Part 1: Microbiology and

Microorganisms
Part 1 – Microbiology and Microorganisms

I. Introduction and Major Themes of

Microbiology
• What Is Microbiology and Why Is It Important?

• Structure and Activities of Microbial Cells

• The Species Concept and Classification

• Molecular Phylogeny and the Tree of Life

What Is Microbiology?
• The study of organisms too small to be seen with the
naked eye?
• ex. Bacteria, viruses, single celled eukaryotes

• Some microorganisms are visible to the naked eye:

• ex. Fungi, algae

• Some microbes are multicellular:

• ex. Myxobacteria, slime molds.
What Is Microbiology?
• Microbiology is defined by techniques
• Culture media for isolation and growth of organisms in
pure culture

• Biochemistry to study cell components

• Molecular and genetic techniques.

Why Is Microbiology Important?
• Microbes are the oldest form of life

• Largest mass of living material on Earth

• Carry out major processes for biogeochemical cycles
• Can live in places unsuitable for other organisms
• Other life forms require microbes to survive.
Structure and Activities of Microbial Cells
• All cells have the following in common:
• Cytoplasmic membrane
Barrier that separates the inside of the cell from the
outside environment
• Cytoplasm
Aqueous mixture of macromolecules, ions, and proteins
• Ribosomes
Site of protein synthesis
Structure and Activities of Microbial Cells
• Genetic material
• All cells store their genetic information as DNA
• The information is divided into functional units called genes
• Genome
• A cell's full complement of genes
• Chromosome
• A genetic element carrying genes essential to cellular
function
• Plasmid
• A piece of DNA that carries non-essential genes (ex.
Genes for antibiotic resistance).
1.2 Structure and Activities of Microbial Cells
• Metabolism
• The sum of all chemical reactions that occur in a cell to
carry out the processes of life
• Catabolism – reactions that break complex molecules into
simpler ones
• Release energy
• Anabolism – reactions that build complex molecules from
simpler ones
• Require energy
• Enzymes
• Proteins that increase the rate at which chemical reactions
occur, by lowering the activation energy required.
Structure and Activities of Microbial Cells
• ATP
• The major energy carrier in
the cell
• Used to store energy
released during catabolism
• Supply energy needed for
anabolism

• Proton motive force (PMF)

• A gradient of protons (H+) across a membrane
• Potential energy that can be used to drive cellular functions
• Some cells use the PMF to generate ATP
• Others use ATP to generate the PMF!
3 categories of microorganisms – based on structure
1. Eukaryotes
• Membrane bound nucleus

• Membrane bound organelles

• Complex internal organization

• Division by mitosis and meiosis.

2 major groups of eukaryotic
microbes
Protists – unicellular or multi-
cellular without differentiation into
tissues
• Protozoa – animal-like
microorganisms
• Algae – photosynthetic
plant-like microorganisms
• Slime molds and water
molds – filamentous
Fungi – Unicellular (yeasts),
filamentous (molds), or multi-
cellular (mushrooms).
2. Prokaryotes
• Visually simple, which is why microbiologists rely on metabolic,
biochemical and genetic properties to classify them

• No membrane bound nucleus or organelles

• Generally smaller (approx 1 µm diameter)

• Simple internal structure

• Divide by binary fission

• Most are unicellular.

Figure 1.3
2 major groups of prokaryotes:
Bacteria (eubacteria)
• Genetically diverse
• Extremely diverse metabolic styles
• Includes both pathogens and non-
pathogens
Archaea (archaebacteria)
• Genetically and biochemically
distinct from bacteria
• Also have diverse metabolism
• Never pathogenic
• Many are famous for living in
extreme environments.
3. Viruses
• Acellular infectious particles

• Extremely small

• Obligate intracellular parasites

• Lack independent metabolism

• No ribosomes

• No ribosomal RNA

• Cannot be classified with other

microbes

• Viruses that infect bacteria are

called bacteriophages.
Evolution and Diversity of
Microbial Cells
• First anaerobic life appeared
between 3.8 and 3.9 billion years
ago
• Photosynthetic bacteria
oxygenated the Earth about 2
billion years ago
• Allowed the evolution of
modern eukaryotic
microorganisms
• First plants and animals
appeared about 0.5 billion years
ago
• LUCA = Last universal common
ancestor.
Classifying organisms based on evolutionary relationships
• Comparing small subunit (SSU) rRNA genes
Prokaryotes – 70S ribosomes
• 16S SSU rRNA
Eukaryotes – 80S ribosomes
• 18S SSU rRNA
• rRNA genes change slowly over time
• Examines genetic differences rather than morphological differences.
Basic steps involved in sequencing rRNA genes
Step 1: DNA is collected from a pure culture
Step 2: The SSU rRNA gene is amplified using the polymerase chain
reaction (PCR)
PCR – a technique used to synthesize many identical copies of a short
sequence of DNA
Step 3: The gene is sequenced
Step 4: Sequence is aligned with sequences from other organisms
• Number of differences is used to calculate evolutionary distance
Phylogenetic tree – A graphic representation of the evolutionary distance
between organisms.
Molecular Phylogeny and the Tree of Life
• Phylogenetic tree based on 16S or 18S ribosomal DNA sequences
• All organisms can be grouped into 3 distinct domains of life:
Bacteria, Archaea and Eukarya

• Microorganisms are far more genetically diverse than plants and

animals.

LUCA
More recent tree of life:
• Most of the tree is
made up of the domain
Bacteria
• Phyla shown in purple
have not yet been
isolated in pure culture
• The entire domain
Eukarya exists as a
branch of the Archaea
• The closest relatives of
the eukarya are the
asgard Archaea
• The ‘Asgard
superphylum’.
A new view of the tree of life. Nat Microbiol 1 (2016)
The Species Concept in Microbiology
• The species is the fundamental unit of biological diversity. But,
what is a species?
• Phylogenetic species concept:
• “A group of strains that share certain diagnostic traits, are
genetically cohesive and have a unique recent common
ancestor”
• In practice, species of Bacteria and Archaea should have:
• Most (but not all) characteristics in common
• Greater than 97% sequence similarity in the 16S rRNA gene
• High degree of genome similarity
• DNA-DNA hybridization
• Whole genome sequences.
Classification and Nomenclature
• Microbiologists use Hierarchical classification
• Groups of organisms are placed in successively larger groups
• In practice: Species, genus and phylum are commonly used.
Classification and Nomenclature
• Binomial species names

Escherichia coli

Genus (capitalized) Specific epithet (not capitalized)

• Strains can be identified by symbols after the species name

• ex. E. coli K12, E. coli O157:H7

Rules
1. Names are latinized.
2. Italicized or underlined.
3. Genus capitalized, epithet is not.
4. Genus name may be abbreviated the second time it’s used: E. coli.
5. Trivial names can be used, but do not follow these rules.
Part 1 – Microbiology and Microorganisms

II. Microbiology in Historical Context

• The Discovery of Microorganisms

• Pasteur and Spontaneous Generation

• Koch, Infectious Disease, and Pure Cultures

 The Discovery of Microorganisms
• Robert Hooke (1635–1703)

• The first to describe microbes

• Used a compound microscope – uses 2

lenses to magnify the image

• Allowed magnification up to 30x

• Used it to observe:
• Cells in cork

• Bread mold filaments – 1st microbe

• Beginning of cell theory – all living

things are composed of cells.
 The Discovery of Microorganisms
• Antoni van Leeuwenhoek (1632–1723)

• Built microscopes that magnified specimen by 50-300x

• Observed single celled microorganisms – called them “animalcules”

• First discovery of bacteria.

 Pasteur and Spontaneous Generation
• Louis Pasteur (1822–1895)

• Studied wine and beer production

• Yeasts convert sugar to alcohol in the absence of oxygen

• Fermentation – “La vie sans air”

• Bacteria can sour wine by converting alcohols to acid

• Developed a method of gentle heating to kill unwanted bacteria –

Pasteurization.
 Pasteur and Spontaneous Generation
• Prepared meat infusions inside of long swan-necked flasks
• Boiled the infusion to sterilize it
• As long as the flask remains upright, dust and microbes cannot
enter, and the infusion remains sterile
• Led to the development of methods for controlling the growth
of microorganisms (aseptic technique).
Koch, Infectious Disease, and the Rise of Pure
Cultures
• Robert Koch (1843–1910)
• Studied anthrax – responsible for epidemics in livestock
• He isolated bacteria from the carcass of a diseased animal –
Bacillus anthracis
• Injected healthy animals with the bacterium
• Animals became ill with anthrax
• Re-isolated B. anthracis from the test subjects and showed that
it was identical
• Established a set of criteria for relating a specific microbe to a
disease
• Koch’s postulates.
Figure 1.29
Solid Media and Isolation of Pure Cultures
• Koch realized that solid media provided a simple way to obtain
pure cultures
• Broth medium solidified with agar
• Polysaccharide derived from marine algae
• Melts at ~ 97°C and polymerizes (solidifies) at ~ 43°C
• Cannot be degraded by most microorganisms
• Typical Petri plate = nutrient broth medium + 1.5% agar.

Nutrient agar
g/l
Peptone 5
Beef extract 3
NaCl 5
Agar 15
Bring up to 1 liter with dH2O
Isolating pure cultures
The streak plate technique
• One edge of a plate is inoculated with a
concentrated sample of bacteria
• Sample is diluted by streaking it across the
surface of the plate
• To deposit individual cells on the plate
(separate from other cells)
• Plate is incubated
• Individual cells grow to form colonies
Colony – a mass of cells that (ideally) arose
from one single cell
• Can be used to create a pure culture.
The spread plate and pour plate techniques
• Sample is diluted before plating
• Diluted sample can be spread over the
surface of the plate with a sterile
spreader
• Separate cells grow into colonies
on the surface of the plate
• Or can be mixed with molten agar
(~ 45°C)
• Colonies form embedded inside the plate.
The standard plate count
• Spread and pour plates allow you to calculate the concentration of
bacteria in a population (bacterial titre)

titre = # colonies
(volume)(dilution)

• titre is expressed in cfu/ml

• cfu = colony forming unit.

Countable plates
• We normally count plates with between 30 – 300 colonies
< 30 – not statistically significant
> 300 – colonies grow into each other – inaccurate counts
• When we have more than one countable plate…
• Calculate titre from each and take the average.
Part 1 – Microbiology and Microorganisms

III. Microscopy

• Light Microscopy

• Improving Contrast in Light Microscopy

• Imaging Cells in Three Dimensions

• Probing Cell Structure: Electron Microscopy

Microorganisms vary greatly in
size and shape
Some Principles of Light Microscopy

• Compound light microscope uses visible light to

illuminate cells

• Many different types of light microscopy:

• Bright-field

• Phase-contrast

• Dark-field

• Fluorescence
 Some Principles of Light Microscopy
• Bright-field microscope
• Slight differences in contrast make specimen
visible against surroundings

• Two sets of lenses

form the image
• Objective lens
(magnifies 10x -100x)
• Ocular lens
(magnifies 10x - 20x)
Some Principles of Light Microscopy

• Total magnification =
ocular X objective

• ex. 10x ocular, 40x objective

• Magnification = 10 × 40 =
400x

• Maximum magnification
achievable with a light
microscope is ~ 2,000x
 Some Principles of Light Microscopy
• Magnification: the ability to make an image larger

• Resolution: the ability to distinguish two objects as

separate and distinct

• Light must pass between

two points for them to be
viewed as separate objects
• As wavelength decreases
resolution improves
• Limit of resolution for light
microscope is about 0.2 μm
• Resolving power.
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Improving Contrast in Light Microscopy
• Microorganisms can be very difficult
to view with a light microscope

• Staining improves contrast

• Dyes are organic compounds

that bind to specific cellular
materials

• Examples of common stains are

methylene blue, safranin, and
crystal violet.
Simple staining
• One dye used to color specimen
• Chromophore – colored portion of a dye

Two types:
• Basic dye – positively charged
chromophore Crystal violet –
basic
• Binds to negatively charged
molecules on cell surface
• Acidic dye – negatively charged
chromophore
• Repelled by cell surface
• Used to stain background Nigrosin – acidic
• Negative stain.
Differential stains: the Gram stain

• Separates bacteria into 2

groups based on cell wall
structure
Gram positive – cells that
retain a primary stain
• Purple
Gram negative – cells that
lose the primary stain
• Take color of counterstain
• Red or pink.
 Other differential stains:
• Acid fast stain
• Detects mycolic acid in the cell wall
of the genus Mycobacterium
• Mycobacterium – retains primary stain
• Fuchsia (pink)
• Anything else on slide – color of
counterstain
• Blue Endospore stain
• Endospores retain primary
• Green
• Cells counterstained
• Pink
Ex. Bacillus anthracis spores.
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 Improving Contrast in Light Microscopy
• Phase-contrast microscopy
• Phase ring amplifies differences in the refractive index of
cell and surroundings

• Improves the contrast of a sample without the use of a stain

• Allows for the visualization of live samples

• Resulting image is dark cells on a light background.

Improving Contrast in Light Microscopy
Dark field microscopy
• Specimen is illuminated with a hollow
cone of light

• Only refracted light enters the

objective

• Specimen appears as a bright object

on a dark background

• Used to observe bacteria that don’t

stain well
• ex. Treponema pallidum – the
causative agent of syphilis.
 Improving Contrast in Light Microscopy
Fluorescence microscopy
• Used to visualize specimens that
fluoresce
• Emit light of one color when illuminated
with another color of light

Cells may fluoresce naturally

ex. Photosynthetic Cyanobacteria have
chlorophyll
• Absorbs light at 430 nm (blue-violet)

• Emits at 670 nm (red)

Or after staining with Fluorescent dye

• ex. DAPI specifically binds to DNA.
 Imaging Cells in Three Dimensions
• Differential interference contrast (DIC) microscopy
• Uses a polarizer to create two distinct beams of polarized light

• Gives structures such as endospores, vacuoles, and granules

a three-dimensional appearance

• Structures not visible by bright-field microscopy are sometimes

visible by DIC.

Nucleus
 Imaging Cells in Three Dimensions
• Confocal scanning laser microscopy (CSLM)

• Uses a computerized microscope

coupled with a laser light source
to generate a three-dimensional
image

• Computer can focus the laser on

single layers of the specimen

• Different layers can then be

compiled for a three-dimensional
image

• Resolution is 0.1 μm for CSLM.

Probing Cell Structure: Electron Microscopy
• Electron microscopes use electrons instead of photons to
image cells and structures

• Wavelength of electrons is
Electron
much shorter than light à source

higher resolution
Evacuated
• Two types of electron chamber

microscopes: Sample
port

• Transmission electron
microscopes (TEM)
Viewing
screen
• Scanning electron
microscopes (SEM)
Transmission electron microscope (TEM)
• Electron beam focused on
specimen by a condenser
lens

• Magnets (not glass)

• Electrons that pass through

the specimen are focused
by two sets of lenses

• compound
microscope

• Electrons strike a
fluorescent viewing screen.
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Transmission electron microscopy (TEM)
• High magnification and resolution (0.2 nm)
• Specimen must be very thin (20 – 60 nm)
• Unstained cells do a poor job of scattering electrons
• Must be stained with metals à lead or uranium
• Bind to cell structures to make them more electron dense
• Allows visualization of internal structures.
Difference in Resolution Between Light and
Transmission Electron Microscope

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Probing Cell Structure: Electron Microscopy
• Scanning electron microscopy (SEM)
• Specimen is coated with a thin film of metal
• An electron beam scans the object
• Scattered electrons are collected by a detector, and an
image is produced
• Allows an accurate 3D image of specimen’s surface.

SEM of
bacteria cells
Part 1 – Microbiology and Microorganisms

IV. Cells of Bacteria and Archaea

• Cell Morphology

• Cell Size and the Significance of Being Small

Cell Morphology = cell shape
Coccus (pl. cocci)
• Roughly spherical
• ex. Streptococcus pyogenes

Bacillus (pl. bacilli)

• Rod shaped
• ex. E. coli

Spirillum (pl. spirilla)

• Spiral shaped
• ex. Spirillum volutans
 Cell Morphology = cell shape
• Cells with unusual shapes
• Spirochete
ex. Treponema pallidum

• Budding and appendaged

bacteria
ex. Caulobacter crescentus

• Filamentous bacteria
ex. Streptomyces griseus
Characteristic arrangements
• Cells of some prokaryotes remain
together after cell division, forming
characteristic arrangements

• Examples:

• Members of the genus

Staphylococcus form grape-like
clusters (ie. Staphylococci)

• Some cyanobacteria form long

chains (ex. Anabaena)

• Many coccoid bacteria form tetrads

(ex. Micrococcus and Deinococcus).
Cell Morphology

• Morphology typically does not predict physiology, ecology,

phylogeny, etc. of a prokaryotic cell

• May be selective forces involved in setting the morphology

• Optimization for nutrient uptake (small cells and those

with high surface-to-volume ratio)

• Swimming motility in viscous environments or near

surfaces (helical or spiral-shaped cells)

• Gliding motility (filamentous bacteria).

Cell Size and the Significance of Being Small

• Size range for prokaryotes: 0.2 µm to >700 µm in diameter

• Size range for eukaryotic cells: 10 to >200 µm in diameter

• Despite the overlap in size, it can be said that prokaryotes

are very small compared with eukaryotes
Prokaryote cell size
Average:
• E. coli ~ 1.0 x 3.0 µm
• Staphylococcus aureus ~ 1.0 µm
diameter

Very small:
• Mycoplasma genitalium ~ 0.3 µm

Very large:
• Epulopiscium fishelsonii ~ 80 x 600 µm.
 Cell Size and the Significance of Being Small

• Small cells have more surface area relative to cell volume

• higher S/V ratio

Provides several advantages:

• Supports greater nutrient
exchange per unit cell volume

• Allows faster growth

• High population numbers

• Increased rate of evolutionary

change.
Cell Size and the Significance of Being Small

• Lower limits of cell size

• Cellular organisms < 0.15 µm in diameter are unlikely

• Open oceans tend to contain small cells (0.2–0.4 µm in

diameter)

• Many pathogenic bacteria are also small, and have

small genomes

• Missing many genes whose functions are supplied to

them by host.