LB + Mg + Glucose for better Gram-N growth (agar + antibiotics possible) 1L

Weigh out ingredients and add to clean bottle

• 10g Tryptone

• 5g Yeast Extract

• 5g NaCl (this is for Lennox formulation, 10g can be used for Miller Formulation)

• 2.46g MgSO4 * 7H20 (10mM)

• 10g D-glucose (1%)

• 15g Agar if making plates (1.5%)

• Add stir bar to bottle and 1000mL of ddH20

• Stir until any chunks are dissolved

• Autoclave at temp (121C) for 40 minutes

  • If adjusting recipe to 500mL or below 30min autoclave is enough

Let cool (putting on a stir plate speeds this up) before pouring plates using aseptic technique

- Can be poured in a laminar flow hood or biosafety cabinet

- Heating the lip of the bottle well before pouring will help in preventing bubbles (they get popped as they pass over the hot lip)

- Shaking the bottle will cause bubbles, try and not do this, use the stir plate

- The thickness of the plates will affect how long they can be used for growth until they begin to dry, the thicker the longer they will last but will use more material

• If adding antibiotics, add them just prior to pouring not to the hot media out of the autoclave

  eg: if wanting 25ug/mL chloramphenicol in final media, add 1mL of 25mg/mL stock solution

Plates should be left out overnight and checked for growth the next day to be sure they are sterile

- Labelling plates with media type before putting themaway will make life easier in the future!

Lightly spraying and wiping down outsdie of plates with 70% ethanol will help keep them sterile while being stored

Put plates away in the fridge (4C)

- They will keep well for a month, and ive successfully used plates much older than that but its best to only make as much as you will use in a month

- If antibiotics were added the 1 month limit is more strict as the antibiotic will loose efficacy over time