Griffitts:Common media

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Bacterial Media and Additives

LB broth (1 L)

• 1 L dH₂0
• 10 g Bacto tryptone
• 5 g Bacto yeast extract
• 5 g NaCl
• 1 mL 2N NaOH
• 12 g agar (optional)
  

Autoclave

LB-Top broth (500 mL)

• 500 mL dH₂0
• 5 g Bacto tryptone
• 2.5 g Bacto yeast extract
• 2.5 g NaCl
• 0.5 mL 2N NaOH
• 2 g agar (optional)
  

Autoclave Add 2.5 mM CaCl₂ before phage infections

TY broth (1 L)

• 1 L dH₂0
• 6 g Bacto tryptone
• 3 g Bacto yeast extract
• 0.5 g CaCl₂·2H₂0
• 12 g Bacto agar (optional)
  

Autoclave Note: for motility assays, mage agar 0.24%

Standard Minimal Medium (SMM)(250 mL)

To 250 mL sterile dH₂0 or water agar add:

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

Autoclave

100X SMAJ (50 mL)

• ~30 mL ddH₂0
• 8 g KH₂PO₄
• 12 g K₂HPO₄
• 2 g NaCl
• 4 g NH₄Cl
  

(takes a while to dissolve)
Filter sterilize

100X MCBT (50 mL)

• 50 mL ddH₂0
• 2.46 g MgSO₄·7H₂0
• 1 mg CoCl₂
• 1 mg biotin
• 3 mg thiamine
  

Filter sterilize Note: Biotin takes a while to dissolve

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

• 15 g carbon source (e.g. sucrose, mannitol, glucose, lactose, sodium succinate).
• QS to volume with ddH₂0

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

Plant Media and Additives

Standard Nodulation Medium (SNM) (1 L)

• 1 L dH₂0
• 0.15 g CaCl₂·2H₂0
• 0.25 g MgS0₄·7H₂0
• 0.35 g KH₂PO₄
• 2 mL 500X minor salts (see below)
• pH to 7.0 (~850 μL 2N KOH
• 13 g plant agar
  

Microwave to dissolve agar
Distribute 8.2 mL per 18×150 tube
Cap tubes
Autoclave
Slant the racks and allow to cool

500X minor salts (200 mL)

• 1.9 g Na₂EDTA·2H₂0
• 1.4 g FeSO₄·7H₂0
• 300 mg ZnSO₄·7H₂0
• 300 mg H₃BO₃
• 300 mg MnSO₄·H₂0
• 30 mg Na₂MoO₄·2H₂0
• 3 mg CuSO₄
• 3 mg CoCl₂

QS to volume with ddH₂0
Filter sterilize