also acts as a condenser.
• A mercury vapor arc lamp or other source produces an intense beam of light that
passes through an exciter filter.
• The exciter filter transmits only the desired wavelength of light.
• The excitation light is directed down the microscope by the dichromatic mirror.
• This mirror reflects light of shorter wavelengths (i.e., the excitation light) but
allows light of longer wavelengths to pass through.
• The excitation light continues down, passing through the objective lens to the
specimen, which is usually stained with molecules called fluorochromes.
• The fluorochrome absorbs light energy from the excitation light and emits
fluorescent light that travels up through the objective lens into the microscope.
• Because the emitted fluorescent light has a longer wavelength, it passes through
the dichromatic mirror to a barrier filter, which blocks out any residual excitation
light.
• Finally, the emitted light passes through the barrier filter to the eyepieces.
• Bacterial pathogens can be identified after staining with fluorochromes or
specifically tagging them with fluorescently labeled antibodies using
immunofluorescence procedures.
• In ecological studies, fluorescence microscopy is used
to observe microorganisms stained with fluorochrome-
labeled probes or fluorochromes that bind specific cell
constituents.

• In addition, microbial ecologists use epifluorescence

microscopy to visualize photosynthetic microbes, as
their pigments naturally fluoresce when excited by light
of specific wavelengths.

• It is even possible to distinguish live bacteria from dead

bacteria by the color they fluoresce after treatment with
a specific mixture of stains.

• Another important use of fluorescence microscopy is

the localization of specific proteins within cells.
 Confocal Microscopy
• When three dimensional objects are viewed with traditional light microscopes, light
from all areas of the object, not just the plane of focus, enters the microscope and is
used to create an image, results in murky and fuzzy image.
• This problem has been solved by the confocal scanning laser microscope (CSLM), or
simply, confocal microscope.
• The confocal microscope uses a laser beam to illuminate a specimen that has been
fluorescently stained.
• A major component of the confocal microscope is an opening (that is, an aperture)
placed above the objective lens.
• The aperture eliminates stray light from parts of the specimen that lie above and below
the plane of focus.
• Thus the only light used to create the image is from the plane of focus, and a much
sharper image is formed.
• To generate a confocal image, a computer interfaced with the confocal microscope
receives digitized information from each plane in the specimen.
• This information can be used to create a composite image that is very clear and
detailed or a three-dimensional reconstruction of the specimen.
• Images of x-z plane cross sections of the specimen can also be generated, giving the
observer views of the specimen from three perspectives.
CSLM is widely used in microbial ecology, especially for
identifying specific populations of cells in a microbial habitat or
for resolving the different components of a structured microbial
community, such as a biofilm or a microbial mat.

In general, CSLM is particularly useful anywhere thick specimens

need to be examined for their microbial content with depth.
 Electron Microscopes
Use Beams of Electrons to Create Highly Magnified Images

• For centuries the light microscope has been the most important instrument for
studying microorganisms.
• However, even the best light microscopes have a resolution limit of about 0.2 μm,
which greatly compromises their usefulness for detailed studies of many
microorganisms.
• Viruses, for example, are too small to be seen with light microscopes (with the
exception of some recently discovered giant viruses). Bacteria and archaea can be
observed, but because they are usually only 1 to 2 μm in diameter, only their
general shape and major morphological features are visible.
• The detailed internal structure of microorganisms therefore cannot be effectively
studied by light microscopy. Recall that the resolution of a light microscope
increases as the wavelength of the light it uses for illumination decreases.
• In electron microscopes, electrons replace light as the illuminating beam.
• The electron beam can be focused, much as light is in a light microscope, but its
wavelength is about 100,000 times shorter than that of visible light.
• Therefore electron microscopes have a practical resolution roughly 1,000 times
better than the light microscope; with many electron microscopes, points closer than
0.5 nm can be distinguished, and the useful magnification is well over 100,000×
 Transmission Electron Microscope
• A transmission electron microscope (TEM) uses a heated tungsten filament in the electron gun to generate a
beam of electrons that is focused on the specimen by the condenser.
• Since electrons cannot pass through a glass lens, doughnut-shaped electromagnets called magnetic lenses
focus the beam.
• The column containing the lenses and specimen must be under vacuum to obtain a clear image because
electrons are deflected by collisions with air molecules.
• The specimen scatters some electrons, but those that pass through are used to form an enlarged image of the
specimen on a fluorescent screen that interfaces with a computer monitor.
• Denser regions in the specimen scatter more electrons and therefore appear darker because fewer electrons
strike that area of the screen; these regions are said to be “electron dense” and in contrast, electron-transparent
regions are brighter.
• The TEM has distinctive features that place harsh restrictions on the nature of samples that can be viewed and
the means by which those samples must be prepared.
• Specimens must be viewed in a vacuum and only extremely thin slices (20 to 100 nm) of a specimen can be
viewed because electron beams are easily absorbed and scattered by solid matter.
Specimen preparation for TEM
• To prepare specimens, they are first fixed with chemicals such as glutaraldehyde and osmium tetroxide to stabilize cell
structure.
• The specimen is then dehydrated with organic solvents (e.g., acetone or ethanol).
• Next the specimen is soaked in unpolymerized, liquid epoxy plastic until it is completely permeated, and then the plastic is
hardened to form a solid block.
• Thin sections are skillfully cut from the block with a glass or diamond knife using a device called an ultramicrotome.
• As with bright-field light microscopy, cells are usually stained so they can be seen clearly with a TEM.
• The probability of electron scattering is determined by the density (atomic number) of atoms in the specimen.
• Biological molecules are composed primarily of atoms with low atomic numbers (H, C, N, and O), and electron scattering is
fairly constant throughout an unstained cell or virus.
• Therefore specimens are further prepared by soaking thin sections with solutions of heavy metal salts such as lead citrate
and uranyl acetate.
• The lead and uranium ions bind to structures in the specimen and make them more electron opaque, thus increasing
contrast in the material.
• Heavy osmium atoms from the osmium tetroxide fixative also stain specimens and increase their contrast.
• The stained thin sections are then mounted on tiny copper grids and viewed.
Negative staining and Shadowing
• In negative staining, the specimen is spread out in a thin film with either
phosphotungstic acid or uranyl acetate.
• Just as in negative staining for light microscopy, the specimen appears bright
against a dark background, in this case because the heavy metals do not penetrate
biological material.
• Negative staining enables visualization of viruses and cellular microbes, but
unlike thin sections, internal structures cannot be discerned.
• In shadowing, a specimen is coated with a thin film of platinum or other heavy
metal by evaporation at an angle of about 45 degrees from horizontal so that the
metal strikes the microorganism on only one side.
• In one commonly used imaging method, the area coated with metal appears dark
in photographs, whereas the uncoated side and the shadow region created by the
object are light.
• This technique is particularly useful in studying virus particle morphology,
bacterial and archaeal flagella, and DNA.
• The process of chemical fixation and dehydration can introduce artifacts that can
alter cellular morphology.
• This can be minimized or avoided by using a freeze-etching procedure.

• When cells are rapidly frozen in liquid nitrogen, they become very
brittle and can be broken along lines of greatest weakness, usually down
the middle of internal membranes.

• The exposed surfaces are then shadowed and coated with layers of
platinum and carbon to form a replica of the surface.

• After the specimen has been removed chemically, this replica is studied
in the TEM, providing a detailed view of intracellular structure
 Scanning Electron Microscope
• Transmission electron microscopes form an image from radiation that has passed through a specimen.
• The scanning electron microscope (SEM) produces an image from electrons released from an object’s surface.
• The surfaces of microorganisms are visualized in great detail; most SEMs have a resolution of about 10 nm.
• Specimen preparation for SEM is relatively easy, and in some cases, air-dried material can be examined
directly.
• However, microorganisms usually must first be fixed, dehydrated, and dried to preserve surface structure and
prevent collapse of cells when they are exposed to the SEM’s vacuum.
• Before viewing, dried samples are mounted and coated with a thin layer of metal to prevent the buildup of an
electrical charge on the surface and to give a better image.
• To create an image, the SEM scans a narrow, tapered electron beam back and forth over the specimen.
• When the beam strikes a particular area, surface atoms discharge a tiny shower of electrons called secondary
electrons, and these are trapped by a detector.
• Secondary electrons strike a material in the detector that emits light when struck by electrons (the material is
called a scintillator).
• The flashes of light are converted to an electrical current
and amplified by a photomultiplier and the signal is
digitized and sent to a computer, where it can be viewed.
• The number of secondary electrons reaching the detector
depends on the nature of the specimen’s surface.
• When the electron beam strikes a raised area, a large
number of secondary electrons enter the detector; in
contrast, fewer electrons escape a depression in the surface
and reach the detector.
• Thus raised areas appear lighter on the screen and
depressions are darker.
• A realistic three-dimensional image of the microorganism’s
surface results.
• A single instrument can house both transmission and
scanning electron microscopes (S/TEM).
 Electron Cryotomography
• Electron cryotomography, a technique that since the 1990s has been providing exciting
insights into the structure and function of cells and viruses.
• Cryo- refers to sample preparation and visualization.
• Samples are prepared by rapidly plunging the specimen into an extremely cold liquid
(e.g., ethane) and the sample is kept frozen while being examined.
• Rapid freezing of the sample forms vitreous ice rather than ice crystals.
• Vitreous ice is a glasslike solid that preserves the native state of structures and
immobilizes the specimen so that it can be viewed in the high vacuum of the electron
microscope.
• Tomography refers to the method used to create images.
• The object is viewed from many directions, referred to as a tilt series.
• The individual images are recorded and processed by computer programs, and finally
merged to form a three-dimensional reconstruction of the object.
• Three dimensional views, slices, and other types of representations of the object can be
derived from the reconstruction.
• The ultrastructure of bacterial and archaeal cells has been the focus of numerous studies
using electron cryotomography.
• Some of these studies have revealed new cytoskeletal elements, such as those associated
with magnetosomes—the inclusions used by some bacteria to orient themselves in
magnetic fields.
 Advanced Microscopy - Scanning Probe Microscopy
Can Visualize Molecules and Atoms
• These microscopes measure surface features of an object by moving a sharp probe over the object’s surface.
• One type of SPM is the scanning tunneling microscope.
• It can achieve magnifications of 100 million times, and it allows scientists to view atoms on the surface of a
solid.
• The scanning tunneling microscope has a needlelike probe with a point so sharp that often there is only one
atom at its tip.
• The probe is lowered toward the specimen surface until its electron cloud just touches that of the surface
atoms.
• When a small voltage is applied between the tip and specimen, electrons flow through a narrow channel in
the electron clouds.
• The arrangement of atoms on the specimen surface is determined by scanning the probe tip back and forth
over the surface while keeping the probe at a constant height above the specimen.
• As the tip follows the surface contours by moving up and down, its motion is recorded and analyzed by a
computer to create an accurate three-dimensional image of the surface atoms.
• The surface map can be displayed on a computer screen or plotted on paper.
• The resolution is so great that individual atoms are observed easily. Even more exciting is that the
microscope can examine objects when they are immersed in water.
• Therefore it can be used to study biological molecules such as DNA.
• The microscope’s inventors, Gerd Binnig and Heinrich Rohrer, shared the 1986 Nobel Prize in Physics for
their work, together with Ernst Ruska, the designer of the first transmission electron microscope.
 Atomic Force Microscope
• A second type of SPM is the atomic force microscope, which
moves a sharp probe over the specimen surface while keeping
the distance between the probe tip and the surface constant.
• It does this by exerting a very small amount of force on the tip,
just enough to maintain a constant distance but not enough
force to damage the surface.
• The vertical motion of the tip usually is followed by measuring
the deflection of a laser beam that strikes the lever holding the
probe.
• Unlike the scanning tunneling microscope, the atomic force
microscope can be used to study surfaces that do not conduct
electricity well.
• The atomic force microscope has been used to study the
interactions of proteins, to follow the behavior of living bacteria
and other cells, and to visualize membrane proteins such as
aquaporins.