Isolation, Cultivation, and Nutrition of Bacteria
Last revised: Friday, January 31, 2003
Reading: Ch. 5 in text

Isolation, Cultivation, and Nutrition of Bacteria

Nutritional categories

1. Energy source
   1. if organic molecules (e.g. sugars, amino acids) = ORGANOTROPH
   2. if inorganic molecules (e.g. H₂S, NH₃) = LITHOTROPH
   3. if light = PHOTOTROPH
2. Macronutrients: Cell requires ~ 30 elements. Needs 6 in large amounts (CHNOPS), some only in trace amts, some in between
   1. C-source
      1. can be CO₂ = AUTOTROPH
      2. can be organic molecules = HETEROTROPH
      • Note: many bacteria can form all organic molecules from any of a wide variety of C-sources. E.g., Pseudomonas strains (soil organisms) can grow on hydrocarbons, variety of sugars, fats, polysaccharides, etc.
      • Full description of microbe typically refers to both E- and C-source; lithotrophic autotroph (chemolithotroph); organotrophic heterotroph, etc. Will see many examples of these.
   2. N-source
      • Typical bacterium is 15% nitrogen (in proteins, nucleic acids, cell wall).
      • Most bacteria obtain either amino groups (-NH₂) or ammonia (NH₃), or nitrate (NO₃^--).
      • Some bacteria can fix atmospheric nitrogen (N₂) to form ammonia (NH₃)
   3. P-source
      • generally available as phosphate ion (PO₄^---) or organic molecules (e.g. RNA).
      • Many bacteria secrete phosphatase enzymes to remove phosphate from organic molecules, obtain it for their own growth
      • Often limiting for growth.
   4. S-source
      • required for 2 amino acids and for some vitamins.
      • Generarally available as sulfate (SO₄^--) or sulfide (S^--).
3. Micronutrients
   • K, Mg, Ca, Fe required in small but significant amts. Act as cofactors for many enzymes, cell structures (e.g., Mg⁺⁺ required by ribosomes for protein synthesis).
   • Na⁺ is not required by many microbes (unlike animals, where nerve system requires Na⁺ and K⁺). Marine microbes require sodium.
   • Trace elements: Co, Zn, Mo, Cu, Mn, Ni, W, Se -- usually required by a small number of enzymes.

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Membrane transport

• Membranes are permeability barriers; they maintain "inside" and "outside" of cell.
• Active cells must continually move food, nutrients in, wastes out. Requires specific transport carriers.
• Types of transport proteins
• Passive Diffusion. Lipophilic solutes cross the membrane freely by dissolving in the lipid bilayer. This is passive diffusion. Examples: ethanol, glycerol, fatty acids.
• Facilitiated Diffusion. Protein carrier is requred, and will speed up movement of substrate across membrane. No energy required, no preferential direction. If molecule is more concentrated outside than inside cell, net movement will be out of cell. Not common in bacteria, because these would allow needed molecules to escape from cell in many environments.
• Active Transport -- substance is imported without chemical change, and energy is expended. Cells can maintain concentration differential to prevent substances from moving out of cell.
  • Energy may be supplied in form of ATP
  • More commonly, cells use energy in form of proton gradient or other ion gradient (e.g., sodium gradient). After proton gradient is established (to be discussed later in section on energetics), symport proteins allow substate to brought in along with entry of proton.
  • Symporter -- protein carries two substances across membrane in same direction (e.g. proton flux is coupled with motion of substrate). Active transport -- requires energy from preexisting concentration gradient.
  • Antiporter -- protein carries one substance in one direction, second substance in opposite direction. Active transport -- requires energy from preexisting concentration gradient.
• Group translocation -- substance is chemically altered while being transported.
  • Example: phosphotransferase system used in sugar transport. Sugars are common energy sources, bacteria have evolved complex transport system to bring them across membrane whenever possible.
  • In E. coli, 24 proteins involved, can transport a variety of sugars (at least 4 proteins needed for any given sugar).
  • View diagram of PTS system
  • Mechanism involves alternate phosphorylation (adding P_i group) and dephosphorylation (removing P_i group) of series of carriers. Initial phosphate donor is high-energy molecule PEP, passes P_i group to Enzme I HPr protein Enzyme II_a Enzyme II_b Enzyme II_c. Only the very last of these proteins is located in membrane; others are all found in cytoplasm. Sugar (e.g., glucose) is carried into cell by Enzyme II_c, and at same time phosphate group is added to make glucose 6-P, hence group translocation (sugar is modified by added P_i group)
  • View animation of group translocation transport mechanism

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Culture Media

• Media must include source of C, N, P, S, 4 of the 6 major nutrients (CHNOPS), as well as micronutrients. These are usually present as trace contaminants in water, on glassware, or in chemicals used to make media.
• Media can be liquid or solid. Use for different purposes:
  1. Liquid media: easiest to prepare and use. Good for growing quantities of microbes needed for analysis or experiments. Unless inoculated with pure culture, cannot separate different organisms.
  2. Solid media: usually made by adding agar, a seaweed extract, to appropriate liquid. 1.5% agar is standard for plates. Agar melts at 80-90 deg. C, will remain liquid until temperature cools to 40-42 deg. C. Very few microbes can degrade agar, so it is normally not a source of C, and acts as inert gelling medium.
• Many types of culture media; see BBL manual and DIFCO manual for formlations
  1. Synthetic of Defined Media: usually relatively simple media, all components are known. Useful for photoautotrophs, also in some experimental situations where want to select mutants unable to use certain compounds, or for radioisotope labeling. Example: you want to select a microbe that can obtain all its nitrogen from atmospheric N₂. You would prepare synthetic medium with sources of C, P, and S, but no N source. Organisms would be unable to grow unless they can fix nitrogen from air.
  2. Complex Media: composition of media not completely known. Often made from inexpensive organic materials such as slaughterhouse wastes (tryptic digests called tryptone, trypticase, etc.), soybeans, yeast wastes from brewing (rich source of vitamins), animal blood, etc. All our standard laboratory media in MCB 229 are complex media, such as Tryptone agar, TSA (trypticase soy agar), Nutrient agar, etc.
  3. Selective Media: media favors the growth of one or more microbes. Example: bile salts inhibit growth of most gram-positive bacteria and some gram-negative bacteria, but enteric bacteria adapted to life in animal gut can grow well. Include bile salts in some media such as EMB, MacConkey agar (will use later in this course) to select for enterics.
  4. Differential Media: media allows distinguishing between different bacteria that grow. Ex: MacConkey agar has color indicator that distinguishes presence of acid. Bacteria that ferment a particular sugar (e.g., glucose in culture media) will produce acid wastes on plates, turn pH indicator red. Bacteria that cannot ferment the same sugar will grow but not affect pH, so colonies remain white.
• Note that it is possible to design a medium that is both selective and differential.

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