Fundamentals of Microbiology

Unit 1: 7 days
 January 7 : Intro to Microbiology
th

• What are microbes?

• What do microbes do?

 Microbes in our lives
• Disease
• Spoiled food
• Food chain base
• Decomposers
• Digestion
• Vitamin synthesis
• Industrial synthesis of chemicals
• Food production
• Genetic engineering
• Sewage treatment
• Bioremediation
 Microbes in our lives
• Only a small number of microorganisms are
pathogenic

• Health care workers must still understand

microbes in order to protect patients from
normally harmless, but opportunistic
organisms
Microbes in our lives
 Historic Changes in Microbiology
• The field is only a few hundred years old, but
bacterial DNA has been found in 3,000 year
old Egyptian mummies

• Arguably the most important discovery in

biology was in 1665 by Robert Hooke and a
crude microscope
 Historic Changes in Microbiology
• Hooke reported that the smallest structural
unit of life was a “little box” or “cell”

• This launched cell theory, and all future

research into cells was based on this first
discovery

• Now cell theory states that “all living things

are composed of cells”
 Historic Changes in Microbiology
• A Dutch merchant and amateur scientist
named Antoni Von Leeuwenhoek was the first
to use stains and really see microorganisms

• He looked at rainwater, peppercorn water, and

scrapings from his teeth under a magnifying
lens and drew pictures of the ‘animalcules’
that he observed
 Historic Changes in Microbiology
• How did these tiny organisms arise?
• Were they born?
• Or did they spontaneously generate?
 Historic Changes in Microbiology
• This is not an unreasonable question 150
years ago

• People believed that toads were born out of

wet soil, that flies came from manure, and
that maggots could arise from decaying
corpses
 Historic Changes in Microbiology
• Italian physician Francesco Redi opposed this
‘spontaneous generation’ viewpoint

• He began trying to disprove that maggots

came from rotting meat in 1668
 Historic Changes in Microbiology
• First Redi took three jars and put decaying
meat in them and then sealed them tightly
• Next he took three more jars, put more
decaying meat in them, and left them open

• Maggots appeared in the open jars, because

flies could land and lay their eggs
 Historic Changes in Microbiology
• Critics said that fresh air was required for
spontaneous generation
• Redi repeated the experiment, but this time
instead of sealing the jars he spread a fine
mesh across the tops

• Again, maggots only appeared in the open jars

 Historic Changes in Microbiology
• Redi’s results were a serious blow to the belief
that complex living material could be
generated from non-living material

• However, people still believed that Van

Leeuwenhoek’s animalcules were simple
enough to spontaneously generate
 Historic Changes in Microbiology
• The debate about microorganisms raged back
and forth with many experiments being done
to determine whether microbes could
spontaneously arise from nutrient fluids
 Historic Changes in Microbiology
• Sometimes heating and sealing prevented
growth, but many argued that severe heating
destroyed the ‘life force’ in the fluids

• Others argued that the was not enough

oxygen (recently proven to be required by
many life forms) in the sealed flask to allow for
microbe generation
 Historic Changes in Microbiology
• In 1858 German scientist Rudolf Virchow
brought forth the concept of biogenesis –
living cells can arise only from preexisting
living cells
• In 1861 Louis Pasteur definitively answered
the debate with a series of clever experiments
– Pasteur’s discoveries form the basis of aseptic
technique today
 The Golden Age of Microbiology
• From 1857 to 1914
• Rapid breakthroughs in the field:
– Fermentation – using microbes to produce alcohol
– Pasteurization – heating to kill most microbes
– Germ Theory of Disease – microbes may cause
illness in both plants and animals
– Koch’s Postulate – sequence of steps to relate a
microbe to a specific disease
– Vaccination
 The Golden Age of Microbiology
• People:
• Edward Jenner – vaccination of smallpox
• Paul Ehrlich – creator of synthetic drugs
• Alexander Fleming – penicillin
 New Areas of Research
• Immunology
• Virology
• Parasitology
• Bacteriology
• Mycology
• Genetic engineering
• Molecular biology
 January 8th: Naming Microbes and Major
Chemical Principles
• The system for scientific nomenclature was
first developed in 1735 by Carolus Linnaeus
• They are Latinized and italicized
• Each organism is given two names:
– Genus – always capitalized
– Species epithet
• Once the species name has been listed it can
be abbreviated with the first letter of the
genus and the species epithet
 Naming Microbes
• Names can honor a researcher, describe the
organism, or even denote the location where
it is found

• Staphylococcus aureus = staph (clustered

colonies), coccus (spherical cells), aureus
(golden in color)
• Escherichia coli = Theodor Escherich, coli
(found in the colon)
 Naming Microbes
• In 1978 Carl Woese developed a classification
system for bacteria
• Eubacteria – bacteria with peptidoglycan cell
walls
• Archaea – bacteria lacking peptidoglycan cell
walls
• Eukarya – all eukaryotes (protists, fungi,
plants, and animals)
 Bacteria
• Very small
• Relatively simple
• Single celled
• Prokaryotic = pre-nucleus
• Several shapes
• Several arrangements
• Binary fission asexual reproduction
• Mostly heterotrophic, a few autotrophs
 Fungi
• Eukaryotic
• Nucleus contains DNA
• Unicellular or multicellular
• Heterotrophic
• Cells walls made from chitin
• Sexual or asexual
• Unicellular species are oval in shape
 Protozoa
• Eukaryotic
• Unicellular
• Classified based on locomotion
– Amoebas – move extensions of their cytoplasm
– Flagellates
– Ciliates
• Sexual or asexual
• Parasitic or free living
 Algae
• Eukaryotic
• Photosynthetic
• Sexual or asexual
• Cell walls – typically of cellulose
• Abundant in fresh water, soil, and in
conjunction with plants
 Viruses
• Very small - most are only visible with an
electron microscope
• Only one type of nucleic acid
– can be DNA or RNA
• Have a protein coat
• Can have a lipid envelope
• All parasitic
• Reproduce by hijacking cellular machinery
 Multicellular Animal Parasites
• Not strictly microorganisms
• Medically important
• Worms
• Often have a microscopic stage of their life
cycle
 Chemistry Review!
• Atomic structure
• About 26 elements are commonly found in
living cells
• Valence electrons and bonding
– Ionic
– Polar Covalent
– Non Polar Covalent
– Hydrogen Bonds
 Chemistry Review!
• Molecular mass
• Moles
• Chemical reactions
– Exothermic
– Endothermic
– Reversible
• Collisions required
• Enzymes lower activation energy
 Chemistry Review!
• Inorganic compounds:
– Small
– Ionically bonded
– Water
– Acids and bases
• Hydrogen and Hydroxide ions
– Salts
• pH
• Buffers
 Chemistry Review!
• Organic compounds:
– Always contain carbon and hydrogen
– Carbon atoms can bond 4 times
– Mostly or entirely covalent
– Many are large
– Carbon skeleton
– Functional groups
• Monomers and polymers
 Chemistry Review!
• Carbohydrates:
– C H and O
– Include sugars and starches
– Isomers – same formula, different structure
• Glucose and fructose
• Lipids:
– Insolubility in water
– Simple lipids – glycerol and 3 fatty acids
– Saturated – no double bonds
– Steroids – carbon ring group
 Chemistry Review!
• Proteins:
– Built by amino acids
– C H O and N, sometimes S
– 20 amino acids occur naturally
• Adenosine Triphosphate:
– Stores chemical energy for various cellular activities
– When the bond in the terminal phosphate group is
broken, energy is released
– The energy from decomposition reactions is used to
regenerate ATP from ADP
 Chemistry Review!
• Nucleic Acid:
– Macromolecules
– DNA and RNA
– Pentose, phosphate, and a nitrogen containing
base
– Right handed double helix
– A,T,C, and G
– RNA has ribose
– Genes consist of sequences of nucleotides
 January 9th: Observing Microbes and
Microscopes
• Microorganisms are measured in micrometers
(10 -6 m) and nanometers (10 -9 m)
 Compound Light Microscope
• This is the most common instrument used in
microbiology

• It is abbreviated LM

• Two sets of lenses

– Ocular
– Objective
 Compound Light Microscope
• Total magnification is calculated by multiplying
the magnification of the two lenses together

• A typical maximum magnification is 2000x

• A typical maximum resolution is 0.2 μm
 Compound Light Microscope
• Stains are used to add more definition to the
image

• Immersion oil is used with the oil immersion

lens to reduce light loss between the slide and
the lens
 Compound Light Microscope
• Brightfield illumination is used for stained
smears

• Unstained cells are more productively

observed using darkfield, phase-contrast, or
DIC microscopy
– These types of microscopy require modified
compound microscopes
 Darkfield Microscopy
• Shows a light silhouette of an organism against
a dark background

• This is the most useful technique for detecting

extremely small organisms
 Phase-Contrast Microscopy
• Brings direct and refracted light waves
together in phase to form an image

• Allows for detailed observation of living

organisms
 Differential Interference Contrast
• Provides a colored image

• 3D

• Allows detailed observation of living cells

 Fluorescence Microscopy
• First, specimens are stained

• Then they are viewed through a compound

microscope using ultraviolet light

• Microorganisms appear as bright objects

against a dark background
 Confocal Microscopy
• A specimen is stained with a fluorescent dye
and illuminated one plane at a time

• Using a computer to process the images, 2D

and 3D images of cells can be produced
 Electron Microscopy
• A beam of electrons is used, instead of light

• Electromagnets control the focus,

illumination, and magnification

• Thin sections of organisms can be seen

 Scanning Tunneling and Atomic Force

• STM and AFM produce 3D images of the

surface of a molecule
 Preparing Smears for Staining
• Staining means coloring a microorganism with
a dye to make some structures more visible

• Fixing uses heat or alcohol to kill and attach

microorganisms to a slide

• A smear is a thin film of material used for

microscopic examination
 Preparing Smears for Staining
• Bacteria are negatively charged

• Colored positive ions of a basic dye will stain

bacterial cells

• Colored negative ions of an acidic dye will

stain the background of a bacterial smear
– This is called a negative stain
 Simple Stains
• An aqueous or alcohol solution of a single
basic dye

• Used to make cellular shapes and

arrangements visible

• Mordant – used to improve bonding between

the stain and the specimen
 Differential Stain
• Differentiate bacteria according to their
reaction to the stain
– E.g. Gram Stain and Acid Fast Stain
 Gram Stain
• Uses:
– Crystal violet (purple stain)
– Iodine (mordant)
– Alcohol (decolorizer)
– Red counter stain

• Gram –Positive bacteria retain the purple stain

• Gram-Negative get decolorized and then
counter stained pink
 Acid Fast Stain
• Acid-fast microbes retain carbolfuchsin after
acid-alcohol decolorization
– Appear red
– E.g. genera Mycobacterium and Nocardia

• Non-acid-fast microbes take up the methylene

blue counterstain
– Appear blue
 Special Stains
• The endospore stain and flagella stain are
special stains that color and isolate certain
parts of bacteria

• Negative staining is used to make microbial

capsules visible
 January 13th, 14th, 21st, and 22nd: Functional
Anatomy of Prokaryotic Cells
• What is a prokaryote?
 Prokaryote vs. Eukaryote
• Similar in their chemical composition and
chemical reactions

• Prokaryotes lack membrane enclosed

organelles
– Including a nucleus

• Peptidoglycan is found in prokaryotic cell

walls, but not in eukaryotic cell walls
 The Prokaryotic Cell
• Bacteria are unicellular

• Most multiple by binary fission

• Species are differentiated by morphology,

chemical composition, nutritional
requirements, biochemical activities, and
source of energy
 Size, Shape, and Arrangement
• Most bacteria are 0.2 to 2 μm in diameter

• Most bacteria are 2 to 8 μm in length

 Size, Shape, and Arrangement
• Three basic bacterial shapes are commonly
found:
– Coccus (sphere)
– Bacillus (rods)
– Spiral (twisted)
 Size, Shape, and Arrangement
• Pleomorphic bacteria can assume several
shapes

• Monomorphic bacteria have a shape

determined by heredity
– Most bacteria are monomorphic
 Size, Shape, and Arrangement
• Diplococci – coccus that remain in pairs
• Streptococci – coccus that form chains
• Tetrads – remain in groups of four, divide in
two planes
 Size, Shape, and Arrangement
• Sarcinae – form cubelike groups, divide in
three planes
• Staphylococci – form grapelike clusters or
broad sheets
 Size, Shape, and Arrangement
• Diplobacilli – rods in pairs
• Streptobacilli – chains of rods
• Coccobacilli – oval shaped cells
 Size, Shape, and Arrangement
• Vibrios – curved rods
• Spirilla – helical or corkscrew shaped, fairly
rigid
• Spirochetes – helical and flexible
 External Structures
• Found outside of the cell wall
 Glycocalyx
• Capsule, slime layer, or extracellular
polysaccharide

• Gelatinous polysaccharide and/or polypeptide

covering
 Glycocalyx
• Enable adherence to surfaces

• Prevent desiccation

• May provide nutrients

 Flagella
• Relatively long filamentous appendages

• Consist of:
– Filament
– Hook
– Basal body
 Flagella
• Prokaryotic flagella rotate to push the cell

• Motile bacteria exhibit taxis

– Positive taxis is movement towards an attractant
– Negative taxis is movement away from a repellant
 Axial Filaments
• Also called an endoflagellum

• Spiral cells that move with an axial filament

are called spirochetes

• Similar to flagella, but wrap completely

around the cell
 Fimbriae and Pili
• Short thin appendages

• Fimbriae – help cells adhere to surfaces

• Pili – join cells together for the transfer of DNA

from one cell to another
 The Cell Wall
• Outer protective layer
 Composition and Characteristics
• Surrounds the cell membrane and protects the
cell from changes in water pressure

• Consists of peptidoglycan (a polymer

consisting of short chains of amino acids)

• Penicillin interferes with peptidoglycan

synthesis
 Composition and Characteristics
• Gram-positive cell walls contain many layers of
peptidoglycan and also contain teichoic acid

• Gram-negative bacteria have a lipoprotein-

lipopolysaccharide-phospholipid outer
membrane surrounding a thin peptidoglycan
layer
 Composition and Characteristics
• The outer membrane protects the cell from
phagocytosis and from penicillin, lysozyme,
and other chemicals
 Composition and Characteristics
• Porins – proteins that allow small molecules to
pass through the outer membrane
 Composition and Characteristics
• Channel proteins – allow specific other
molecules to move through the outer
membrane
 Composition and Characteristics
• The lipopolysaccharide component of the
outer membrane consists of sugars that
function as antigens

• Also has lipid A, which is an endotoxin

 Cell Walls and Gram Staining
• The crystal violet – iodine complex combines
with peptidoglycan

• The decolorizer removes the lipid outer

membrane of gram-negative bacteria and
washes the crystal violet out
 Atypical Cell Walls
• Mycoplasm is a bacterial genus that naturally
lacks cell walls

• Archaea have pseudomurein (rather than

peptidoglycan)
 Damage to Cell Walls
• Lysozyme destroys gram-positive cell walls
– The remaining cell contents are called a protoplast

• Lysozyme damages gram-negative cell walls,

but does not destroy them
– The remaining cell contents are called a
spheroplast
 Damage to Cell Walls
• Protoplasts and spheroplasts are susceptible
to osmotic lysis

• Antibiotics interfere with cell wall synthesis

 Internal Structures
• Found within the cell wall
 Plasma Membrane
• Encloses the cytoplasm
• Is a phospholipid bilayer
• Has protein imbedded (fluid mosaic model)
• Selectively permeable
 Plasma Membrane
• Carries enzymes for metabolic reactions
– Nutrient breakdown
– Energy production
– Photosynthesis

• For photosynthesis these enzymes are located in

infoldings of the plasma membrane that extend
into the cytoplasm called thylakoids (or
chromatophores)
 Plasma Membrane
• Some plasma membranes contain one or
more large irregular folds, called mesosomes

• The function of these is much debated, but it

is currently thought that they are artifacts, not
true cell structures
• Thought that they develop by the process
used for preparing the specimen for electron
microscopy
 Plasma Membrane
• Can be destroyed by alcohols and polymixins
– Disrupt the membrane’s phospholipid bilayer
– Cause leakage of internal contents
 Movement of Materials
• May be passive
– From high concentration to low
– No energy expended
– Continues until equilibrium is reached

• May be facilitated diffusion

– Molecules are carried across membranes by
carrier proteins (permeases)
– Still from high to low concentration
 Movement of Materials
• Osmosis
– Movement of water
– Selectively semipermeable membrane
– Continues until equilibrium is reached

• Active transport
– Materials move from low concentration to high
– The cell must expend energy
 Cytoplasm
• The fluid component inside the plasma
membrane

• Contains:
– Mostly water (80%)
– Inorganic and organic molecules
– DNA
– Ribosomes
– Inclusions (reserve deposits)
 The Nuclear Area
• Contains the DNA of the bacterial
chromosome

• Can also contain plasmids

– Extra chromosomal DNA circles
 Ribosomes
• The cytoplasm of a prokaryote contains many
70S ribosomes

• Consist of rRNA and protein

• Location of protein synthesis

• Can be inhibited by certain antibiotics

 Inclusions
• Reserve deposits
• Found in both prokaryotic and eukaryotic cells
• Include:
– Metachromatic granules (inorganic phosphate)
– Polysaccharide granules (glycogen or starch)
– Lipid inclusions
– Sulfur granules
– Carboxysomes (e.g. ribulose)
– Magnetosomes (Fe3O4)
– Gas vacuoles
 Endospores
• Resting structures formed by some bacteria
• Allow for survival during harsh environmental
conditions

• The formation process is called sporulation

• Return process is called germination
 Eukaryotic Cells
• Have membrane bound organelles
• Some eukaryotic organisms can still be
unicellular
 Flagella and Cilia
• Flagella:
– Few and long in relation to cell size
• Cilia:
– Numerous and short
– Move substances along surface of the cell
• Used for motility
• Consist of an arrangement of 9 pairs and 2
single microtubules
 Cell Wall and Glycocalyx
• The cell walls of many algae and some fungi
contain cellulose
• The main material of fungal cell walls is chitin
• Yeast cell walls consist of glucan and mannan
• Animal cells are surrounded by a glycocalyx
– Strengthens the cell
– Provides means of attachment to other cells
 Plasma Membrane
• Similar to the prokaryotic membrane
– Phospholipid bilayer with proteins
• Contain carbohydrates attached to the
proteins
• Contain sterols
• Can move molecules through endocytosis
– Phagocytosis
– Pinocytosis
 Cytoplasm
• Includes everything inside the plasma
membrane and external to the nucleus
• Resembles chemical composition of
prokaryotic cytoplasm
• Has a cytoskeleton
• Exhibits cytoplasmic streaming
 Organelles
• Specialized membrane enclosed structures

• Located in the cytoplasm of eukaryotic cells

 Organelles
• Nucleus –
– Contains DNA
– Most characteristic organelles

• Nuclear envelope –
– Connected to a system of parallel membranes
called the endoplasmic reticulum
 Organelles
• Endoplasmic reticulum –
– Provides a surface for chemical reactions
– Serves as a transporting network
– Stores synthesized molecules
– Location of lipid synthesis

• Rough endoplasmic reticulum –

– Location of protein synthesis
– Location of protein transport
 Organelles
• Ribosomes –
– 80S ribosomes found in the cytoplasm or attached
to the rough ER

• Golgi complex –
– Flattened sacs (called cisterns)
– Functions in membrane formation
– Protein secretion
 Organelles
• Mitochondria –
– Primary sites of ATP production
– Contain 70S ribosomes and DNA
– Multiply by fission

• Chloroplasts –
– Contain chlorophyll and enzymes
– Contain 70S ribosomes and DNA
– Multiply by fission
 Organelles
• Lysosomes –
– Formed from Golgi complexes
– Store powerful digestive enzymes

• Centrioles –
– Pair of cylindrical structures
– Involved in cell division
– Found near the nucleus
 Organelles
• Vacuoles –
– Membrane enclosed cavities
– Derived from Golgi complex
– Usually found in plant cells
– Store various substances
– Bring food into the cell
– Increase cell size
– Provide rigidity to leaves and stem
 Evolution of Eukaryotes
• Endosymbiont theory
• Organelles evolved from symbiotic
prokaryotes living inside other prokaryotic
cells