Some short notes about physiology of media making:
Rich Media (L broth, MHB, BH, Nutrient Broth): For running physiological experiments, note that these lack buffering capacity. They also have lower levels of certain cations such as Mg or Fe.
Minimal media: Make sure all essential components are present. Read below.
Following should be considered when making media.
- Nitrogen source: Use Ammonia and/or a defined amino acid mix. If the nitrogen source need not to be defined, add casamino acids, tryptone, BSA…etc.
- Carbon source: Not necessary if you are adding a metabolizable amino acids or other nitrogen source. Note that some amino acids cannot be used as the sole carbon source for some bacteria (glutamate for E. coli K12). Carbon source is necessary if only NH4 is present as the N source. Succinate or other TCA cycle intermediates are preferred for Pseudomonads and EM or ED pathway intermediates are preferred for enterobacteria (preferably Glc or Gly).
- Essential cations: K+, Mg++ and Fe++ (Na+ is needed by certain bacteria). Other cations are trace elements and no need to add. They are invariably present in rich broth as well as chelates in phosphates.
- Essential anions: (i) Sulfur: best given as fresh Cys but commonly given as SO4 ion. (10-20 mM NH4SO4 is the common chemical to use if you need NH3 as the nitrogen source. Otherwise, use 10-20mM Na2SO4 ). (ii) PO4: normally supplied as either Na or K salts. Again, 10-20mM is sufficient unless PO4 is used in a dual role as a buffer. Then, use 75-150mM PO4, provided as K or Na or mixture of both ionic solutions.
- Vitamins: wildtype bacteria can make all their vitamins, unless you are using a minimal media with a auxotroph (eg: DH5alpha and other cloning or conjugation helper E. coli need Thiamine). Vitamins are used as trace elements and does not effect the growth rate significantly if you are using rich media.
- Nucleic acid bases: Bacteria will make them in minimal media, and they will salvage it (from Yeast extract or from BHI) in rich media. If needed, best to give as nucleoSIDES (Adenosine or Guanosine, cytidine). Nucleosides are easier to dissolve up to 10mM solutions. Add KOH pellets if you need at higher concentrations but make sure to increase the ionic strength of the buffer as well.
- Trace elements: Mn, Mo, Bo, Cu and other elements are usually present as chelates in the KPO4 salts. Calcium can be added to minimal media <250uM if needed.
- Fe: Best given as the ferrous (Fe++) salt as it is immediately available and natural chelators are not required. In fact, Ferrous ion will suppresses the secretion of chelators such as pyoverdine. Fe++ will suppress Fur. But Ferrous is unstable (oxidizes into the ferric form) so using FeSO4 is quite useless. Therefore, use Ferrous Ammonium Sulfate (FeNH4SO4) at 10-100micromolar, and it is relatively stable. Make this solution at 100mM and filter sterile. Can be kept at -80C but not at -20C. Ferric ion (fe+++) can be given but bacteria will need chelators such as pyoverdine to scavenge this ion. Usually supplied as Ferric chloride (FeCl3) at 10-100uM.
- Buffers to use: Potassium or Sodium Phosphate buffers (50-150mM Phosphate ion) are popular because they supply essential ions as well. Other buffers are MOPS or HEPES for pH 7 and MES for low pH work (at 50mM) and Bis-Tris Propain for high pH. Never use TRIS buffers for biological work.
- Making media for live fluorescent microscopy. Avoid any protein digests or yeast extract. Use BSA and ammonia for the nitrogen source. Don’t use vitamins.
A major factor that will increase the growth rate is the quality of Nitrogen source and other macromolecules such as lipids and nucleotides. More protenacious the source, better the growth, specially if rich in Gln and Glu. Eg: Brain heart infusion support better growth than tryptone/yeast based LB broth. Don’t use a catabolic repressing sugar with another sugar (such as Glc and Gly combination). Adding extra yeast extract will also increase the growth rate.
Acetate toxicity: Too much glucose will cause acetate toxicity in E. coli.
Trace element mix and the vitamine mix became popular after Neidhardt, published his paper about bacterial growth media.
Neidhardt, F. C., J. Ingraham, and M. Schaechter. 1990. Physiology of the Bacterial Cell: A Molecular Approach. Sinauer Associates, Sunderland, MA. 520 pagess.
Neidhardt, F. C. (Ed. in Chief), R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (eds). 1996.Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology. 2 vols. 2898 pages.