Microbiology

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Microbiology
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Prokaryote Architecture

• Simple in shape, but genetically and
  biochemically advanced

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General Prokaryote Shapes

• Coccus round or spherical
• Bacillus rod shaped
• Spirila / Vibrio spiral or twisted, corkscrew,
  halfmoon

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• Diplo groups of 2
• Strepto - chains
• Staphlo grape like clusters

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Microscopy

• Compound Light Microscope
• Helps to determine cell size and shape
• Some internal structures may be seen
• Usually need special dye

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Microscopy

• Electron Microscope (to 150,000X)
• Transmission (TEM) helps to see internal
  cellular features (DNA, cell wall/membrane,
  ribosomes, etc.)
• Scanning (SEM) helps to see external features
  (cell surface, envelope, flagella, etc.)

TEM HIV on lymph
SEM RBCs in clot
SEM E.coli on sm intestines
SEM intestinal tape worm
TEM flu virus
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Typical Prokaryotic Components

• Cell membrane selectively permeable barrier
  separating inside of the cell from its
  environment
• Cell wall rigid structure surrounding cell
  membrane
• Gives structural support
• Protection from lysing (breaking)
• Made of peptidoglycan (sugar protein polymer)
• Sensitive to PCN

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• 3. Ribosomes combination of RNA protein
• Site of protein synthesis
• Sequence of RNA nucleotide bases is used to
  identify species
• 4. Chromosome DNA of cell
• Almost always only 1 per cell
• May be 2-4 copies in an actively growing cell
• 5. Inclusions single structure of molecules of
  C, P, S, N
• Stockpiles of necessary nutrients for future use
  in metabolism

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• Flagella - structure that allows cell to be
  mobile in an aqueous habitat
• Classified based on how many flagella

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2 Major Groups of Bacteria

• Gram Positive Bacteria

• Gram Negative Bacteria

• About 10 of all bacteria
• Thick cell wall
• Contains lots of peptidoglycan
• Purple

• About 90 of all bacteria
• Thin cell wall
• Small amounts of peptidoglycan
• Pink

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Prokaryotic Cell Walls

• Diagrams of the cell wall structure of
  Gram-negative (left) and Gram-positive bacteria.
  Key peptidoglycan layer (yellow) protein
  (purple) teichoic acid (green) phospholipid (
  brown) lipopolysaccharide (orange).

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Gram Staining

• Simple staining technique used to differentiate
  the 2 groups of bacteria
• Uses the differences in the cell walls of
  different bacteria
• Specific Steps to process
• Heat fix slide
• Crystal violet (1 min)
• Iodine (1 min)
• Alcohol (5-10 secs)
• Safranin (1 min)

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Microbial Growth
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• Bacteria vary from minutes to years in
  reproduction time!
• Microbial growth increase in the number of
  cells in a population (group of individuals of
  the same species)
• right conditions must exist.
• DNA replication, transcription, and translation
  have to occur
• Proteins, lipids, polysaccharide synthesis all
  occur simultaneously

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• Binary Fission one cell to two cells

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• Exponential Growth population increases in
  number of cells in a fixed time period (1 ? 2? 4?
  8? 16 ? 32)
• Ideal growing conditions must be present
• Steady nutrient supply space
• Unchecked / unlimited growth

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Growth Curve of Bacteria

• Assumes abundant space, food, and no
  competition!

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• Lag (acclimation) Phase culture is transferred
  to fresh media
• Requires time to adjust for growth to begin
  (synthesize DNA, enzymes)
• Log (exponential) Phase time of rapid growth
• Exponential increase
• Unlimited resources, ideal growing conditions

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• Stationary Phase log ends as nutrients space
  are used up and waste products build up
• Balance b/w reproduction and death
• Cells began to encounter environmental stress
• Lack of water, nutrients, space
• Build up of waste
• Changes in oxygen and pH
• Death Phase cells cease metabolism they become
  inactive or die due to limiting factors in the
  environment
• Some dead cells are alive but enter into
  suspended animation or form spores
• Both can grow again

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• Have the growth curve because we can measure the
  number of total cells in broth culture (blood,
  tissue, water)
• Microscopy
• Spread Plating
• Turbidity

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Microscopy

• Direct cell count by counting the cells within a
  grid (field) then extrapolating to total volume

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Spread Plating

• Plate counts after a serial dilution, then count
  colonies, and extrapolate total volume

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• A plate count may be done on plates prepared by
  either the pour plate method or the spread plate
  method.
•  

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Turbidity

• Use a spectrophotometer
• Use a broth culture in a tube and insert in
  machine
• Amount of light blocked by cells is proportional
  to the number of cells.

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Which method is better?
Pros Cons
Direct Count Easy Dead Living
Plate Count Only living counted Time-consuming
Turbidity Easy Also get dead cells

• Best approach is to use turbidity after a plate
  count
• Use 2 methods _at_ 1st, then turbidity

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Nutrition Metabolism

• Catabolism breakdown of chemicals to release
  energy
• Anabolism biosynthesis building of larger
  molecules
• Requirements for Growth
• Physical
• Temperature ( -15? C to 125?C)
• pH (-0.05 to 13)
• Salt (0 to 30)
• Osmotic pressure
• Chemical
• micro macro elements
• Oxygen (0-21)

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Temperature

• Cold
• psychrophiles (cold loving) organisms that
  grows best -15?C up to 15?C. Dont grow above
  25?C
• Psychrotolerant (cold tolerant) - organisms that
  grows best gt20?C, but can grow at lower temps
• Middle
• Mesophile organism that grows best b/w 20?C
• Hot
• Thermophiles (hot loving) organisms that grow
  best above 45?C but below 80?C
• Hyperthermophiles (extreme thermophiles) grow
  best above 80?C

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pH

• Acidophile organisms that grow best below pH of
  6
• many foods, such as sauerkraut, pickles, and
  cheeses are preserved from spoilage by acids
  produced during fermentation
• Neutrophile (neutral pH) anything b/w 6 8
• Where most bacteria grow best
• Alkalinophile (basic) grows best pH above 8

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Salt

• Halotolerant tolerant to salt, dont require
  salt, but can grow in presence of salt
• Halophiles require some salt for growth (up to
  10)
• Extreme halophiles- require at least 10 salt for
  growth

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Osmotic Pressure

• Microbes obtain almost all their nutrients in
  solution from surrounding water
• Tonicity
• isotonic
• hypertonic
• hypotonic

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• Many chemicals needed for growth of these
  organisms

Elements C O N H P S Na K Ca Mg Fe Cu, Zn, Mo, B, Se, Cl, Ni, Co, etc.
of cell(dry wt) 50 20 14 8 3 1 1 1 .5 .5 .2 .2
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Major Elements Uses in Cells

• MACRONUTRIENTS
• H (8), O (20) H20
• Carbon (C) 50 major constituent of all
  macromolecules uptaken by cells as organic
  carbon or as CO2
• Nitrogen (N) 14 major element in proteins and
  nucleic acids uptaken as NH3, NO3-, N2,
  organic molecules
• Phosphorus (P) 3 major element in ATP,
  phospholipids, nucleic acids uptaken as PO43-
• Sulfur (S) 1 used in amino acids (cysteine,
  methionine) vitamins uptaken as SO42- HS

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• MICRONUTRIENTS
• Potassium (K) 1 transport of small molecules
  across the cell membrane, helps in enzyme
  function, involved in protein synthesis
• Sodium (Na) 1 can be used by enzymes in
  membrane transport, but it is not required by all
  species
• Magnesium (Mg2) 0.5 stabilizes DNA and helps
  in enzyme function, such as DNA polymerase and in
  ATP productions
• Metals (Fe2, Fe3, Cu, Zn) used in electron
  transport, used by proteins involved in electron
  transport processes (metabolism)

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• Can remember these by
• CHOPKNS CaFe all except Mg Na

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Oxygen

• There are 4-5 different oxygen requirements for
  bacteria
• Obligate aerobe
• Facultative aerobe
• Microaerophile
• Aerotolerant anaerobe
• Obligate anaerobe

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Obligate aerobe

• Require full level of O2 (20-21) to grow

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Facultative aerobes

• Grows best in O2, but can grow without O2

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Microaerophiles

• Grow at O2 levels lt20, but require less

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Aerotolerant anaerobes

• Dont require O2, but can grow in presence of O2

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Obligate anaerobes

• No O2 required, O2 is toxic

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1. Obligate aerobic
2. Obligate anaerobic
3. Facultative aerobe
4. Microaerophile
5. Aerotolerant

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What does all this mean?

• You need to know what the organism you are
  culturing requires for growth so you can study
  and treat them!!

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Growth control
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Methods to Control Growth

1. Heat Sterilization
2. Radiation
3. Filtration
4. Antimicrobial Agents

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Heat Sterilization

• Sterilization destruction of all viable life
• Incineration (dry heat) glassware, metal
  objects
• 160? 550?C
• Denatures proteins and makes organic molecules
  unstable
• Takes seconds to hours
• Pasteurization (low heat over time) milk,
  fluids
• 63? 72?C
• Kills up to 99 organisms in milk
• 15 seconds 30 minutes
• Autoclave (moist heat) glassware, metal
  objects, liquids (sm. Vol.), plastics
• 121? 15 psi pressure is used to increase temp.
• Minutes to hours

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Radiation

• Ionizing radiation (gamma rays) breaks DNA
  disrupts important genes death
• Used for plastics, antibiotics, food
• Ultraviolet radiation (ultraviolet rays) DNA
  RNA absorbed and forms strong bonds between
  thymine prevents DNA replication
• Sterilizes water, air, and surfaces

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Filtration

• Size of pores or matrix of fibers capture cells
  while air or fluid passes through

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Antimicrobial Agents

• cide death, bacterialicidal or fungacidal
• static growth inhibiting, bacteriostatic,
  algastatic, etc.
• Antimicrobial agents are chemicals (natural,
  synthetic) used to control microorganisms
• Examples
• alcohol (denatures proteins dissolves lipids
• Halogens (bleach, iodine)
• Antibiotics natural chemicals produced by
  microbes to inhibit growth of other organisms

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Antibiotics

• Kills 3 ways
• Destruction of cell membrane
• Disrupts cell wall synthesis (peptidoglycan)
• Interferes with protein synthesis or nucleic acid
  synthesis
• The trick is to harm the microbe without harming
  the host
• Bacterial antibiotics are not usually a problem
  with humans (unless they become resistant)
• Fungal antibiotics much riskier since the
  mechanism of action is eukaryote-specific

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Target Mechanisms
Cyclohexamide Fungi, blocks translation
PCN Gram pos, blocks cell wall synthesis
Chloramphenicol Broad spectrum, blocks translation
Polymyxins Broad spectrum, disrupts cell membrane
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Protein Synthesis

• DNA Replication
• DNA passed from parent to progeny
• Binary fission
• gt2 copies of the chromosome occur in actively
  growing cells
• Transcription
• RNA copies of DNA (genes)
• mRNA
• Translation
• mRNA decoded into proteins and enzymes
• Takes place at ribosomes using tRNA and rRNA
• tRNA carries amino acids to ribosome, compliments
  mRNA
• rRNA joins amino acids together, part of the
  ribosome structure reads the mRNA (structure
  catalytic role)

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• In prokaryotes, there is one circular chromosome
• Bases range from 500,000 to 10,000,000

Yeast Chromosome
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Fidelity of Replication

• Assuming a genome of 5,000,000 bases and an error
  rate of 1 in 1,000,000,000 bases. How many
  changes has occurred?
• Mutation is a change in the base sequence of DNA
  that is inherited
• Mutation rate is the number of base changes for 1
  cell

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Answer

• 5,000,000 /1,000,000,000
• 0.005

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• DNA can fix its mistakes!!

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DNA Replication

• Considered semi-conservative
• Half of chromosome is copied (template) to make a
  complimentary strand
• Other strand is copied simultaneously
• Each resulting cell in binary fission has ½ the
  original DNA and ½ newly synthesized DNA

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• ALWAYS proceeds from the 5 to 3 direction
• Each new nucleotide as added to 3 OH group

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• Transcription mRNA copy of DNA (tRNA and rRNA
  also transcribed)
• Translation reading of the mRNA information
  to form a protein
• Occurs at the ribosomes (in cytoplasm)
• Up to 1,000,000 in active cells
• Ribosomes are 66 rRNA and 34 protein
• tRNA, rRNA, mRNA are all involved in
  translation

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• The language of DNA exists in 3 letter words
  that we call codons
• The sequence of DNA determines sequence of amino
  acids in proteins and enzymes
• Change in DNA sequence change in amino acid
  sequence (often mutation)

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Reading Frame

• TAC AGG TCC GCA TAT

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2 types of mutations

• Base change - ATC GTC
• May or may not be fatal
• 3 outcomes
• Positive mutation enhances cell survival
• Neutral mutation no change on cell survival
  ability
• Negative mutation detrimental effect on cell
  survival ability, usually leads to death
• Frame shift (insertion or deletion)
• Almost always negative!!!
• Some mutations can be fixed by DNA repair enzymes

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Altering Microbial Genomes

• Main mechanisms of genomic change
• Mutation replication errors, radiation,
  chemical stress
• Transformation DNA from environment
• Transduction viral DNA to bacteria
• Conjugation bacterial DNA to bacteria
• Transposition jumping genes transposable
  elements

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• Genetic recombination
• Genetic elements contained in 2 separate entities
  are added together
• recA protein responsible for swapping DNA
  sequences

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Transformation

• DNA source is free in the environment
• Many gram positive and gram negative bacteria and
  some archaea can take up free DNA, but not all
  species do
• Competence ability to accept DNA from outside
  of the cell
• Uptaken DNA can be a fragment or a plasmid
• DNA is recombined with chromosomal DNA or is left
  as a plasmid

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Transduction

• DNA is donated from a bacterial cell to another
  via a virus
• Virus infects a cell (bacterial), degrades its
  DNA, multiplies, and lyses cell
• Some new viruses have host DNA
• Virus infects new cell and incorporates old host
  DNA into it (Forest Rowher)

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Conjugation

• Cell to cell contact involving either the
  transfer of a plasmid or chromosome (more rare)
• Plasmid DNA that isnt part of the chromosomes
  and isnt necessary for cell survival
• May help in cell survival in the presence of
  unusual foods (pesticides, solvents), antibiotic
  resistance, or toxic metals
• extra source of genes on plasmids allow the
  food to be degraded, the antibiotic to be
  blocked, or te metal to be detoxified/blocked

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• Conjugation involves a pilus (tube or channel
  between 2 cells)
• Plasmid is replicated and transferred to a new
  cell at the same time
• Donor cell has plasmid
• Recipient cell doesnt have the plasmid

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(PCR) polymerase chain reaction

• PCR invented in 1983 by Kary Mullis of Lenoir
• Technique allows DNA to be copied outside of the
  cell
• Biochemical reaction in a heated tube
• Mimics cellular processes consists of a
  repeated series of temperature changes (thermal
  cylcing)

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3 steps

1. Denatureation
2. Annealing
3. Elongation Extension

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Denaturation

• 94 degrees C
• DNA melting from double stranded to
  single-stranded molecules
• Takes the place of helicase and binding proteins

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Annealing

• 45 65 degrees C
• Allows primers to find their complements on the
  DNA template
• PCR primer usually 15-25 nucleotides in length
  and specific to a gene or gene fragment

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Elongation Extension

• Polymerase finds the primers and adds nucleotides
  to the 3 OH groups, complementing the template
• Polymerase from Thermus aquaticus is used
• Taq adapted for high temperature function

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• Each cycle of PCR (94 then 45-65 then to 72
  degrees C) results in doubling of DNA copies
• http//www.cnpg.com/video/flatfiles/539/

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• http//www.maxanim.com/genetics/PCR/PCR.htm
• http//www.sumanasinc.com/webcontent/animations/co
  ntent/pcr.html

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Applications of PCR

• Identify unknown microbial species
• Crime investigations
• Genome sequencing
• Gene screening, medicine development, gene therapy

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Molecular Chronometers

• How we tell the evolutionary relatedness of life
• Linus Pauling and Carl Woese searched for
  biological molecules that were common to all life
  that could be used to reconstruct evolution
• Found that 16s and 18s rRNA eventually were found
  to be the most useful

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Phylogenetic Tree of Life
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Molecular Chronometer

• Evolutionary time keeping
• Groups of organisms are arranged on tree drawings
  together and separate distinctly related
  organisms
• Evolutionary distance the sum of the physical
  distance on an evolutionary tree
• Trees are drawn by comparing the nucleic acid
  sequence

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Molecular Chronometer should have these features

• The molecule should be found in all groups
  studied
• The molecules should have the same function in
  all organisms
• Sequence alignments should be easily done
• ATC GCC TTT
• ACT CGG TTT (2/3 align)
• Rate of change has to be slow enough to measure
  time excessive mutations not good b/c they mask
  evolutionary change.

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• We use rRNA as a molecular chronometer b/c it
  fits the above criteria and includes all life
  (ALL life needs ribosomes to make protein)
• Molecular Chronometer is very powerful tool
  to tie all life together!!

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Small subunit rRNA of the three domains of life.
Bacteria (Mitochondria Chloroplasts) 16SrRNA
Archaea 16SrRNA
Eucarya 18SrRNA
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• Construction of tree requires massive computer
  calculations to determine the best fit of the data

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Phylogenetic Distance Matrix Tree

• Nodes represent divisions in taxonomic units.
• Relative evolutionary distance is the sum of
  line distance.
• Scale is in units of fixed-point mutations per
  sequence position.
• Trees can be rooted or unrooted (rooted trees
  require more complex calculations as there are a
  greater combination of possible outcomes)

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Prokaryotes and Humans

• Pathogen an organism that gives injury to host
• Virulence measure of the ability of a pathogen
  to inflict damage
• Infection growth or a population in/on tissue
• Most bacteria associated with humans belong
  there!! (There is more microbial cells than human
  cells on your body)

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Bacteria on Skin

• Mostly gram positive bacteria
• 102 106 /cm2 increases when moist

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Bacteria in Mouth

• Think teeth and gums (cavities)
• W/I saliva 108 cells/mL

Streptococcus mutans
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Nasal / Throat

• Moist, nutrient-rich environments
• Mucous phlegm are nutrition sources for
  microbes

Streptococcus aureus
Haemophilus influenzae
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Stomach

• Very acidic environment (pH lt2.0)
• Contains lt10 cells/mL of stomach fluid

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Small Intestines

• Increase in micrfobial numbers
• Very rich food supply
• pH increase from stomach
• Probiotics in livestock

Enterococcus faecalis
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Large Intestines

• fermentation vat
• 1010 1011 bacterial cells/gram
• Aids in digestion
• Provides vitamin b12, biotin, riboflavin, vitamin
  k

Escherichia coli
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Urethra

• Relatively free of bacteria
• Lower areas have some bacteria fungi (yeast)

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Vagina

• Very moist environment
• Nutrient rich
• Bacterial and fungal yeast have steady ecosystem
  that deters invading species (pH around 4)

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Foreskin

• Beneath foreskin creates great microbial habitat

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• Oppertunistic pathogens free living organism
  but can attack host
• Candida albicans
• Kaposi's Sarcoma
• Clostridium difficile
• Accidental pathogens produces toxins or causes
  disease inadvertenly (unusual circumstances)
• Clostridium tetani
• Obligate pathogen cant live outside the body
• Treponema pallidum syphilis