MICRO 3

• Binary Fission: most common mechanism of cell replication in bacteria. Forms 2 daughter cells.
• Asymmetrical Division: budding (yeast) or production of spores (fungi)
• Generation Time:

  • In Eukaryotes: time between the same time point of a life cycle in 2 successive generations
  • In Prokaryotes: time it takes for the population to double through a single round of binary fission "Doubling Time"
• The number of microbial cells increases exponentially.

   Nn = N0 * 2n
• Bacterial Growth Curve

  A) Lag

  B) Exponential

  C) Stationary

  D) Death

  
• Phases of the Microbial Growth Curve:

  1. Lag: cells adjust to a new environment following inoculation
  2. Exponential: cells are growing as fast as possible. most susceptible to antimicrobials here.
  3. Stationary: waste products accumulate, nutrients run out, there is no net growth of the population.
  4. Death: more cells dying than dividing as toxic waste accumulates.
• Chemostat can be used to continuously add nutrients and remove waste products, in order to sustain microbial growth.
• Direct Cell Count: literally counting cells in a liquid culture or colonies on a plate.
• Direct Microscopic Cell Count:

  • transfer a known volume of a culture on a slide with a small grid, Petroff-Hausser chamber.
  • count several squares and average for reliable numbers
  • downside: tedious and no living/dead differentiation
• Fluorescent Staining and Counting:

  • stains differentiate between living and dead cells
  • downside: more expensive and more steps
• Automated Cell Counter:

  • electronic counter automatically detects changes in resistance between 2 electrodes
  • downside: very dense samples can be underestimated, and no differentiation between living and dead cells
• Viable Plate Count:

  • determine Colony Forming Units
  • must contain 30-300 colonies in order to be countable
  • downside: may have to plate several dilutions to get a countable plate
• Most Probable Number:

  • used when the concentration is too low to reliably detect with previous methods
  • often used with water samples
  • growth in each tube is determined on a binary basis (yes/no)
• Serial Dilution:

  • dilute from original sample 1 part, into 9 parts of saline and mix
  • each dilution is 10% of the previous dilution
• CFU/mL= (number of colonies on the plate) / (pipetted volume in mL)(10^-x)
• Spectrophotometry: gives a measure of the turbidity (cloudiness) of a sample.

  • linearly related to cell count, but does not yield a direct cell count.
  • a higher value of the output = more stuff
  • output of the spectrophotometer = light transmitted - light detected
  • Indirect Cell Count
• Dry Weight Determination: cells in a suspension are centrifuged, washed, then dried, and weighed.

  • Indirect Cell Count
• Biofilm Structure:

  • maintains integrity and function of the biofilm
  • allows for the movement of nutrients, waste, gases, and water
  • protects organisms from desiccation and predation
• Biofilm Formation:

  1. reversible attachment of planktonic cells
  2. first colonizers become irreversibly attached
  3. growth and cell division
  4. production of EPS and formation of water channels
  5. attachment of secondary colonizers and dispersion of microbes to new sites
• Planktonic: free-floating, especially in aquatic environments.

  Sessile: cells attached to a surface.
• Quorum Sensing:

  • mechanism by which cells within a biofilm can coordinate their activities in response to environmental stimuli.
  • cell density is detected and microorganisms release autoinducers, that trigger a cascade of reactions
• 2 Major Signaling Molecules:

  • Gram Positive: small peptides
  • Gram Negative: N-acetylated homoserine lactones
• Biofilms in Human Health:

  • Beneficial: layers of normal microbiota lining the intestinal and respiratory mucosa ward off potential pathogen infections.
  • Harmful: plaque formation on teeth can cause dental and periodontal disease.
• Oxygen Concentrations:

  • Minimum Permissive: lowest oxygen concentration that will allow for the growth of a given organism
  • Optimum: the concentration that is the most ideal for a given organism.
  • Maximum Permissive: the highest tolerated concentration of oxygen.
• Oxygen Use

  A) Obligate Aerobes

  B) Obligate Anaerobes

  C) Facultative Anaerobes

  D) Aerotolerant Anaerobes

  E) Microaerophiles

  
• Anaerobic Environment Examples:

  • deep soil sediments
  • still waters
  • animal intestinal tracts
• Anaerobic Jars: small, sealed containers with chemical packs that remove oxygen and release CO2.

  Anaerobic Chambers: larger containers, devoid of O2 that will have gloves attached so that researchers can handle samples.
• Effects of Cold on Bacteria:

  • slow the reaction rate of chemical processes like metabolism
  • no growth while metabolism is stopped
  • generally does not lead to cell death
• denaturation from high temperatures can be temporary or permanent -> cell death
• Minimum Growth Temperature: lowest temperature that will allow some measurable growth, enzymes inactive. Cells may product antifreeze to prevent cell damage and death.
• Optimum Growth Temperature: the temperature at which optimum growth occurs. not all enzymes work at the max rate, but the temp is suitable fo metabolism as a whole.
• Maximum Growth Temperature: highest temperature at which the microbial population will continue to grow. Above this temp, enzymes are permanently denatured and cells die.
• Thermophiles

  A) Psychrophiles

  B) Mesophiles

  C) Thermophiles

  D) Hyperthermophiles

  
• pH Ranges

  A) Acidophile

  B) Neutrophile

  C) Alkaliphile

  
• Growth Vs. Temperature Curve is asymmetrical.
• Listeria monocytogenes:

  • common foodborne pathogen that can grow at refrigeration temps. 4 °C
  • mostly a threat to immune compromised and pregnant people
• Thermophilic Organisms:

  • proteins contain additional secondary structures
  • increased ionic and covalent bonds to resist denaturation
  • DNA has a higher proportion of GC bonds
  • increased stability in high temps
• Psychrophilic Organisms:

  • proteins are rich in hydrophobic residues and have increased flexibility
  • antifreeze proteins and solutes that decrease freezing temp of cytoplasm
  • unsaturated lipids in the cell membrane to increase fluidity
  • increased fluidity in low temps
• Acidophiles: microorganisms whose optimum pH is below 5.5

  • high concentration of H+required for the membrane to be stable
  • lactic acid producing
  • used in food production to prevent spoilage
  • found in stomach acid and vagina in humans
• Neutrophiles: microorganisms whose optimum pH is between 5.5-7.9

  • can live and grow in/on the human body
  • many human pathogens and spoilage bacteria
  • intestinal microflora