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Microbiology. Prokaryote Architecture. Simple in shape, but genetically and biochemically advanced. General Prokaryote Shapes. Coccus – round or spherical Bacillus – rod shaped Spirila / Vibrio – spiral or twisted, corkscrew, halfmoon. Diplo – groups of 2 Strepto - chains

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prokaryote architecture
Prokaryote Architecture
  • Simple in shape, but genetically and biochemically advanced
general prokaryote shapes
General Prokaryote Shapes
  • Coccus – round or spherical
    • Bacillus – rod shaped
    • Spirila / Vibrio– spiral or twisted, corkscrew, halfmoon
Diplo – groups of 2
  • Strepto - chains
  • Staphlo – grape like clusters
  • Compound Light Microscope:
    • Helps to determine cell size and shape
    • Some internal structures may be seen
    • Usually need special dye
  • 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 – RBC’s in clot

SEM – E.coli on

sm intestines

SEM – intestinal

tape worm

TEM – flu virus

typical prokaryotic components
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

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

Flagella - structure that allows cell to be mobile in an aqueous habitat

    • Classified based on how many flagella
2 major groups of bacteria
2 Major Groups of Bacteria

Gram Positive Bacteria

Gram Negative Bacteria

About 90% of all bacteria

Thin cell wall

Small amounts of peptidoglycan


  • About 10% of all bacteria
  • Thick cell wall
  • Contains lots of peptidoglycan
  • Purple
prokaryotic cell walls
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).

gram staining
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)

***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

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
growth curve of bacteria
Growth Curve of Bacteria

**Assumes abundant space, food, and no competition!


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

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

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

  • Microscopy
  • Spread Plating
  • Turbidity
  • Direct cell count by counting the cells within a grid (field) then extrapolating to total volume
spread plating
Spread Plating
  • Plate counts after a serial dilution, then count colonies, and extrapolate total volume

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

  • 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.
which method is better
Which method is better?
  • Best approach is to use turbidity after a plate count
  • Use 2 methods @ 1st, then turbidity
nutrition metabolism
Nutrition & Metabolism
  • Catabolism – breakdown of chemicals to release energy
  • Anabolism – “biosynthesis” – building of larger molecules
  • Requirements for Growth
    • Physical
      • Temperature ( -15ْ C to 125ْC)
      • pH (-0.05 to 13)
      • Salt (0% to 30%)
      • Osmotic pressure
    • Chemical
      • micro & macro elements
      • Oxygen (0-21%)
  • Cold
    • psychrophiles (cold loving) – organisms that grows best -15ْC up to 15ْC. Don’t grow above 25ْC
    • Psychrotolerant (cold tolerant) - organisms that grows best >20ْC, but can grow at lower temps
  • Middle
    • Mesophile organism that grows best b/w 20ْC
  • Hot
    • Thermophiles (hot loving) – organisms that grow best above 45ْC but below 80ْC
    • Hyperthermophiles (extreme thermophiles) grow best above 80ْC
  • 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
  • Halotolerant – tolerant to salt, don’t 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
osmotic pressure
Osmotic Pressure
  • Microbes obtain almost all their nutrients in solution from surrounding water
  • Tonicity
    • isotonic
    • hypertonic
    • hypotonic
major elements uses in cells
Major Elements & Uses in Cells


  • 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


  • 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)

Can remember these by:

CHOPKNS CaFe all except Mg & Na

  • There are 4-5 different oxygen requirements for bacteria
    • Obligate aerobe
    • Facultative aerobe
    • Microaerophile
    • Aerotolerant anaerobe
    • Obligate anaerobe
obligate aerobe
Obligate aerobe
  • Require full level of O2 (20-21%) to grow
facultative aerobes
Facultative aerobes
  • Grows best in O2, but can grow without O2
  • Grow at O2 levels <20%, but require less
aerotolerant anaerobes
Aerotolerant anaerobes
  • Don’t require O2, but can grow in presence of O2
obligate anaerobes
Obligate anaerobes
  • No O2 required, O2 is toxic

Obligate aerobic

  • Obligate anaerobic
  • Facultative aerobe
  • Microaerophile
  • Aerotolerant
what does all this mean
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!!
methods to control growth
Methods to Control Growth
  • Heat Sterilization
  • Radiation
  • Filtration
  • Antimicrobial Agents
heat sterilization
Heat Sterilization

Sterilization – destruction of all viable life

  • Incineration (dry heat) – glassware, metal objects
    • 160ْ – 550ْC
    • Denatures proteins and makes organic molecules unstable
    • Takes seconds to hours
  • Pasteurization (low heat over time) – milk, fluids
    • 63ْ – 72ْC
    • Kills up to 99% organisms in milk
    • 15 seconds – 30 minutes
  • Autoclave (moist heat) – glassware, metal objects, liquids (sm. Vol.), plastics
    • 121ْ & 15 psi; pressure is used to increase temp.
    • Minutes to hours
  • 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
  • Size of pores or matrix of fibers capture cells while air or fluid passes through
antimicrobial agents
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
  • 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
protein synthesis
Protein Synthesis
  • DNA Replication
    • DNA passed from parent to progeny
    • Binary fission
    • >2 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)

In prokaryotes, there is one circular chromosome

  • Bases range from 500,000 to 10,000,000

Yeast Chromosome

fidelity of replication
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

5,000,000 /1,000,000,000


dna replication
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

ALWAYS proceeds from the 5’ to 3’ direction

    • Each new nucleotide as added to 3’ – OH group

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

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)
reading frame
Reading Frame


2 types of mutations
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
altering microbial genomes
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

Genetic recombination

  • Genetic elements contained in 2 separate entities are added together
  • recA – protein responsible for swapping DNA sequences
  • 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
  • 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)
  • Cell to cell contact involving either the transfer of a plasmid or chromosome (more rare)
  • Plasmid – DNA that isn’t part of the chromosomes and isn’t 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

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 – doesn’t have the plasmid
pcr polymerase chain reaction
(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)
3 steps
3 steps:
  • Denatureation
  • Annealing
  • Elongation & Extension
  • 94 degrees C
  • DNA “melting” from double stranded to single-stranded molecules
  • Takes the place of helicase and binding proteins
  • 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
elongation extension
Elongation & Extension
  • Polymerase finds the primers and adds nucleotides to the 3’ OH groups, complementing the template
  • Polymerase from Thermusaquaticusis used
    • Taq – adapted for high temperature function

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/


  • http://www.sumanasinc.com/webcontent/animations/content/pcr.html
applications of pcr
Applications of PCR
  • Identify unknown microbial species
  • Crime investigations
  • Genome sequencing
  • Gene screening, medicine development, gene therapy
molecular chronometers
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
molecular chronometer
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
molecular chronometer should have these features
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
    • 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.

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!!****

small subunit rrna of the three domains of life
Small subunit rRNA of the three domains of life.


(Mitochondria & Chloroplasts)


Archaea 16SrRNA

Eucarya 18SrRNA

phylogenetic distance matrix tree
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)
prokaryotes and humans
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)***

bacteria on skin
Bacteria on Skin
  • Mostly gram positive bacteria
  • 102 – 106 /cm2; increases when moist
bacteria in mouth
Bacteria in Mouth
  • Think teeth and gums (cavities)
  • W/I saliva 108 cells/mL

Streptococcus mutans

nasal throat
Nasal / Throat
  • Moist, nutrient-rich environments
  • Mucous & phlegm are nutrition sources for microbes

Streptococcus aureus


  • Very acidic environment (pH <2.0)
  • Contains <10 cells/mL of stomach fluid
small intestines
Small Intestines
  • Increase in micrfobial numbers
  • Very rich food supply
  • pH increase from stomach
  • Probiotics in livestock


large intestines
Large Intestines
  • ‘fermentation vat’
  • 1010 – 1011 bacterial cells/gram
  • Aids in digestion
  • Provides vitamin b12, biotin, riboflavin, vitamin k

Escherichia coli

  • Relatively free of bacteria
  • Lower areas have some bacteria & fungi (yeast)
  • Very moist environment
  • Nutrient rich
  • Bacterial and fungal yeast have steady ecosystem that deters invading species (pH around 4)
  • Beneath foreskin creates great microbial habitat

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 – can’t live outside the body
    • Treponemapallidum – syphilis