Dr Syahira part

• Studying the nutrition of microorganisms

  • Crucial for biotechnological applications, health and disease, food safety and preservation, environmental applications
• Microbial nutrition


  Supplying the cells with the chemical tools they need to make monomers
• Cells consist of macromolecules and water (70–80% of the wet weight)
• Besides water, cells consist primarily of macromolecules - proteins, nucleic acids, lipids, and polysaccharides
• Monomers


  The building blocks of macromolecules - amino acids, nucleotides, fatty acids, and sugars
• Macronutrients


  Essential for providing energy, required in large amounts
• Micronutrients


  Required in lesser or just trace amounts, including trace elements and growth factors
• Different organisms need different complements of nutrients, required in different amounts
• Macronutrients - Carbon


  • All cells require carbon, most prokaryotes require organic compounds, about 50% of the dry weight of a bacterial cell, autotrophic microorganisms build their cellular structures from carbon dioxide (CO2)
• Macronutrients - Nitrogen


  • A bacterial cell is about 13% nitrogen, present in proteins, nucleic acids, and several other cell constituents, bulk of nitrogen available in nature is ammonia (NH3), nitrate (NO3-), or nitrogen gas (N2), all prokaryotes can use NH3 as their nitrogen source, many can also use NO3-, and some can use organic nitrogen sources, such as amino acids, N2 can only be used as an N source by nitrogen-fixing prokaryotes
• Other macronutrients


  • Hydrogen and Oxygen, Phosphorus, Sulfur, Potassium, Magnesium, Calcium, Sodium
• Defined media


  Prepared by adding precise amounts of pure inorganic or organic chemicals to distilled water, major importance is the carbon source
• Complex media


  Made from digests of microbial, animal, or plant products, such as casein, beef, soybeans, yeast cells, disadvantage is that the nutritional composition is not known precisely
• Classes of culture media

  • Defined media
  • Complex media
  • Enriched medium
  • Selective medium
  • Differential medium
• Complex medium is easiest to prepare and supports growth of both Escherichia coli and Leuconostoc mesenteroides
• The simple defined medium supports growth of E. coli but not of L. mesenteroides, growth of L. mesenteroides in a defined medium requires the addition of several nutrients not needed by E. coli
• The fourth medium supports growth of the sulfur bacterium Thiobacillus thioparus, this medium would not support growth of any of the other organisms because T. thioparus is both a chemolithotroph and an autotroph and thus has no organic carbon requirements
• Different microorganisms can have vastly different nutritional requirements, for successful cultivation, it is necessary to understand an organism's nutritional requirements and then supply it with the nutrients it needs in both the proper form and amount
• Viable count


  Measures the cells in the culture that are capable of reproducing
• Optical density (turbidity)


  A quantitative measure of light scattering by a liquid culture, increases with the increase in cell number
• Growth cycle of cells

  1. Lag phase

  2. Exponential phase

  3. Stationary phase

  4. Death phase
• Lag phase


  • Growth begins only after a period of time
  • Depends on history of the inoculum and nature of the medium and growth conditions
  • No lag and exponential growth begins immediately if an exponentially growing culture is transferred into the same medium under the same conditions
• Exponential phase


  • Cell population doubles at regular intervals
  • Cells are typically in their healthiest state
  • Rates of exponential growth vary greatly and are influenced by environmental conditions and genetics
• Stationary phase


  • No net increase or decrease in cell number, growth rate is zero
  • Energy metabolism and biosynthetic processes continue at a reduced rate
  • Some cells may divide but no net increase due to others dying (cryptic growth)
• Death phase


  • Occurs as an exponential function
  • Rate of cell death is much slower than the rate of exponential growth
• Batch culture


  Simplest fermenter operation, closed system, all nutrients added before inoculation, nothing added during except O2, antifoam, acid/base
• Advantages of batch culture

  • Very basic
  • Inexpensive
  • Can be used for different reactions
  • Chance of contamination is minimum
• Disadvantages of batch culture

  • Batch variability
  • Downtime and high proportion of unproductive time
  • Gives low product yield
  • Higher costs and not economic
• Continuous culture


  Open system, fresh medium added at constant rate, spent medium removed at same rate, maintains steady state
• Chemostat


  Most common type of continuous culture, allows independent control of growth rate and cell density
• Advantages of continuous culture

  • Productivity and growth rate can be optimised
  • Longer periods of productivity with less downtime
  • Can take advantage of cell immobilization
  • Effects of environmental factors more easily analysed
• Disadvantages of continuous culture

  • FDA does not accept for therapeutic products
  • Not all products produced optimally
  • Contamination can be a major problem
  • Culture mutation can easily occur
• Microscopic counts


  Counting cells on slides or in liquid samples using counting chambers or flow cytometers
• Viable counts


  Counting colonies formed from viable cells on agar plates, using spread-plate or pour-plate methods
• Spectrophotometry


  Measuring turbidity (optical density) of cell suspension, which is proportional to cell mass and number
• Turbidity measurements can be used as a substitute for total or viable counting methods, but a standard curve must be prepared first
• Culture


  A population of bacteria grown in the laboratory in solid (colonies) and liquid culture media (bacterial broth)
• Pure culture


  Contains only one single type of microorganisms
• Mixed culture


  Contains two or more different microorganisms
• How to start your laboratory culture

  1. Define your objectives

  2. Select the microorganism

  3. Choose a growth medium

  4. Prepare culture equipment

  5. Prepare the growth medium

  6. Inoculate the medium

  7. Incubate the culture

  8. Monitor growth and development

  9. Subculture for maintenance

  10. Storage and preservation

  11. Safety precautions