M9

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.

Protocol 2

1. In a 4 liter Erlenmeyer flask, combine:

• 12.8 grams Na₂HPO4
• 3.0 grams KH₂PO₄
• 0.5 grams NaCl
• 1.0 gram NH4Cl

2. Bring to approximately 950 ml with ddH₂O and pH to 7.4 with NaOH.

3. Autoclave for 20 minutes on slow exhaust. Store at 4oC when finished.

4. Dissolve the following in 5-10 ml of ddH₂O in a test tube and cold filter, using a 10 ml sterile syringe and a 0.22 micron filter, into the autoclaved media:

• 2.0 grams glucose
• 0.4940 grams MgSO₄.7H₂O
• 0.0152 grams CaCl₂.2H₂O
• 0.0100 grams Thiamine
• 0.0100 grams FeSO₄.7H₂O

5. Or, dissolve each in a total volume of approximately 50 ml of ddH2O and cold filter that into the media.

6. After filtering each chemical into the media, rinse the test tube with ddH₂O and filter the ddH₂O into the media also. When finished, rinse the filter really well with ddH₂O and bring to 1 liter.

7. Prior to inoculation, add the appropriate antiobiotic/s.

Protocol 3

1. Make M9 salts
To make 10X M9 Salts aliquot 800ml H2O and add

• 60 g Na₂HPO₄.7H₂O
  
• 30 g KH₂PO₄
  
• 5 g NaCl
  
• 10 g NH₄Cl
  add water to 1000 ml and autoclave
  

Stir until dissolved
Adjust to 1000ml with distilled H2O
Sterilize by autoclaving

2. Make medium

Measure ~700ml of distilled H2O (sterile)

• Add 100ml of 10X M9 salts
• Add 2ml of 1M MgSO₄ (sterile)
• Add 20 ml of 20% glucose (or other carbon source)
• Add 100 ul of 1M CaCl₂ (sterile)

Adjust to 1000 ml with distilled H2O