module 3 and 4

• Nutrients required for microorganism growth

  • Carbon
  • Nitrogen
  • Phosphorous
  • Vitamins
  • Trace elements

  
• Cell composition


  • 70-80% cell wet weight
  • 55% cell dry weight
  • 3.1% cell dry weight
  • 20.5% cell dry weight

  
• Carbon sources for heterotrophic microorganisms

  • Necessary for synthesis of new cell materials
  • Glucose, lactose
• Carbon sources for autotrophic microorganisms

  CO2 is reduced to glucose
• Nitrogen sources


  • Makes up 13% of the cell- proteins, nucleic acids, other cellular materials
  • Necessary for protein synthesis and amino acid production
  • Fastidious organisms usually require the 20 amino acids in the medium
  • Some organisms may act as nutrient parasites
  • May be obtained from tryptone, peptone, etc.
• Other nutrients required


  • Phosphorous
  • Sulfur
  • Potassium
  • Magnesium
  • Calcium
  • Sodium
• Cell Division


  • Binary fission
  • Cell duplication
  • Unilateral growth

  
• Phosphorous


  ATP synthesis, phospholipids, and nucleic acids
• Sulfur


  Protein synthesis, other cell components
• Cell Division


  • Budding
  • Daughter cells develop from a mother cell
  • Unequal cell growth
• Biofilms are a production of polysaccharide matrix and a community of microorganisms
• Magnesium


  Ribosomes, nucleic acids, membrane stability
• Lag Phase


  • Adapting to new environment
  • Metabolically active but no replication
  • Duration depends on nutrients available and species
• Logarithmic/Exponential Phase


  • Cell divides at a rapid rate
  • Increased cell population
  • May be maintained via chemostat
  • Generation time of organism may be determined
• Calcium


  Cell wall stability, heat stability for endospores
• Chemostat


  Factors that govern cell density: Dilution rate, Concentration of limiting nutrient
• Sodium


  Depends on the nutrient requirement of the organism
• Stationary Phase


  • Equal rate of cell replication & cell death
  • Limited nutrients
  • Increasingly toxic environment
  • Highly acidic pH
  • Limited oxygen availability
• Death Phase


  • Depleted nutrients
  • Overwhelming amount of toxic waste
  • Cells may undergo involution
  • Endospores may form
• Trace metals and growth factors

  • Act as cofactors in enzymatic reactions
  • Trace metals (Fe, Cu, Co, Zn, Mg)
  • Facilitate redox reactions, cellular respiration, etc
  • Growth factors (Folic acid, vitamin B12, vitamin K)
  • Some pathogens obtain vitamins from host
• Serial Dilutions


  1. Done to reduce the bacterial load

  2. Series of tubes containing a diluent (NSS, PBS)

  3. Followed by plate counting

  4. Colony growth on plate reflects numbers of cells in the sample

  5. Consider colony counts of 30-300

  
• Direct Cell Counts


  • Sample placed into a Petroff-Hauser counting chamber (haemocytometer)
  • Counts the number of cells in the grids
  • Compute for the cell concentration per sample
• Liquid and solid media

  • Bacterial colonies are indicative of purity
  • More than 1 colony type = contaminated

  
• Types of culture media

  • Defined
  • Complex
• The great plate count anomaly is that direct cell counts reveal higher numbers compared to viable plate counts because direct count considers both live and dead cells and not all cells are culturable
• Defined media


  Contains precise amounts of highly purified organic/inorganic components
• Complex media


  • Contains dehydrated digests of animal, plant, or microbial products
  • Contains imprecise nutritional composition

  
• Most Probable Number Technique

  Basis for positive results: Gas production, Turbidity
• Types of complex media

  • Enriched medium
  • Selective medium
  • Differential medium
• Spectrophotometry


  • Optical density (OD) measures the amount of light unscattered (absorbance)
  • The more light absorbed, the higher the OD
  • Measures the amount of bacterial cells present in the medium
• Enriched medium


  Complex base with additional nutrients (eg. blood, serum, etc)

  
• pH


  • Neutrophiles: Growth at pH 5.5-7.9
  • Acidophiles: Growth at pH <5.5
  • Alkaliphiles: Growth at pH >8
• Selective medium


  Contains compounds that inhibits the growth of one bacterial group but supports the growth of another
• Temperature


  • Psychrophiles: Cytoplasmic membrane with unsaturated, short chain FA, Lipids with polyunsaturated FA, Cold-shock proteins, Cryoprotectants
  • Thermophiles: Cytoplasmic membrane with saturated fatty acids, Heat-stable proteins, Protein structures
• Temperature Classes


  • Psychrophiles
  • Mesophiles
  • Thermophiles
  • Hyperthermophiles
• Differential medium


  • Contains an indicator that determines if a biochemical reaction occurred
  • Useful for differentiating among organisms
• Oxygen Classes


  • Aerobic
  • Anaerobic
  • Microaerophilic
  • Facultative anaerobic
• Selective media


  • MacConkey Agar (MAC)
  • Eosin Methylene Blue (EMB)
  • Mannitol Salt Agar (MSA)
• Reactive Oxygen Species (ROS)


  Triplet and singlet oxygen, Superoxide anion, Hydrogen peroxide
• Differential media


  • MacConkey Agar (MAC)
  • Eosin Methylene Blue (EMB)
  • Mannitol Salt Agar (MSA)