Minimal Medium

Recipe I

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.

Recipe 2

1x M9 salts
2mM MgSO4
0.1mM CaCl2
0.4% carbon source (e.g. glycerol, glucose, etc.)
In sterile H2O

For 1L of media

500ml 2xM9 salts ∗
20ml 0.1M MgSO4 ∗
200μl 0.5M CaCl2 ∗
469.8ml sterile deionized H2O ∗ (if carbon source to be added)
479.8ml sterile deionized H2O ∗ (if no carbon source to be added, useful as a control for sugar metabolism)

Combine above solutions using sterile technique. (May notice some precipitation during preparation but precipitate should go back into solution once volume is brought up to 1l with sterile H2O.)
Add antibiotic as appropriate and store at 4°C

5X M9 salts contains:

64 g Na2HPO4.7H2O
15 g KH2PO4
2.5 g NaCl
5 g NH4Cl

dissolved in deionized water to a final volume of 1 L

M9-glu contains glucose: In the basic recipe take 800 ml sterile water and supplement with 100 ml 20% (w:v) sterile filtered glucose before adding M9 salts, magnesium and calcium salts. [This and following instructions refer to the basic M9 medium recipe.]

M9-CA contains casamino acids: In the basic recipe take 800 ml sterile water and supplement with 100 ml 10% (w:v) autoclaved casamino acids (in water) before adding M9 salts, magnesium and calcium salts.

M9-CAglu contains both: In the basic recipe take 700 ml sterile water and supplement with 100 ml 20% glucose and 100 ml casamino acids before adding M9 salts, magnesium and calcium salts.

Recipe 3 for 15N labeling

Expression Protocol in M9 Minimal Media via T7 Promoter

The following protocol has been used successfully to 15N or 13C/15N label proteins using  pET/BL21(DE3) expression system: Preparing M9 minimal media begins with preparing a 5x stock solution of M9 salts. Generally, M9 salts contain a nitrogen source in the form of NH4Cl. If a labeled nitrogen source,  5x salts should be prepared without NH4Cl. Standard 5 X M9 Minimal Media salts minus nitrogen source For 1L 5xM9 salts:

64g Na2HPO4-7H2O
15g KH2PO4
2.5g NaCl
H2O to final 1L volume and autoclave

To prepare 500mL M9 minimal media;

100mL of 5xM9 salts
1 mL 1M MgSO4
50 uL 1M CaCl2
5 mL 100x Basal Medium Eagle Vitamin Solution (Gibco)
2.5 mL filter sterilized NH4Cl (0.2 g/mL) or 0.5g dry
10 mL 20% d-glucose or 2g dry
glass distilled & autoclaved H2O to final volume of 500mL

pH solution to 7.3 and filter sterilize (0.2u filter).

Introduce media to a pre-autoclaved, wide-bottom (baffled) 2L flask and add ampicillin to a final concentration of 70-100 ug/ml.

Grow 5mL overnight culture in same media to inoculate 500mL M9.

Shake culture at 37oC until an OD600 of 07+/-0.2 then induce protein expression with the addition of IPTG (0.01-0.1 mM final concentration)

Recipe 4

Minimal growth medium 200.0 ml
5 × M9 Salts 20.0 ml
D-glucose Stock (20 g/100 ml) (0.2_m filter sterilized)a 10.0 ml
Basal Vitamins Eagle Mediab 2.0 ml
1 M MgSO4 (autoclaved) 0.1 ml
1 M CaCl2 (autoclaved)
5 × M9 Salts (autoclaved) 15.0 g
KH2PO4 64.0 g
Na2HPO4 •7H2O 2.5 g
NaCl 5.0 g
NH4Cl*  (pH adjusted to 7.2 with NaOH)

• For isotopic labeling 15 NH4Cl and 13C D-glucose were substituted for unlabeled NH4Cl and D-glucose respectively. For incorporation of deuterium, 75% 2 H2O was used in place of deionized water for preparing the solution.
  
  

Basal Vitamins Eagle Media can be purchased from Life Sciences Technology.
The above recipe is a modification of M9 minimal media (Sambrook et al., 1989).

Recipe 5

Optimized high-cell-density IPTG-induction minimal medium

50 mM Na2HPO4.7H2O
25 mM KH2PO4 (pH 8.0–8.2)
10 mM NaCl
5 mM MgSO4
0.2 mM CaCl2
0.25 x Metals
0.25 x Vitamins
0.1% NH4Cl or 15NH4Cl
1.0% Glucose
or 13C-Glucose

Optimized autoinduction minimal medium
50 mM Na2HPO4
50 mM KH2PO4
(pH 8.0–8.2)
50 mM 15NH4Cl
5 mM Na2SO4
2 mM MgSO4
0.5–22 x metals
(Based on Studier’s recipe)
0.4% 13C-glycerol
0.05% 13C-glucose
0.01% ß-lactose
12x Vitamins
(100 x BME vitamins
stock solution, SIGMA, MO)

Recipe 6

Add the following amounts per liter of medium:
Na2HPO4 6 g (12.8 g if the 7H2O hydrate form is used)
KH2PO4 3g
NaCl 0.5 g
NH4Cl 1.0 g
Adjust pH to 7.0.
Autoclave at 121 °C, 15-20 minutes.
Let medium cool, then add:
1 mL 0.1 M CaCl2
1 mL 1 M MgSO4 · 7 H2O
10 mL 40% glucose (or 20 mL 20% glucose)
+ vitamin solution (see below)
+ antibiotics

0.1 M CaCl2
(MW=111 g/mol, 0.111 g CaCl2 in 10 mL MQH2O, filter sterilize)

1 M MgSO4 · 7 H2O
(MW=246 g/mol, 2.46 g MgSO4 · 7 H2O in 10 mL MQH2O, filter sterilize)

40% Glucose (or 20% glucose – can be autoclaved)

Vitamins:

Option 1 (CVS vitamins)
Vitamin prep: smash 1 vitamin and mix with 20 ml of H2O. Mix, bath sonicate, and remove insoluble junk by low speed centrifugation. Filter with steriflip. Store at -20 °C. Use 2 mL per L minimal media.

Option 2.(MEM vitamins)
Use commercially available MEM vitamin solution, 10 mL to 1L minimal medium.