PART 1: Microbiology and Microorganisms.

Introduction and Major Themes of Microbiology

• What Is Microbiology and Why Is It Important?

• Structure and Activities of Microbial Cells
• Evolution and Diversity of Microbial Cells
• The Species Concept and Classification
• Molecular Phylogeny and the Tree of Life
Activity 1.

What is Microbiology?
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
• Biochemical to study cell components
• Molecular and genetic techniques.

Figure 1.1
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.

Figure 1.9
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

Figure 1.2
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).
3 categories of microorganisms – based on structure
1. Eukaryotes
• Membrane bound nucleus

• Membrane bound organelles

• Complex internal organization

• Division by mitosis and meiosis.

Figure 1.2
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
• No membrane bound nucleus or organelles

• Generally smaller (approx 1 mm diameter)

• Simple internal structure

• Divide by binary fission

• Most are unicellular.

Figure 1.2
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
• Most 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.
Activity 2: Name that organism ie whether it’s a prokaryote,
eukaryote or virus based on cell image
A)
B)
C)
D)
D)
F)
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.

Figure 1.4
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.

Figure 1.6
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.

Figure 1.6
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.

Figure 1.6
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
• In the very near future: 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

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.
II. Origins of Microbiology

• The Discovery of Microorganisms

• Pasteur and Spontaneous Generation
• Koch, Infectious Disease, and Pure
Culture Microbiology
 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: Figure 1.13
• 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.

Figure 1.14
 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).

Figure 1.17
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 1.20
Koch, Infectious Disease, and Pure Cultures
• 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
Activity: Can you remember the scientist and what
they did in the past to influence microbiology?
ROBERT KOCH
• Studied anthrax – responsible for epidemics in livestock
• He isolated bacteria from the carcass of a diseased animal –
Bacillus anthracis
• Established a set of criteria for relating a specific microbe to a
disease
• Koch’s postulates.
 Robert HOOKE
• 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.
 ANTONI VAN LEEUWENHOEK
• Built microscopes that magnified specimen by 50-300x

• Observed single celled microorganisms – called them “animalcules”

• First discovery of bacteria.

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.

To follow formula:
159 = 159 * 103 = 1.59 x 105
1x10-3
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.
Activity: What is the bacterial titer? i.e cfu/ml

Plate 1 Plate 2 Plate 3 Plate 4 Plate 5

 Volume # of Titre
PLATE Dilution Significant?
Plated Colonies (CFU/ml)