Minimal Media contains the essentials for bacterial species to grow. The
media is often used to define if a particular microbial species is a
heterotroph, namely an organism that does not have any nutritional
requirements beyond core sources of carbon (sugars) and nitrogen (to
synthesize amino acids and nucleic acid bases). Auxotrophs, or organisms
with nutritional requirements will not be able to grow on minimal media.
Important components are:
- A source of carbon, commonly the sugar glucose (C6H12O6).
- A source of nitrogen, needed to synthesize amino acids for proteins and bases for nucleic acids. Ammonium chloride is commonly used (NH4Cl), particularly given that the majority of bacterial species cannot fix nitrogen (convert atmospheric N2 into biologically usable ammonia or amine groups).
- Ions/electrolytes - like us, bacterial cells need core nutrients such as sodium, chloride, potassium, calcium, phosphorus and magnesium. These components are provided in the "M9 salts" solution
Reagents/Materials:
- Erlenmeyer flask of volume at 2X for amount of media to be made (i.e. 1L flask to make 500mL)
- M9 minimal salts solution (5X concentrate)
- To 800mL of distilled/deionized water add:
- 64g sodium phosphate, penta-hydrate -- Na2HPO4-7H2O
- 15g potassium phosphate (dibasic) -- KH2PO4
- 2.5g table salt -- NaCl
- 5.0g ammonium chloride -- NH4Cl
- Stir until dissolved.
- Adjust volume to 1000mL (1L).
- Sterilze by autoclaving or filter sterilization if autoclave is not available.
- Distilled water
- 1M solution of magnesium sulfate (MgSO4).
- 20% solution wt/wt of glucose (20g gulcose in 80g of water). Add powdered glucose (also called "dextrose") to water solution slowly while mixing, else the glucose will clump and not easily dissolve. Filter sterilize when finished - do not autoclave as the heat will carmelize the sugar.
- 1M solution of calcium chloride (CaCl2)
- To make solid media, include agar base and sterile Petrie plates.
Method
- To prepare 1L of media, add:
- 200mL 5X M9 salts solution
- to ~800mL of distilled water
- Add 15g of agar media if agar plates are to be poured.
After autoclaving, swirl to mix evenly and "temper" at room temperature until you can place your hand on the flask for 2 second (an adult should do this!), then add the following:
- 2mL of 1M MgSO4 solution
- 0.1mL of 1M CaCl2 solution
- 20mL of 20% glucose
Swirl to mix evenly.
Liquid media should be distributed to sterile bottles or tubes with a pipette and pipettor. Small bottles can hold 250mL of media for use when needed, for instance, or aliquots of 5mL or 10mL can be transferred to sterile, capped test tubes.
If plates are to be poured, ~25mL of media should be added per Petrie dish and allowed to solidify at room temperature. Poured/solid plates can be stacked and replaced in the original plastic sleeve in which they came. Label the sleeve with the type of media and date poured.