Griffitts:Common media

Bacterial Media and Additives

LB Broth (1 L)

• 1 L dH₂O
• 10 g Bacto tryptone
• 5 g Bacto yeast extract
• 5 g NaCl
• 1 mL 2N NaOH
• 12 g agar (optional)
  
  • 3 g agar + 250 mL broth per bottle

Autoclave
Note: Add 3 mL 1 M CaCl₂ before phage transductions

LB-Top agar (500 mL)

• 500 mL dH₂O
• 5 g Bacto tryptone
• 2.5 g Bacto yeast extract
• 2.5 g NaCl
• 0.5 mL 2N NaOH
• 2 g agar
  
  • Autoclave
    

Note: Add 1.5 mL 1 M CaCl₂ before phage transductions

Swarm agar for E. coli (400 mL)

• 400 mL dH₂O
• 4 g Tryptone
• 2 g NaCl
• 0.88 g Agar (for 0.22%)
  

Autoclave

TY broth (1 L)

• 1 L dH₂O
• 6 g Bacto tryptone
• 3 g Bacto yeast extract
• 0.5 g CaCl₂·2H₂O (this is equal to 3.4 mM)
• 12 g Bacto agar (optional)
  • 3 g agar + 250 mL broth per bottle
    

Autoclave
Note: for motility assays, make agar 0.22%

TY top agar (500 mL)

• 500 mL dH₂O
• 3 g tryptone
• 1.5 g yeast extract
• 0.25 g CaCl₂·2H₂O (=3.4 mM)
• 2 g agar
  

Autoclave
Note: You do not need to add any additional calcium when performing phage transductions

PH medium (1 L)

• 1 L dH₂O
• 4 g peptone
• 0.5 g yeast extract
• 0.5 g tryptone
• 0.5 g CaCl₂·2H₂O
• 0.2 g MgSO₄·7H₂O
• 12 g agar (optional)
  • 3 g agar + 250 mL broth per bottle
    

Autoclave

PSY medium (1 L)

• 1 L dH₂O
• 20 g peptone
• 20 g sucrose
• 5 g yeast extract
• 12 g agar (optional)
  • 3 g agar + 250 mL broth per bottle
    

Autoclave
NOTE: For culturing Bradyrhizobium japonicum, supplement with 0.1% arabinose

YEM Broth (1 L)

• 1 L dH₂O
• 10 g mannitol
• 1 g Bacto yeast extract
• 500 mg K₂HPO₄
• 200 mg MgSO₄·7H₂O
• 100 mg NaCl
• 12 g agar (optional)
  
  • 3 g agar + 250 mL broth per bottle

Autoclave

Water agar (250 mL)

• 250 mL dH₂O
• * 3 g agar
  

Autoclave

Standard Minimal Medium (SMM) (250 mL)

To 250 mL sterile dH₂O or water agar add:

• 2.5 mL 100X SMAJ
• 2.5 mL 100X MCBT
• 2.5 mL 30% carbon source
• antibiotics (optional)
  

Autoclave
Note: Very mucoid growth results from SMAJ at 1/2X—especially in sucrose

100X SMAJ (50 mL)

• ~30 mL ddH₂O
• 8 g KH₂PO₄
• 12 g K₂HPO₄
• 2 g NaCl
• 4 g NH₄Cl
  

(takes a while to dissolve)
QS to volume with ddH₂O
Filter sterilize
Store at room temperature

100X MCBT (50 mL)

• 50 mL ddH₂O
• 2.46 g MgSO₄·7H₂O
• 1 mg CoCl₂
• 1 mg biotin
• 3 mg thiamine
  

Filter sterilize
Note: Biotin takes a while to dissolve
Store at room temperature

100X (30%) carbon source (50 mL)

• 15 g carbon source (e.g. sucrose, mannitol, glucose, lactose, sodium succinate).
• QS to volume with ddH₂O

Filter sterilize
Store at room temperature
Note: mannitol and lactose are soluble at 30% only upon heating

100X (2%) calcofluor (40 mL)

• 0.8 g calcofluor
• QS to volume with ddH₂O

Filter sterilize
Store at room temperature

Plant Media and Additives

Standard Nodulation Medium (SNM) (1 L)

Agar slants

• 1 L dH₂O
• 0.35 g KH₂PO₄
• 2 mL 500X magnesium sulfate
• 2 mL 500X calcium chloride
• 2 mL 500X minor salts
• pH to 7.0 (~850 μL 2N KOH)
• 13 g plant agar
  

Microwave to dissolve agar
Distribute 8.2 mL per 18×150 disposable tube
Cap tubes
Autoclave for 10 minutes
Wet the tubes (this way they'll stick to the rack instead of sliding out) Slant the racks carefully, making sure the agar reaches the bottom of the cap Allow to cool for at least one hour and preferably overnight

Broth

• 1 L dH₂O
• 0.35 g KH₂PO₄
• pH to 7.0 (~850 μL 2N KOH)
  

Autoclave for 15 minutes
Add:

• 1 mL 500X magnesium sulfate
• 1 mL 500X calcium chloride
• 1 mL 500X minor salts

500X magnesium sulfate (50 mL)

• 50 mL ddH₂O
  
• 6.25 g MgSO₄·7H₂O
  

Filter sterilize

500X calcium chloride (50 mL)

• 50 mL ddH₂O
  
• 3.75 g CaCl₂·2H₂O
  

Filter sterilize

500X minor salts (200 mL)

• 1.9 g Na₂EDTA·2H₂O
• 1.4 g FeSO₄·7H₂O
• 300 mg ZnSO₄·7H₂O
• 300 mg H₃BO₃
• 300 mg MnSO₄·H₂O
• 30 mg Na₂MoO₄·2H₂O
• 3 mg CuSO₄
• 3 mg CoCl₂

QS to volume with ddH₂O
Some heating may be required to get it all into solution
Filter sterilize

500X Potassium nitrate (40 mL)

• 40 mL ddH₂O
• 4.04 g potassium nitrate (KNO₃)

Filter sterilize