BIOL 251: Final Exam Study Guide
Everything covered in class.
The exam is supposed to be 50% lectures and 50% diseases – don’t stress
about details too much, just big ideas.
Likely on the test.
Lecture 1: Intro to Microbiology
∙ Microorganisms (microbes): organisms too small to be
observed by the unaided eye.
∙ Limit of resolution: an optical system’s capacity to
resolve point objects separately.
∙ The human eye in its peak condition can see about .1mm
o For visualizing details, you need a microscope. Higher detail/resolution = lower limit of
∙ Types of organisms: Prokaryotes (anucleic Bacteria and Archaea), fungi (single/multicelled, eukaryotic), protozoa (singlecelled, euk, microscopic algae (single/multicelled,
euk), viruses (acellular), and tiny parasites (euk). Bacteria is the domain; “bacteria” –
Don't forget about the age old question of sarah myruski
uncapitalized and unitalicized – is a generic
term for microbes.
∙ Importance of Microbiology: In the News
o Public health issues, emerging diseases, newly discovered pathogens (EV-D68 and C105 do the same thing as polio), White Nose Syndrome
(increased humidity grows particular fungi and kills
frogs and bats), immunizations, food allergies,
antibiotic resistance (superbugs).
∙ We could not exist without microbes. They are important
o Health: microbes help maintain life; only a few are
An adult human is made up of around 30 trillion cells and another 40 trillion bacterial cells.
These human microbes form the microbiome or microbiota, the normal amount called flora (the first line of defense). Bacteria, such as E.
coli, aid digestion, synthesize vitamins, and
prevent the growth of pathogenic microbial
1) Only a few microbes are pathogenic (disease
producing); most are beneficial and necessary. 2) Health products, like insulin, are made using
3) LECTURE-ONLY MATERIAL If you want to learn more check out uf majors quiz
∙ Poor oral health is coordinated to
∙ The bacterium Porphyromonas
gingivalis secretes a toxic protein called
gingipain found in the brain, spinal fluid,
and saliva of Alzheimer’s patients.
∙ Blocking this protein is shown to halt
1) Regulates the atmosphere (~78% N2; 21% O2; 1%
Ar & trace gases)
o O2 generated by photosynthesis comes
mostly from microbes (photosynthetic algae)
o N2 is also generated by microbes, which
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of other wastes
o Water quality: microbes purify water.
o Bioremediation: microbes eat oil spills and
o Food Industry
1. Produce fermented foods (e.g. vinegar and cheese) 2. Food spoilage and food poisoning
o Economic impact: bad food chains can go out of business; knowledge of this can save
o You can save moldy maple syrup by
scooping out the affected part and
boiling the rest.
o Toxins that cause botulism can be so
potent as to be absorbed immediately by
o Chemical Industry
1. Produce valuable chemical products.
∙ It’s important to study microbiology for health, the
economy, and the environment.
1. Prevent diseases, epidemics, and pandemics
2. Prevent food spoilage (saves a lot of money) 3. Enhance positive traits of microbes for human use 4. Enhance degradation of pollution (bioremediation)
∙ The First Observations: History of Microbiology ∙ 1623-1673: Anton van Leeuwenhoek: The first
microbes are observed; “animalcules.”
∙ 1665: Robert Hooke: living things are composed of little boxes (cells); cell theory – all living things are
composed of cells.
o Spontaneous generation: the hypothesis that life arises from nonliving matter; “vital force” We also discuss several other topics like blanks plus
used for life.
o Biogenesis: the hypothesis the living cells can
come only from preexisting living cells.
∙ 1668: Francesco Redi: filled jars with decaying meet; maggots only appeared in the open jars (flies laid If you want to learn more check out what is a graphical link of competitive advantage, ksfs, and supporting activities?
∙ 1745: John Needham: boiled nutrient broth then placed in covered flasks; microbial growth;
∙ 1765: Lazzaro Spallanzani: boiled nutrient broth in sealed flasks; no microbial growth; biogenesis, but
deniers claimed air was necessary for spontaneous
generation to occur.
∙ 1861: Louis Pasteur: growth appears in nutrient broth heated in flask unsealed; S-shaped flasks let air in, kept microbes out, no growth in; biogenesis – We also discuss several other topics like ion defintion
microbes are present in the air.
∙ 1858: Rudolf Virchow: cell theory; cells arise from preexisting cells – against germ theory of disease (hypothesis that disease came from the cells of the
Golden Age of Microbiology (1857-1914)
o 1861: Louis Pasteur shows microbes are present in
Proof for biogenesis: s-shaped flasks keep microbes out and let air in. Broth showed no
signs of life and proves biogenesis.
o 1835: Agostino Bassi shows silkworm disease was
correlated to fungus.
o 1865: Pasteur shows silkworm disease was caused by
o 1840s: Semmelweis advocates handwashing to prevent transmission of puerperal fever (childbed fever) from physicians not cleaning their hands between obstetrical patients.
o 1860s: Joseph Lister (namesake of Listerine) used chemical antiseptic (phenol) to prevent surgical
o 1876: Robert Koch discovered anthrax was caused by a bacterium and developed Koch’s Postulates (still used today) to determine if a specific microbe causes
a specific disease.
The microbe must be:
∙ Found in abundance in all diseased
organisms and not in healthy ones.
∙ Isolated from the test organism and grown
in pure culture.
∙ Cause disease when introduced to a
∙ Re-isolated from the inoculated, diseased
test host and identified as identical to the
original causative agent.
o 1796: Edward Jenner inoculated a person with cowpox virus and made them immune to small pox
(protection = immunity).
∙ Modern Chemotherapy
o Treatment of disease with chemicals =
o Chemotherapeutic agents = synthetic drugs or
Antibiotics: chemicals produced by bacteria or fungi that inhibit/kill other microbes.
∙ 1928: Fleming accidentally discovers the
first true antibiotic; penicillium fungus
makes an antibiotic, penicillin, that killed
∙ 1940s: Penicillin was tested and mass
produced in time for WWII.
First synthetic drugs: quinine from tree bark
was used to treat malaria.
∙ 1910: Paul Ehrlich’s “magic bullet” could destroy a pathogen without harming the
host; a synthetic arsenic drug, salvarsan,
∙ 1930s: sulfonamides (sulfa drugs) are
synthesized (first antibiotic) and cause
allergies in some.
∙ Microbes Classification and Nomenclature
o Classification: process by which scientists group
living organisms based on similarities.
o Taxonomy: science of defining groups of life. o Nomenclature: science of naming organisms. ∙ Classification of Biological Organisms
o 1964: Robert Whittaker designs 5 kingdom scheme ∙ Plants, animals, fungi, protists, monera –
no tree of life
o 1980s: Carl Woese develops three domains based on specific genetic information – phylogeny (evolutionary relationships).
Bacteria, Archaea, and Eukarya
∙ Virus = possible 4th domain
LUCA: Last Universal Common Ancestor
Node = last common ancestor between two branches
∙ Biological Hierarchy
∙ Naming microorganisms – common names don’t translate
o Binomial naming: Genus + specific (species) epithet Species epithet can be used more than once,
but the genus can only be used once (e.g. Escherichia coli).
First use, you must spell it out. Second use, you can abbreviate the genus (e.g. E. coli).
Lecture 2: The Microscope
∙ Resolution (resolving power): the ability to distinguish be two separate objects; the distance between the objects (e.g. a resolving power of .02 μm means can distinguish
two points .02 μm apart.
∙ Know these units and be able to convert them (μm to nm
1mm = 10-3m; 1μm = 10-6m.
1pm = 10-12m = 10-9mm
1nm = 10-9m = 10-6mm
1μm = 10-6m = 10-3mm
1000nm = 1μm
.001μm = 1nm
1. Light microscopy (LM): good for cells and bacteria, not
2. Electron microscopy (EM): cells, cellular structures,
3. Atomic force microscopy (AFM): good for even molecules. 4. Combinations and specialized microscopy
∙ Limit of resolution (resolving power): ability to distinguish two separate objects with distance between them (e.g.
resolving power of .2 μm means distance of .2 μm). ∙ Shorter wavelength of light means greater resolution. o Electrons have much lower wavelength than light,
thus they produce images with lower resolution. ∙ Microscopes and Magnification
o Higher magnification/resolution = lower limit of
∙ Anatomy of a Microscope
∙ Light Microscopy
o Compound Light M.
Uses visual light and a 2 lens system
Total magnification = ocular lens (10X) x
objective lens (4X, 10X, 40X, or 100X)
Light may refract after passing through a specimen, stopping it from passing through the
∙ Refractive index: measure of a medium’s light
∙ Immersion oil keeps light from refracting; lowers the refractive index because it has the same
index as glass.
Brightfield Illumination: objects are either
colored or dark
∙ Requires staining; little detail; dark objects are visible against a bright (grey)
Light reflected off specimen doesn’t enter the
o Darkfield m.
Light objects, dark background; internal
Opaque disk placed in condenser; only light reflected off the specimen enters the objective lens.
o Phase-Contrast M.
Brings two sets of light (direct and diffracted) to form an image; grey background; living
organisms, internal structures, and visible cilia.
o Differential Interference Contrast (DIC)
M. – Nomarski Illumination – False Color Similar to phase contrast; 3D image; uses two
light beams + prisms to split
beams; organelles; no
o Fluorescence M.
Uses UV light: short wavelength = better
Fluorescent substances absorb UV light and emit longer visible wavelengths; naturally or
dyed fluorescent; dark background.
o Confocal m.
Fluorochromes; short wavelength (blue) light excites a single plane; each plane
is illuminated; 3D image
constructed to look at one plane
at a time.
o Scanning Acoustic M. – False Color
soundwaves; studies biofilms; Rz =
∙ Electron M.
o Electrons have shorter wavelength, greater
resolution, higher magnification; used for small images (e.g. virus).
o Transmission EM – False Color
Beam of electrons passes through ultra-thin specimen sections, an electromagnetic lens,
then a projector lens.
Specimens stained with toxic heavy-metal salts for contrast – beam is dangerous (shown on a
10,000 – 100,000x magnification; Rz = 10 pm o Scanning EM
Beam of electrons scans surface of the
specimen; secondary electrons produce a 3D
image, false color
1,000 – 10,000x magnification; Rz = 10 nm
∙ Scanning Tunneling M.
o Tungsten probe has resolution of 1/100 of an atom; scans details of a specimen
∙ AFM – 12 nm
o 3D images; metal-diamond probe
∙ Light Microscopy Procedures: Staining is Important o Staining: coloring microbes with dye to emphasize
o Smear: thin film of material containing microbes o Fixed: attaching the microbes to the slide with heat,
∙ Prokaryotic staining: the cell wall is negative and can only
be stained with basic stains (+ cation chromophore). One dye = simple stain – highlights entire
microbes to visualize structures.
Mordant: chemical that makes the specimen
look larger by keeping the stain in the cell.
∙ Differential stains: distinguishing prokaryote types o Gram stain: reflection of cell wall chemistry; Gram
positive (thick peptidoglycan cell walls) vs Gram negative (thin peptidoglycan cell walls and
Apply crystal violet (primary stain)
Apply iodine (mordant)
Alcohol wash (decolorization) – destroys LPS
Apply safranin (counterstain)
o Acid-fast stain – Mycobacterium and Nocardia Binds bacteria with waxy material in cell wall;
acid-fast can’t be decolorized; IDs tuberculosis
Carbolfuchsin (primary stain)
Methylene blue (counterstain)
∙ Structural Stains
o Capsule stain
Capsule: uncharged jelly covering (no dyes). Requires negative staining; acid dye (anion)
stains the background black and creates a halo. o Endospore stain
Endospores: resistant, dormant structures
∙ Primary stain: malachite green with heat
∙ Decolorize cells: water
∙ Counterstain: safranin
∙ Spores appear green within red/pink cells
o Flagella staining: difficult
Flagella: structures of locomotion (motility)
Use mordant and carbolfuchsin stain.
∙ Domains: Bacteria vs Archaea
Prokaryotic (pre-nucleus), single-celled
Peptidoglycan cell walls (rigidity and antibiotic
Binary fission or budding
Organic/inorganic chemicals or photosynthesis for
No peptidoglycan cells walls
Extremophiles (e.g. methanogens , halophiles,
∙ Virology: Study of Viruses
o Iwanowski (1892) and Stanley (1935) discovered the cause of mosaic disease to be a virus (aka a
∙ Mycology: Study of Fungi
o Eukaryotic with a distinct nucleus
o Chitinaceous cell walls
o Absorbs organic chemicals for energy (can’t make
o Yeast (unicellular) and molds and mushrooms
Molds consist of mycelia, composed of hyphae
∙ Protozoology: Study of Protozoa
o Absorb/ingest organic chemicals (not autotrophic like
o May be motile via pseudopods (amoeba), cilia, or
o Free-living or parasitic
∙ Phycology/Algology: Study of Microscopic Algae o Eukaryotic cells
o Found in freshwater, saltwater, and soil
o Cellulose cell walls
o Use photosynthesis to produce oxygen, carbohydrates,
and toxins (if in water)
∙ Parasitology: Study of Multicellular Animal Parasites o Eukaryotic/multicellular animals
o Parasitic flat worms and roundworms = helminths Considered microbes because of microscopic life
Lectures 3 & 4
∙ Prokaryotic vs Eukaryotic Cells
o Prokaryotic (pre nucleus)
One circular chromosome;
Binary fission/budding 70S ribosomes (S =
Svedberg unit; reflection of size)
o Eukaryotic: true
Paired chromosomes in
membrane-bound nucleus Histones
Polysaccharide cell walls (plants) like chitin and
Divides by mitosis
∙ Sizes, Shapes, and Arrangements of Prokaryotic Cells o Sizes: average is 0.2 – 2.0μm diameter x 2-8μm in
o Shapes: monomorphic (a single shape) or pleomorphic
(showing many shapes).
Bacillus (rod-shaped) or bacilli
Coccus (spherical) or cocci
∙ Vibrio (comma-shaped)
∙ Spirillum or spirilla
Groups of four: tetrads
Cube-like groups of eight: sarcinae
∙ Structure of Protists
o Glycocalyx: external to cell wall; viscous, jelly-like,
Two types: capsule (firm, organized) and slime
layer (loose, unorganized)
1. Capsule: provides virulence, prevents
a. Compartmentalization: DNA tightly
coiled in a nucleoid
b. Flagella (propels bacteria backward and
c. Fimbrin (attaches to structures)
2. Cell wall
3. Plasma membrane
∙ Prokaryotic Flagella
o Appendages external to cell that propel and rotate
o Made of flagellin; three parts:
Filament: Outermost region
Hook: Attaches to filament (motor)
Basal body: Embeds flagella into the wall
o Gram-positive bacterium do not have the outer membrane of a gram-negative bacterium; gram negatives have much more anchored flagella (doubled basal body).
o Allows bacteria to move toward or away from stimuli
Chemotaxis – run (to positive) and tumble
o Flagella proteins: H-antigens (anything that stimulates antibiotic resistance) that distinguish serovars (forms of bacterium).
∙ Arrangements of Flagellum
o Peritrichous: covered in flagella
o Monotrichous/polar: one flagellum
o Lophotrichous/polar: bundled at one end
o Amphitrichous: flagella on both ends
∙ Specialized Flagella – Axial Filaments/Flagella o Endoflagella in spirochetes (only between
membranes in gram-negatives); Anchored at one end
o Moves more like a worm/corkscrew in rotation (e.g. syphilis)
∙ Fimbriae: attachment with hair-like appendages ∙ Pili: proteinaceous, two types:
o Twitching motility pili (attaches, loosens, lifts,
o Conjugation pili (constant sex, intercellular DNA
∙ The Cell Wall
o Prevents osmotic lysis, protects the cell membrane,
contributes to pathogenicity
o In Bacteria, it is partially peptidoglycan and therefore
Peptidoglycan (rigid) is a polymer of NAG and NAM in rows linked by polypeptides. Recognize:
∙ ∙ Gram-positive vs Gram-negative
Thick peptidoglycan Teichoic acids (rigidity,
NO outer membrane Cell walls
Lipoteichoic acids link wall
to the plasma membrane Antigen specificity:
NO teichoic acids
Outer membrane: protects against phagocytes and
Lipopolysaccharides (LPS) O polysaccharide:
Lipid A: endotoxin in top
Porins (proteins) form membrane channels
o Know the Gram
o Periplasm (not periplasmic space) in the middle ∙ Contains thin peptidoglycan layer
∙ The Gram Staining Mechanism
o With crystal violet stain, iodine crystals form inside the
o Add decolorizing agent.
Gram +: crystals can’t get out, cells stay purple. Gram -: alcohol dissolves the LPS, crystal violet
leaves through peptide holes, safranin stains cells red/pink.
o Gram + Walls
2 rings in the basal body Exotoxins (botulism) Susceptibility to penicillin Disrupted by lysosome
(enzyme that destroys peptidoglycan)
o Gram – Walls
4 rings in basal body Endotoxin (Lipid A) +
exotoxin (secreted outside
Decreased penicillin susceptibility
∙ Atypical Cell Walls
o Acid-fast in Mycobacteria and Nocardia: use acid
alcohol to decolorize, stain with carbolfuschin.
Contains a waxy lipid (mycolic acid) bound to
o Mycoplasma: walking pneumonia
Lacks cell walls; sterols in naked plasma
o Archaea: wall-less, or walls of pseudomurein (no
peptides (NAM or D-amino acids))
∙ Cell Wall Damage
o Lysozyme hydrolyzes peptidoglycan bonds
o Penicillin inhibits peptide bridges in peptidoglycan
o Two cells susceptible to osmotic lysis:
Protoplast: wall-less gram +
Spheroplast: wall-less gram -
o L forms: wall-less cell that swells into irregular shapes ∙ Cytoplasmic membrane: phospholipid bilayer o Types of proteins:
Peripheral: weakly linked on the surface
Integral: embedded, penetrates the membrane Transmembrane: spans across the membrane ∙ Cell Membrane Structure: Mostly Lipid
o Fluid mosaic model: flexible, adaptive, and self
Membrane is as viscous as olive oil
Proteins move freely for various functions
Phospholipids rotate and move laterally
∙ Cell Membrane Functions
o Selective permeability allows passage for and
against some molecules.
o Contains ATP production enzymes: cellular respiration.
Some have chromatophores, photosynthetic pigments, on the folds of the membrane; no
chromatophores = invagination for surface area. o Cell content leakage via by alcohols, quaternary
ammonium (detergents), and polymyxin antibiotics. ∙ Antibiotics target: peptidoglycan, ribosomes, and the
∙ Movement Across the Membrane
o Passive: high to low concentration; no energy
Simple diffusion: small molecules (e.g. O2) travel until homeostasis or equilibrium is
Facilitated diffusion: ions/larger molecules use a transporter protein; move with the
Osmosis: movement of water across the
membrane from high water concentration (low solute) to areas of low water concentration (high
∙ Uses aquaporins to move through the lipid
∙ Osmotic pressure: pressure needed to
stop water movement across the membrane
(an increase in solute within – water follows
solute – equals greater pressure).
o Isotonic solution: the solute
concentration is equal inside and
outside the cell; water is at equilibrium.
o Hypotonic: the solute concentration is
lower outside than within; water moves
into the cell.
o Hypertonic: the solute concentration
is higher outside than within; water
leaves the cell.
o Active: substances move from low to high
concentration; energy expended.
Active transport: requires a transport protein
∙ Goes against the concentration gradient; the cell needs more of what it already has a lot
Group translocation: requires a transporter protein and a phosphotransferase (phosph- = you
can get energy from it via the phosphate group) ∙ Substance is altered as it crosses the
∙ Cytoplasm: not just a sac; the cell compartmentalizes o Specifically, the substance inside the membrane. o 80% water + proteins, carbs, lipids, and ions o Cytoskeleton: filaments (or microtubules for
∙ Nucleoid: where the DNA is; no membrane or nucleus
o Bacterial chromosome: circular DNA (single, size
varies) thread contains cell’s genetic information o Plasmids: extrachromosomal genetic elements (e.g.
noncrucial genes like antibiotic resistance and toxin
Resistance can be accidentally passed on to
pathogenic microbes during sex at the pili.
∙ Ribosomes (70S): protein synthesis sites
o Made of protein and ribosomal RNA
o Svedberg units (S) are not additive – they are based on
70S = 50S + 30S subunits
∙ Inclusions: structures within prokaryotes
o Metachromatic granules (volutin): polyphosphate
o Polysaccharide granules, lipid inclusions, and sulfur
granules: energy reserves
o Carboxysomes: RuBisCo (most abundant protein in the body, autotrophic) enzyme, used in photosynthesis
o Gas vacuoles: protein-covered cylinders maintain
o Magnetosome: iron oxide (magnetic crystals)
inclusions: destroys H2O2
∙ Endospores: produced by Bacillus and Clostridium.
o Resting, resistant cells of gram-positives for survival o Sporulation: six steps to formation
1. Spore septum isolates new DNA and part of
2. Plasma membrane surrounds DNA, cytoplasm,
and isolated membrane.
3. Spore septum surround isolated portion –
4. Peptidoglycan layer forms between
5. Spore coat forms.
6. Endospore is free from cell.
o Germination: the endospore returns to a vegetative
∙ Eukaryotic Cell Structures
o Cell Wall: found in plants, algae, and fungi
Made of carbohydrates (plants – cellulose; fungi
– chitin; yeasts – glucan and mannan)
o Flagella (long) and cilia (short) – proteins made of
Used for motility
Made of microtubules made of tubulin,
organized as nine pairs in a ring with two
tubules in the center.
∙ Glycocalyx (animal cells): carbs bonded to proteins and
lipids in the plasma membrane
∙ Plasma Membrane: similarities to prokaryotes o Phospholipid bilayer
o Integral and peripheral proteins
o Selective permeability
o Simple and facilitated diffusion, osmosis, and active
o Differences from prokaryotes
Sterols: complex lipids
Carbohydrates: rigidity and recognition
Endocytosis (white blood cells)
∙ Phagocytosis: pseudopods engulf
∙ Pinocytosis: membrane folds in, vesicles
∙ Cytoplasm: inside the plasma, outside the nucleus o Cytosol: fluid portion
o Cytoskeleton: microfilaments and intermediate
filaments – shape and support
o Cytoplasmic streaming: movement of cytoplasm throughout he cell; how an amoeba moves (building
and breaking up the cytoskeleton).
∙ Ribosomes: site of protein synthesis
o Eukaryotes: 80S (60S + 40S) – membrane-bound and free
Has 70S ribosomes in chloroplasts and
o Prokaryotes: 70S density
o Nuclear envelope contains DNA
o Histones – chromatin protein complexes that stabilize
Chromatin condenses into chromosomes
o Communication to the cytoplasm through pores and
∙ Endoplasmic Reticulum (ER): transport network
o Rough ER: studded with ribosomes, protein synthesis o Smooth ER: no ribosomes, synthesizes membranes,
fats, and hormones
∙ Golgi Apparatus (GA): transport organelle (Transmission
o Modifies ER proteins – changed from original mRNA
o Transports proteins to the membrane via secretory
o Cisternae: form secretory and transfer vesicles o Lysosomes: formed inside; contains digestive
o Vacuoles (secretory vesicles): cavities in cell formed from GA; brings food in (phagocytosis); provides
shape and structure.
∙ Mitochondria – 70S ribosomes
o Double membrane, cellular respiration, ATP production
o Inner folds (cristae) and fluid/material within (matrix) Increases surface area for increases # of
∙ Also in prokaryotes
∙ Chloroplasts – 70S ribosomes
o Double membrane, location of photosynthesis o Thylakoid: flattened membranes of chlorophyll
(stacked = Granum)
∙ Other Organelles
o Peroxisomes: oxidize fatty acids, destroys hydrogen
o Centrosomes: protein fibers and centrioles; mitotic
spindle and chromosome segregation
∙ Eukaryote Evolution
o The earliest organism was prokaryotic.
o Life arose 3.5 – 4 billion years ago (byo).
o First eukaryotes: 2.5 byo
o Earth is the same age as the solar system: 4.54 byo o Universe is 13.8 byo.
∙ Origin of Eukaryotes – Endosymbiotic
o The early cell takes up 70S chloroplast (for photosynthesis) from photosynthetic bacteria and 70S mitochondria (for respiration) from aerobic
o Larger cell eats this smaller cell – forms the double membrane.
∙ ∙ Lectures 5 & 6
∙ The Study of Phylogenetic Relationships
o Taxonomy: science of classification – degree and
o Phylogeny and systematics: study of evolutionary
o 1735: Linnaeus – Kingdoms Plantae and Animalia. o 1800s: Nägeli – Bacteria and fungi (kingdom
Haeckel – Kingdom Protista proposed for
bacteria, protozoa, algae, and fungi.
o 1937: The term “prokaryote” introduced to
distinguish cells without a nucleus.
o 1968: Murray – Kingdom Prokaryotae
o 1969: Whittaker – five-kingdom system
o Early 1978: Carl Woese – 3 Domain System o No fossil evidence of prokaryotes
3.5 byo structures of prokaryotes from South
∙ Appearances deceive
Stromatolites: cross section of fossilized
∙ Three Domains – Carl Woese (1978)
o Based on nucleotide sequence in 16S rRNA;
o Eukarya: animals, plants, and fungi
Membrane lipids: straight carbon chains linked
to glycerol by ester linkage
First AA in synthesis: Formylmethionine
o Archaea: methanogens, halophiles, and thermophiles Prokaryotic
Membrane lipids: branched carbon chains
attached to glycerol by ether linkage
First AA in synthesis: Methionine
No RNA loops or arms
o Recognize ester vs ether
o Phylogeny (Evidence of Endosymbiosis)
Originated from infoldings of prokaryotes –
Symbiosis: relationship between two organisms ∙ Bacteria within organism formed organelles
∙ Mitochondria has its own DNA that looks
like a prokaryote’s (circular)
∙ Eukaryotic mitochondria and chloroplasts are 70S, have no
histones, and divide via binary fission
∙ Eukaryotic species: group of closely related organisms that
breed and produce non-sterile progeny.
∙ Prokaryotic species: population of cells with similar (not
genetically identical) characteristics.
o Culture: bacteria grown in laboratory media Mixed: more than one species
Pure: one species (achieved with streak plates)
o Clone: populations of cells derived from a single
o Strain: same species, but not the same parent cell. ∙ Archaea and Bacteria do not have Kingdom classifications. ∙ Virus classification: not part of any domain and not
composed of cells; requires a host cell.
o Viral species: population of viruses with similar
characteristics within a particular ecological niche. ∙ Methods of classifying and identifying microbes o Classification: placing organisms in groups of related
o Identification: matching characteristics of an unknown organism to lists of known organisms
(clinical lab identification).
o Bergey’s Manual of Determinative Bacteriology provides identification schemes for identifying
species of Bacteria and Archaea.
o Approved Lists of Bacterial Names lists species of known classification; peer review decides the names
o Transport media: used to collect and transport
pathogens to a laboratory.
o Morphological characteristics: useful for identifying eukaryotes and some prokaryotes; says little about phylogenetic relationships.
o Differential staining: Gram staining, acid-fast staining (e.g. for tuberculosis); not useful for wall-less
bacteria like mycoplasms.
o Biochemical tests: determines presence of
o Polyphasic: a diverse approach to classification
using multiple techniques.
∙ Biochemical tests: used to be difficult, now easy. o Rapid identification methods: several biochemical
tests occur simultaneously using 20 tubes at a times,
with results assigned a number.
1. One tube with multiple tests.
2. Incubation with growth medium.
3. Assign each result its appropriate number.
4. Compare to a book of identification.
o Serology: science that studies serum and immune
responses in serum
o Microorganisms are antigenic – they stimulate the
body to form antibodies in the serum
o In an antiserum, a solution of antibodies is tested
against an unknown bacterium
o Slide agglutination test: bacteria bind, or agglutinate, when mixed with antibodies produced in response to the bacteria. (Positive result =
o Serological testing: differentiate between species and strains – very specific.
o Enzyme-linked immunosorbent assay (ELISA): Known antibodies and an unknown type of bacterium are added to a well, where the reaction identifies the
bacteria (via agglutination).
Antibody is absorbed to the well. Sample is added, antigen binds to antibody. Enzyme
linked antibody specific to the test antigen is
added, binds to antigen and forms a sandwich. Enzyme’s substrate is added, reaction produces
a visible color change.
o Western blotting: identifies antibodies in a
patient’s serum, confirms HIV infection.
Super quick; for detecting Lyme disease; uses lysing, gel, and electricity for separating
proteins by weight; transfer to filter, add serum, add antibodies; if it sticks, the bacteria is
∙ Phage typing: virus that affects bacteria; tests for determining which phages a bacterium is susceptible to; specific; lysing and plagues (large clearings where the
cells were killed) appear when the test is positive. ∙ Flow cytometry: uses differences in electrical
conductivity between species or fluorescence or size.
∙ Fluorescence-activated cell sorter (FACS): separates
cells that don’t react; two collection tubes.
∙ DNA base composition: separating and IDing by DNA; guanine (G) pairs with cytosine (C); adenine (A) pairs with thymine (T) (more common); two closely related
organisms have similar amounts of various amounts. ∙ DNA fingerprinting: electrophoresis of restriction
enzyme digests of an organism’s DNA; comparing
fragment profiles to find patterns and similar cuts. ∙ Nucleic Acid Amplification Tests (NAATs): organism
with few cells; use of PCR to amplify DNA (up to the entire genome of a single cell) of an unknow microorganism that
can’t be cultured
∙ Nucleic acid hybridization: measures the ability of DNA strands from one organism to hybridize with DNA strands of another organism; greater hybridization = greater
degree of relatedness
o Southern blotting uses this and a DNA probe to identify unknown organisms. Fluorescence = positive
∙ DNA chips, or microarrays, contain DNA probes and detect pathogens via hybridization between the probe and DNA, detected by fluorescence. Any gene; thousands of bacteria at once.
o Manufactures thousands of single-stranded DNA
o Fluorescent in situ hybridization (FISH): fluorescent DNA or RNA probes stain the target microbes;
determines identity, abundance, and relative activity. ∙ Putting classification methods together
o Node: similarity between rRNA sequences.
o Dichotomous keys (based on questions) and cladograms (maps that show evolutionary
relationships among organisms; based on rRNA sequences; >/= 98% means same species).
∙ Lecture 7: Viruses
∙ General characteristics of viruses
o Obligatory intracellular parasites (can’t grow alone) o Contain either DNA or RNA but never both; one
o No ribosomes or ATP-generating mechanisms. ∙ Virus host range: spectrum of host cells a virus can
o Most viruses infect only specific types of cells in one
Determined by attachment sites and receptors 20 nm to 1000 nm in length
∙ Bacteriophages: viruses that infect bacteria ∙ Structure of viruses
o Virion: complete, fully developed viral particle Nucleic acid: DNA or RNA can be single- or
double-stranded; linear or circular.
Capsid: protein coat made of capsomeres.
Envelope: lipid, protein, and carbohydrate coating on some viruses (generally a lipid
bilayer from the host cell; not all virions have
Spikes: projections from the outer surface (like
∙ Morphology of a nonenveloped polyhedral virus o Icosahedrons: Adenoviruses cause colds; 20
equilateral triangle faces.
∙ General morphology
o Helical: hollow, cylindrical capsid
o Polyhedral: many-sided
o Enveloped: indefinite shape, surround by host
∙ Morphology of complex viruses (phages)
o Most are made of proteins; act like syringes injecting
DNA into the bacterial cell.
∙ Viral taxonomy
o Genus name: ends in -virus
o Family name: ends in -viridae
o Order name: ends in -ales
o Viral species: a group of viruses sharing the same
genetic info and host, or what disease they cause Descriptive common names for species;
subspecies are designated by a number.
∙ Growing bacteriophages in the lab
o Viruses must be grown in living cells.
o Bacteriophages are grown in bacteria
Form plaques on the surface of agar; each
plaque corresponds to a single virus in plaque
forming units (PFUs).
∙ Growing animal viruses in the lab
o Complicated and time-consuming
o Done with living animals, embryonated eggs, and cell
∙ Cell cultures: tissue treated with enzymes, cells suspended in culture medium, normal cells grow in a single layer while transformed/continuous cell cultures
grow in pile.
o Cancer cells don’t show contact inhibition. ∙ Viral identification
o Cytopathic effects (CPEs)
Includes cell inclusions (e.g. Negri Bodies in
the rabies virus)
Syncytia: cell fusion; cells with many nuclei. o Serological tests
Western blotting – reaction of the virus with
antibodies (e.g. viral meningitis).
o Nucleic acids
DNA or RNA and double- or single-stranded
∙ Viral multiplication
1. Must invade a host cell
2. Must take over the host’s metabolic machinery ∙ Two general tasks:
3. Replicate nucleic acid
4. Synthesize proteins for the capsid (DNA to mRNA to protein)
∙ Viral one-step growth curve
o Bacteriophage enters the host and the eclipse
o Virions are released from the host, triggering acute
infection and peaking at the spike.
o Host dies.
∙ Bacteriophage multiplication
o Lytic cycle: Phage causes lysis and host cell death. o Lysogenic cycle: Phage DNA incorporated in the host
∙ T-even bacteriophages: the lytic cycle
o Attachment: phage attaches with tail fibers o Penetration: phage lysozyme opens the cell wall;
tail sheath contracts to force the tail core and DNA
into the cell
o Biosynthesis: production of phage DNA and proteins o Maturation: assembly of phage particles
o Release: phage lysozyme breaks the cell wall ∙ Bacteriophage Lambda (λ): The Lysogenic Cycle o Lysogeny: phage remains latent
o Phage DNA incorporates into host cell DNA
o Prophage: inserted phage DNA
o When the host cell replicates its chromosome, it also
replicates prophage DNA – phage conversion ∙
∙ Lecture 8: Metabolism
∙ Metabolism: the buildup and breakdown of nutrients o Chemical reactions provide energy and create life
o Catabolism: break down of complex molecules;
o Anabolism: build-up of complex molecules;
o Metabolic pathways: enzymatically catalyzed chemical reaction sequences (determined by
enzymes, encoded by genes).
∙ Collision theory: chemical reactions occur when atoms,
ions, and molecules collide.
o Activation energy: collision energy required for a
o Reaction rate: frequency of collisions containing enough energy to bring about a reaction (increased by enzymes or increased temperature, pressure, or
∙ Catalysts: speed up chemical reactions without
∙ Enzymes: bio-catalysts (specific substrate, lowers
o Substrate + enzyme’s active site = enzyme
o Substrate is transformed into products, enzyme is
o Turnover number: number of substrates an enzyme
converts to a product per second (1 to 10,000) o Named by adding -ase to the end; grouped based on
∙ Enzyme components
o Apoenzyme: protein portion
o Cofactor: nonprotein component (metal ion) Coenzyme: organic cofactor
o Holoenzyme = apoenzyme + cofactor
o Electron carriers assist enzymes.
∙ Factors of Enzyme Activity
o Extreme temperature and pH = denatured proteins o High substrate concentration (saturation) = enzyme
catalyzes at its max. rate
o Higher temperature and pH are good, but only up to
a certain point (35-40 degrees Fahrenheit, 5 pH) ∙ Inhibitors
o Competitive inhibitors: fill the active site of an
enzyme, competes with the substrate.
o Noncompetitive inhibitors: interact with the allosteric site (somewhere other than the active
site) in allosteric inhibition.
o Feedback inhibition: a reaction’s end-product allosterically inhibits the enzyme that made it;
prevents waste of energy.
∙ Ribozymes: RNA that acts as catalysts by cutting and
∙ OIL-RIG: Oxidation is Loss (of electrons), Reduction is
Gain (of electrons)
o Redox reaction: oxidation paired with reduction o Dehydrogenation: biological oxidation
∙ Generation of ATP
o 3 Main Pathways
o 3 Main Biochemical Mechanisms
Substrate-Level Phosphorylation: Glycolysis ∙ High-energy phosphate is added to ADP
∙ Electrons are transferred from one electron
carrier to another in the electron
transport chain (ETC) system, releasing
energy to generate ATP.
Photophosphorylation: only in light-trapping
∙ Light converted to ATP when chlorophyll
transfers electrons (oxidation) through an
∙ Prokaryotes: no oxygen produced, one
∙ Plants & Cyanobacteria: oxygen produced,
∙ Lecture 9: Microbial Growth
∙ Micronutrients are required for microbial growth, but you
don’t need much of them.
o Organic: vitamins, amino acids, purines, and
If you have to add these, it means the body
doesn’t make it itself.
o Inorganic (trace) elements: required in small
amounts, usually as enzyme cofactors (e.g. metal) Includes iron, copper, molybdenum, zinc, etc. ∙ The Effects of Oxygen on Growth: Recognize Growth
o Obligate aerobe: requires oxygen, grows near the
o Facultative anaerobe: grows via
fermentation/anaerobic respiration when oxygen isn’t available but prefers to have oxygen (enhanced
growth near the top).
o Obligate anaerobes: can’t use and are harmed by
oxygen (e.g. Clostridium tetani and botulin).
o Aerotolerant anaerobes: tolerate but can’t use
oxygen – no enhanced growth (fermenters).
o Microaerophiles: require oxygen concentration lower than air – hardest to grow.
∙ Toxic Byproducts of O2 Respiration
o Single oxygen (1O2-): charged, reactive
o Superoxide radicals: O2-
Toxic thieves that convert normal molecules to
Neutralized to water and oxygen by SOD
o Peroxide anion: O22-
Toxic, destroyed to water by…
∙ Catalase (bubbles)
o Hydroxyl radical: OH – ionizing radiation
∙ Biofilms: non-pure microbial communities that form slime/hydrogels (similar to glycocalyx) to adhere to
o Quorum sensing: bacteria communicate through the biofilm cell-to-cell; uses chemical called an
o Share nutrients
o Protection: shelters bacteria from the environment; 1,000x resistant to microbiocides; more resistant to
o Found in digestive and sewage treatment system;
cleans water but can also clog pipes.
o Involved in 70% of infections, usually involving implanted devices (e.g. catheters, heart valves,
∙ Culture Media
o Liquid: broth or broth culture.
o Solid/semi-solid: agar
Complex polysaccharide used as a solidifying
agent for culture media
Not metabolized by microbes
Liquifies at 100°C; solidifies at 40°C
o Chemically-defined media: the exact chemical
composition is known.
Fastidious organisms that require many
o Complex media: chemical compositive varies, uses extracts and digest of plants or meat (e.g. nutrient
broth or agar).
∙ Composition of Mediums
o Chemically Defined: Chemoheterotroph
∙ Glucose (C source)
∙ Ammonium phosphate, monobasic (nitrogen for protein)
∙ Sodium chloride
∙ Magnesium sulfate (for DNA)
∙ Potassium phosphate, dibasic (for phosphates and buffer to prevent end product poisoning)
∙ 1L – always add 1L of water
o Defined: Leuconostoc mesenteroides
∙ e.g. NaCl
∙ Amino Acids
∙ e.g. alanine, glycine
∙ Purines and
∙ e.g. adenine, uracil
∙ e.g. folate
∙ Trace Elements
∙ Fe, Co, Mn, etc.
∙ Buffer, pH 7
∙ Sodium acetate
∙ 1 L Distilled
∙ Also serves as
o Complex for heterotrophic
∙ Peptone (N, S, and C)
∙ Beef extract (N, S, and C)
∙ Sodium chloride
∙ Agar (solidifying agent)
∙ Anaerobic Growth
o Reducing media: removes oxygen and reduces
Cultivates anaerobic bacteria
Contains chemicals (sodium thioglycolate) that combine with O2 to deplete it (antioxidants,
Heated: drives off O2
In an anaerobic jar, the catalyst palladium
combines hydrogen and oxygen to form water. A colorless methylene blue indicator means
the environment is anaerobic.
∙ Special Culture Techniques
o Capnophiles: microbes that require lots of CO2;
candle jar or CO2 packet.
∙ Selective Media
o Suppress unwanted microbes, encourage desired microbes.
o Contains inhibitors to suppress growth
∙ Differential Media
o Distinguishes colonies of different microbes on the
same plate by measuring reactions.
∙ Selective and Differential: MacConkey’s Agar o Selective: crystal violet and bile salts only grow
o Differential: shows which ones ferment lactose (pink)
and which ones don’t (colorless).
∙ Selective and Differential: Mannitol-Salt Agar o Selective: high salt grows only some microbes. o Differential: ferments mannitol (yellow), doesn’t
ferment mannitol (phenol red stays red).
∙ Differential: Blood Agar
o Tests for virulence, not species; pathogenic = uses
Alpha: incomplete destruction of RBCs – E. coli Beta: complete destruction of RBCs – S.
Gamma: no hemolysis at all
∙ Enrichment Culture
o Encourages the growth of a desired microbe by increasing small numbers to detectable levels; first,
enhance; second, inhibit; detects salmonella.
∙ Culture Media Summary
∙ Biosafety Levels
o BSL-1: no special precautions, basic teaching labs.
o BSL-2: lab coat, gloves, eye protection.
o BSL-3: biosafety cabinets prevent airborne
o BSL-4: sealed, negative pressure, hot zone –
exhaust air is filtered twice, sterilize with UV light. ∙ Obtaining Pure Cultures
o Contains only one species/strain
o Colony: population of cells arising from single
AKA colony-forming unit (CFU) with viable
o Streak plate method: used to isolate CFUs. ∙ Preserving Bacterial Cultures (Theoretically Suspended
o Deep-freezing: -50 to -95 Celsius (generally -80C). o Lyophilization (freeze-drying): frozen (-54 to
-72C) and dehydrated in a vacuum.
∙ Bacterial Growth (Division)
o Increase in number not size
o Binary fission: 1 cell = 2 cells
o Budding: the parent cell is bigger than its single
o Conidiospores (actinomyocyetes)
o Fragmentation of filaments: mold
∙ Generation Time
o Time required for a cell to divide (20 mins to 24
o Binary fission doubles the number of cells each
o Total number of cells = 2number of generations
e.g. if you are five generations out, the total number of cells will be 25 = 32, log1032 =1.51
Take the log10 of the total number for your
∙ Growth curves are represented
logarithmically to make the lines linear and
∙ Phases of Growth (Know the Growth Curve)
1. Lag phase: not growing – shocked/had adapted to
2. Log phase: linear upward
3. Stationary phase: run out of nutrients, slow down
and stop growing b/c dividing = dying.
4. Death phase: population declines due to decrease
in nutrients or increase in toxins; difficult to place. ∙ Direct Measurement
1. Direct microscopic count
Volume of a bacterial suspension is placed on
Average number of bacteria per viewing field Petroff-Hausser bacterial cell counter (grid
squares of diluted microbes)
Total microbes = number counted / volume of
Depends on dilution (for decreasing
cloudiness or killing motile microbes) and
Disadvantage: viable (living) cells aren’t
2. Plate count: determines living cell number Dilute the original inoculum via serial
o Need number of 30-300 CFUs to give a
statistically significant count.
o Find dilution (D): D = amount added /
(amount added + volume of tube)
Total dilution = D x D x D x …
o Find concentration of CFUs (C): C = # colonies x inverse dilution; for two or
more tubes, [#colonies (1) + #colonies
(2)] / 2 (x inv. dil.)
o Count colonies: going forward, multiply your starting tube’s colonies by its
dilution; going backward, divide the
starting tube’s colonies by its dilution.
Bacteria are mixed into an agar dish (pour plate method) or spread on a plate surface
(spread plate method).
3. Filtration: metallic sheen, differential and selective. Solution passed through a filter that collects
Pore size = 0.22 micrometers
Filter is transferred to a Petri dish; grows CFUs
on the surface. Used for water and coliforms. o Coliforms: aerobic/facultative anaerobic,
Gram-negative, non-spore-forming rod-
shaped bacteria that ferment lactose
with gas formation within 48 hours at
E.g. Enterobacteriacea found in the
4. Most probable number (MPN) method
Multiple tubes: dilution through decreasing
amounts of inoculum.
Count the positive tubes and compare with a statistical table (goal is to have more
negative tubes) to find number of viable cells.
∙ Indirect Measurement: Growth Curves, Not CFUs 1) Turbidity: measurement of cloudiness with a
Optical density (OD) increases with an
increase in time and light. Increased OD
means a decrease in light.
A blank is set at 100, allowing all light, as the
2) Metabolic activity: amount of metabolic product
proportional to number of bacteria.
3) Dry weight: filtered, dried, and weighed
(filamentous organisms which grow in length).
∙ Lecture 10: Control of Microbial Growth
∙ Microbial Control
o Sepsis: bacterial contamination (usually in blood).
o Asepsis: absence of significant contamination (e.g.
o Sterilization: removing and destroying all microbial
life (including spores).
o Commercial sterilization: killing C. botulinum
endospores in canned goods.
o Disinfection: destroying harmful organisms on inanimate objects – not too worried about toxicity.
o Antisepsis: destroying harmful microbes from living
tissue – worry about toxicity. Not sterilization.
o Degerming: the removal of microbes from a limited
area (e.g. washing hands).
o Sanitization: lowering microbial counts on eating
o Biocide (germicide): treatments that kill microbes Bacteriocidal – applies to antibiotic drugs and
Fungicide – applies to fungi.
o Bacteriostasis: inhibits growth in microbes Bacteriostatic – applies to antibiotic drugs and
Fungistasis – applies to fungi.
∙ Rate of Microbial Death: The Microbial Death Curve o When microbes die, it’s not all at once. Occurs
o One log decrease = 90% of population killed o Treatment effectiveness depends on:
1) Number of microbes (closer to zero is best)
2) Environment (organic matter – increased
amount = increased difficulty; temperature;
biofilms = resistant)
3) Time of exposure
4) Microbial characteristics
o Gram positive versus Gram negative –
Gram-positives are harder to kill.
o Spore formation
∙ Microbial Control Agents
o Alteration of membrane permeability
o Damage to proteins (enzymes – denaturation) o Damage to nucleic acids (radiation)
∙ Heat: denatures enzymes
o Thermal death point (TDP): lowest temp. at which
all cells in liquid culture are killed in 10 min.
o Thermal death time (TDT): minimal time for all bacteria in liquid culture to be killed at a particular
o Decimal reduction time (DRT): minutes to kill 90%
of population at a given temp.
∙ Moist Heat Sterilization
o Moist heat (boiling, free-flowing steam) denatures
o Autoclave: steam under pressure contacts the item’s surface; heat, steam, and pressure in a double
o 121C at 15 psi for 15 min must reach the core of
o Kills everything; larger item = larger sterilization time.
o Test strips indicate sterility: black = positive. ∙ Heat to Reduce Numbers of Organisms
o Pasteurization: reduce spoilage and pathogens, but
can affect texture/taste.
o Equivalent treatment: 63C for 30min
High-temp short-time (HTST): 72C for 15 sec Ultra-high-temp (UHT): 140C for 4 sec
o Thermoduric: survive; don’t cause disease. ∙ Dry Heat Sterilization
o No autoclave; kills by oxidation.
Flaming, incineration, hot-air sterilization in
Either 160C for 2 hours or 170C for 1 hour.
Instruments should be dry since water
∙ Filtration: no dry heat; use for proteins, insulin. o Passage of substance through a screen-like material. o Used for heat-sensitive liquids.
o High-efficiency particulate air (HEPA) filters
remove microbes (>0.3 micrometer pores).
o Membrane filters remove microbes (>.22
∙ Physical Microbial Control
o Low-temp = bacteriostatic effect
Refrigeration, deep-freezing, and lyophilization. o High pressure denatures proteins
o Desiccation: absence of water prevents metabolism
o Osmotic pressure uses salts/sugars to create hypertonic environment; causes plasmolysis
o Ionizing (X-rays, gamma rays, e-beams): ionizes water to create reactive hydroxyl radicals; damages
DNA by causing lethal mutations.
o Nonionizing (UV light): damages by creating
o Microwaves: kills by heat, not very antimicrobial. o Radiant Energy Spectrum: increase the
wavelength, decrease the energy.
∙ ∙ Effective Disinfection: concentration of disinfectant,
amount of organic matter, pH, and time.
∙ Use-Dilution Tests
o Metal cylinders are dipped in test bacteria and dried. o Cylinders are disinfected for 10 minutes at 20C. o Cylinders are transferred to culture media to
determine if the bacteria survived.
∙ The Disk Diffusion Method
o Evaluates efficacy of chemical agents to kill bacteria o Filter paper disks are soaked in a chemical, placed on
o That concentration of disinfectant works if there is a
zone of inhibition.
Undiluted works very well but has toxic residue. ∙ Phenol and Phenolics
o Injures lipids of membranes, causes leakage.
o Joseph Lister used phenol/carbolic acid in aseptic
surgery which created phenol toxicity.
o Phenol was modified to be O-phenylphenol to be less
∙ ∙ Bisphenols: two phenol groups connected by a bridge. o E.g. hexachlorophene and triclosan – disrupt plasma
membranes and don’t burn people.
∙ ∙ Biguanides: antiseptic and disinfectant; pre-surgery;
disrupts plasma membrane.
∙ Halogens (Disinfectants and Antiseptics)
o Iodine (tincture, iodophor): impairs protein
synthesis, alters membranes.
o Chlorine (bleach, chloramine) : ozidizing agents shut
down cellular enzyme systems; WWI.
∙ Alcohols: denature proteins, dissolve lipids o No effect on endospores and nonenveloped viruses o Require water – 100% doesn’t inhibit growth, prefer
Ethanol and isopropanol
∙ Heavy Metals: nasty; be careful with the concentration.
o Oligodynamic action/effect: very small amounts
exert antimicrobial activity.
o Denatures proteins.
o Silver nitrate (ophthalmia neonatorum), mercuric chloride (milder in paint), copper sulfate (algicide),
zinc chloride (mouthwash).
∙ Surface-Active Agents: removes oils (e.g. acne); just
o Soap: degerming.
o Acid-anionic sanitizers: anions react with the
o Quaternary ammonium compounds: toxic;
bactericidal, denature proteins, disrupt membranes. o Recognize
∙ Chemical Food Preservatives
o Sulfur dioxide prevents wine spoilage.
o Organic acids inhibit metabolism and prevent molds
in acidic foods.
o Nitrites and nitrates prevent endospore germination,
adds red color to meats.
∙ Antibiotics: bacteria that produce compounds to inhibit
o Bacteriocins: proteins that inhibit a bacterium (e.g.
nisin and natamycin – prevent cheese spoilage). ∙ Aldehydes: inactivate proteins by cross-linking with
functional groups (e.g. -NH2)
o Used to preserve specimens and medical equipment.
Formaldehyde and glutaraldehyde (toxic
fixatives that preserve tissues).
∙ Chemical Sterilization: Gaseous sterilants cause alkylation (hydrogen atoms are replaced with a free
o Cross-links nucleic acids and proteins
o “Cold” systems: used for heat-sensitive material Ethylene oxide and formaldehyde
∙ Plasma: fourth state of matter consisting of electrically
o Free radicals destroy microbes; used for hands and
∙ Supercritical Fluids (SCF): CO2 with gaseous and liquid
o Any substance at a temperature and pressure above its critical point where liquid and gas phases do not
o Effuses through solids like a gas, dissolves material
like a liquid.
o Cleans medical implants
∙ Peroxygens and Others
o Oxidizing agents used for contaminated surfaces and
O3 (Ozone) and H2O2 (hydrogen peroxide)
∙ Depending on what we sterilize, our methods change. For instance, glutaraldehyde is fair against endospores, but good against mycobacteria (which is acid fast). ∙ Lecture 11: Genetics
∙ Big Picture
o Genetics: science of heredity
o Central dogma of molecular biology:
DNA -> mRNA -> protein -> function
o Mutations drive evolution (mutated DNA = altered
o Gene expression is controlled by operons (e.g.
differentiates kidney from blood cell.)
o Sex: exchange and recombination of genetic
∙ Types of Mutations
o Base-substitution mutation: a single DNA base
pair is altered.
o Frame-shift mutation: DNA base pairs are added or
∙ Importance of Microbial Genetics
o Alteration of genes and gene expression in bacteria
Find causes of diseases.
Prevent/treat disease (gene therapy).
Manipulate them for human benefit (insulin and
∙ Structure and Function of Genetical Material o Genetics: the study of genes, how they carry and
express information, and how genes are replicated.
o Chromosomes: structures containing DNA that carry hereditary info; containing genes and non-coding
o Genes: self-contained segments of DNA that encode
o Genome: all the genetic info in a cell (prokaryote =
o Genetic code: set of rules determines how a
nucleotide sequence is converted to proteins.
∙ Genotype: genetic makeup of an organism (not
∙ Phenotype: expression of a gene that can be observed as
∙ DNA and Chromosomes
o Bacteria: singular chromosome made of DNA and
o Eukaryotes: >1 chromosome and histones
o Chromosomes contain genes and short tandem
STRs: short repeating sequences of noncoding
∙ Small and specific; forensics.
∙ Flow of Genetic Information
o Vertical gene transfer: flow of genetic info from one generation to the next with daughter cells and
o Horizontal gene transfer: flow of genetic info
without cell division (bacteria).
∙ DNA Replication
o Describe DNA:
Nucleotides are held together by H-bonds between A-T and C-G (requires more energy to
Antiparallel strands (5’ 3’, 3’ 5’)
Sense and antisense strands
o The order of nitrogen-containing bases forms the
genetic instructions of the organism.
o One strand is the template for the production of a second – original strands are relaxed by topoisomerase and gyrase (H-bonds are relaxed,
and the activation energy is lowered).
o Helicase separates the strands, and a replication fork is created – bacterial DNA replication occurs at
o DNA polymerase (aka DNA-dependent DNA
Adds nucleotides to the growing strand. ∙ ALWAYS in the 5’ -> 3’ direction
∙ Initiated by an RNA primer
∙ Leading strand: continuous synthesis ∙ Lagging strand: discontinuous synthesis o Okazaki fragments
Removes RNA primers.
Uses DNA ligase to join Okazaki fragments
o Energy for DNA replication: hydrolysis of two phosphate groups (pyrophosphate).
o Semiconservative replication: each offspring cell
receives one strand of the original.
o Proofreading: DNA polymerase makes replication
∙ Transcription: RNA Synthesis
o RNA: single stranded nucleotide with 5-carbon ribose
Uracil (U) instead of thymine (T); A-U and G-C. o Transcription: DNA -> mRNA
Sense strand: 3’ to 5’, not transcribed into mRNA, contains codons, same nucleotide
sequence as mRNA.
Antisense strand: 5’ to 3’, transcriber to
1) Ribosomal RNA (rRNA): part of ribosomal
2) Transfer RNA (tRNA): transports amino acids
during protein synthesis.
3) Messenger RNA (mRNA): carries coded info from
DNA to ribosomes.
o Prokaryotes Transcription
Antisense strand: template for mRNA
Begins when RNA polymerase (DNA-dependent RNA polymerase) binds to the promoter
sequence on DNA.
Proceeds in 5’ -> 3’ direction.
Stops when it reaches the DNA terminator
Operon: unit of DNA with >1 gene.
∙ Translation: Protein Synthesis
o Translation: mRNA -> protein
o mRNA is translate into protein language; codons are groups of three mRNA nucleotides that code for an
o 61 sense codons encode the 20 essential amino
o Genetic code: degeneracy means many amino acids are coded by several codons (the 3rd position is
flexible enough to enable silent mutations, which is a base pair transfer that results in the same amino
o Begins at a start codon: AUG (Methionine)
o Ends at nonsense codons: UAA, UAG, UGA
o Codons are read sequentially
o tRNA transport amino acids to the ribosome to make
polypeptide chains (joined by peptide bonds).
An anticodon base-pairs with the codons
∙ Process of Translation
1) AUG starts translation; peptide bond begins at P
2) A site: acceptor site that attaches the next amino
acid. E site: where the tRNA exits.
3) The tRNA is released and the ribosome moves along
4) UAG stops translation. The last tRNA is released, the ribosome comes apart, and the polypeptide forms a
∙ In prokaryotes, translation can begin before transcription is completed, and multiple transcriptions can occur
simultaneously; no wasting; efficient.
∙ In eukaryotes, transcription occurs in the nucleus, and
translation occurs in the cytoplasm.
o Exons: regions that code for proteins
o Introns: DNA regions that don’t code for proteins. o Small nuclear ribonucleoproteins (snRNPs)
remove introns to splice exons together.
∙ Bacterial Gene Expression Regulation
o Carefully controlled so energy is not wasted: gene
Constitutive genes: expressed at a constant,
Others: expressed only as needed: inducible, repressible, and catabolite repression –
eukaryotes will not make more of what it’s given
in a medium.
∙ Pre-transcriptional Control
o Repression: inhibits transcription using repressors Default position is on.
o Induction: initiates transcription using inducers Default position is off.
∙ Operon Model
o Promoter: segment of operon where RNA
polymerase initiates transcription of structural genes. o Operator: segment of DNA that controls
transcription of structural genes.
o Operon: group of operatory and promoter sites and
the structural genes they control
o Polycistronic mRNA: eukaryotes have mRNA for
o Inducible operon: structural gene transcription does not occur until an inducer is present, the lac
Repressor active, operon off.
∙ The repressor protein binds to the operator
and prevents transcription from the
Repressor inactive, operon on.
∙ The inducer binds to the repressor protein
and structural genes are transcribed,
producing enzymes needed for lactose
o Repressible operon: structural gene transcription
occurs until a repressor is present, the trp operon. Repressor inactive, operon on.
∙ Transcription and translation proceed,
leading to tryptophan synthesis.
Repressor active, operon off.
∙ The corepressor tryptophan bines to the
repressor protein and binds the operator
and prevents transcription.
∙ Positive Regulation
o Catabolite repression inhibits cells from using
anything other than glucose as their carbon source. o Cyclic AMP (cAMP) builds up when glucose is not
cAMP binds to the lac promoter, initiating transcription during a lag time and allowing the
cell to use lactose.
∙ Epigenetic Control
o Epigenetics: heritable changes in gene
o Methylating nucleotides turn genes off.
o Methylated (off) genes are passed to offspring. o Not permanent, reversible through specific enzymes. ∙ Post-Transcriptional Control (zero waste in
o MicroRNAs (miRNAs) base pair with mRNA to make
o Double-stranded RNA is enzymatically destroyed. o Some mRNA is recycled for nutrients.
∙ Changes in Genetic Material
o Mutation: a permanent change in the DNA base
Can be neutral, harmful, or beneficial.
o Mutagens: agents that cause mutations (e.g.
o Spontaneous mutations: occur in absence of
mutagens (increased with age).
∙ Types of Mutations (Part 2)
1) Base substitution: change in one base in DNA, silent mutation doesn’t always change the amino
2) Missense mutation: base substitution results in a
3) Nonsense mutation: base substitution results in
a nonsense, or stop, codon.
4) Frameshift mutation: insertion/deletion of one or more nucleotide pairs shifts the translational
∙ Chemical Mutagens
o Nitrous acid: causes adenine to bind with cytosine
instead of thymine (A-C not A-T).
o Nucleoside analog: incorporates into DNA instead
of a normal base, causing mistakes in pairing.
o Oxidation of a nucleotides create a mutagen. ∙ Raditation
o Ionizing radiation (X-rays and the sun’s gamma rays) destroy DNA by forming ions that oxidize nucleotides
and break deoxyribose-phosphate backbones.
o UV radiation causes thymine dimers (2 thymines bind on the same strand); know DNA ligase joins old and
o Lethal mutation = cell death.
∙ Mutation Frequency
o Spontaneous mutation rate: one in a billion replicated
base pairs or one in a million replicated genes. o Mutagens increase the mutation rate to 10-5 or 10-3
per replicated gene.
∙ Mutation Identification
o Positive (direct) selection: mutant cells grow or
appear different from the normal cells.
o Negative (indirect) selection: mutant cells cannot
grow or perform a certain function.
o Auxtotroph: mutant that has a nutritional
requirement absent in the parent (replica plating). ∙ Identifying Chemical Carcinogens
o The Ames test: exposes mutant bacteria to mutagenic substances to measure the rate of
reversal (indicates how mutagenic a substance is) –
∙ Genetic Transfer and Recombination
o Genetic recombination: exchange of genes between two DNA molecules; creates genetic
o Crossing over: two chromosomes break and rejoin,
resulting in foreign DNA insertion to the chromosome. o RecA protein catalyzes the joining of two strands. o Vertical gene transfer: genes from an organism to its
o Horizontal gene transfer: genes between cells of the
same generation (no daughter cells)
Responsible for antibiotic resistance
2) Transformation: genes are taken up by one
bacterium from another as naked DNA.
a. Competent cells can transform/uptake
DNA – usually nonencapsulated.
3) Conjugation: horizontal gene transfer.
∙ Conjugation in Bacteria
o Conjugation: plasmids transferred from one
bacterium to another.
o Requires cell-to-cell contact via sex (F) pili.
o The F pilus pulls cells together for the mating bridge
(not reproduction, just DNA transfer).
Donor = F+
∙ Carries the plasmid ( Fertility factor) and
make the F pilus.
Recipient = F
o Mating F+ and F- = 2 F+ cells
o Hfr cells contain the F factor on their chromosomes o When an Hfr donor mates with an F- cell, the
recombinant F- cell gets DNA from Hfr cells, but
doesn’t have enough F factor.
∙ Plasmids: self-replicating, extrachromosomal, circular DNA. ∙ 1-5% the size of a bacterial chromosome (pretty
small, but megaplasmids exist).
∙ Code for proteins that enhance a bacterium’s
o Conjugative plasmid: F-factor; carries genes for
sex pili and transfer of the plasmid.
o Dissimilation plasmid: encodes enzymes for the catabolism of unusual compounds (e.g. certain
o Resistance factor: R-factor; multiple genes for
o Copy number: number of copies of a specific
plasmid per cell.
o Transposons, or jumping genes, are DNA segments that move from one region of DNA to another and may or may
o Insertion sequences (IS) code for transposase that cut and reseal DNA (does this by itself, no other
∙ Complex transposons can carry other genes
∙ BIG IDEA: The IS is the simplest transposon, and it carries the transposase gene, an enzyme that catalyzes transposition (horizontal gene transfer; a chromosomal segment is transferred to a new position on the same or another chromosome). The transposase gene is bound at both ends by inverted
repeat sequences, or transposon recognition sites. o Genes and Evolution: mutations and recombination create
cell diversity; natural selection acts of populations to ensure the survival of the fittest (e.g. antibiotic resistance).
∙ Lecture 12: Principles of Disease
o Pathology: the study of a disease.
o Etiology: the cause of a disease.
o Pathogenesis: the development of a disease. o Infection: the invasion or colonization by pathogens in
o Disease: an abnormal state where the body is not
performing normal functions.
o Normal microbiota: microbes that permanently colonize the host and do not cause disease normally (first line of
o Transient microbiota: not necessarily harmful; present for days, weeks, or months.
o Human Microbiome Project: analyzes the relationship of the microbial communities between
human health and the body.
o Distribution and Composition Factors of the Human
∙ Nutrients (in the gut and on the skin)
∙ Physical/chemical factors (lysosomes of saliva and
tears inhibit microbes)
∙ Host defenses
∙ Mechanical factors (same as physical)
o The skin, eyes, and nose/throat regions are made up of many different types of microbes. Keratin, low pH,
tears, mucus, and cilia help inhibit pathogens. o Relationships Between the Host and the Normal Microbiota o Microbial antagonism (competitive exclusion):
competition between microbes.
o Symbiosis: relationship between normal microbiota and host; varies over time depending on the
Commensalism: one organism benefits, the
other is unaffected.
Mutualism: both organisms benefit (normal
Parasitism: one organism benefits at the
expense of the other (all viruses).
o Opportunistic pathogens: the microbe goes where
it’s not supposed to be.
o Normal microbiota protects the host via:
o Competing for nutrients (e.g. a large amount of E. coli in our microbiome protects against food
o Producing substances harmful to invaders
o Affecting pH and O2 (sweat = acidic skin = dead
microbes, low oxygen = no competition)
o Koch’s Postulates: used to prove the cause of an
1. The same pathogen must be present in every case of
2. The pathogen must be isolated from the diseased
host and grown in pure culture.
3. The pathogen from the pure culture must cause the disease when it's inoculated into a healthy,
susceptible laboratory animal.
4. The pathogen must be isolated from the inoculated animal and must be shown to be the original
o Koch’s Postulates: Problems
o Some pathogens cause several diseases.
o Some pathogens cause disease only in humans. o Some microbes have never been cultured.
o Classifying Infectious Diseases
o Symptoms: changes in the body felt due to the
o Signs: changes in the body that can be measured
(qualitative/quantitative) due to the disease.
o Syndrome: a specific group of signs and symptoms for a disease.
o Communicable disease: a disease that is spread
from one host to another
o Contagious diseases: easily and rapidly spread. o Noncommunicable disease: a disease that is not
o Disease Occurrence
o Incidence: number of people who develop a disease
during a specific time period (only new cases). o Prevalence: number of people who develop a
disease at a specified time, no matter when it first
appeared (new and old).
o Sporadic disease: disease that only occurs
occasionally (typhoid fever).
o Endemic disease: disease is constantly present
o Epidemic disease: disease acquired by many
people in a given area in a short time (influenza). o Pandemic disease: worldwide epidemic (AIDS). o AIDS data: education and data (expansion of surveillance)
increased the number of cases, followed by a decline in number due to treatment and education. It is believed we
know Patient X.
o Severity/Duration of a Disease
o Acute disease: symptoms develop rapidly, disease
o Chronic disease: symptoms develop slowly, last a long time.
o Subacute disease: intermediate between acute and
o Latent disease: causative agent is inactive, but
then activates with symptoms.
o Herd immunity: immunity in most of a population (80% immunity is necessary to protect the
o Extent of Host Involvement
o Local infection: pathogens are limited to a small
area of the body (can become systemic).
o Systemic (generalized) infection: infection
throughout the body.
o Focal infection: a systemic infection that started as
o Sepsis: toxic inflammation from the spread of
microbes from a focus of infection.
o Bacteremia: presence of bacteria in the blood. o Septicemia: aka blood poisoning; growth of bacteria
in the blood that causes infection/inflammation. o Toxemia: toxins in the blood.
o Viremia: viruses in the blood.
o Primary infection: acute infection that creates the
o Secondary infection: opportunistic infection after
the primary or predisposing infection.
o Subclinical disease: no noticeable signs/symptoms
o Predisposing Factors to Disease: What Makes the Body Susceptible