A. Make Luria broth (LB) and LB agar plates

Day 1

LB is a nutrient-rich media that is commonly used to grow bacteria. The addition of agar to LB results in the formation of a gel that bacteria can grow on, as they are unable to digest the agar but can gather nutrition from the LB within. You can also add an antibiotic to the broth. The antibiotic allows for the selection of only those bacteria with the specific antibiotic resistance that is conferred by a plasmid carrying the antibiotic resistance gene.

The following protocols require the use of LB and LB agar plates, both with and without antibiotic selection. You will need to make batches of each. They can be stored in airtight containers or wrapped in plastic bags, at 4°C for up to 2 weeks.

  1. Weigh out the following reagents each into a 1L Erlenmeyer flask (Table 1). Make a 500 mL batch of LB and a 1L batch of LB; you will add antibiotic to one batch at a later stage (see below).

Table 1 Reagents for making LB and LB agar plates

LB LB agar plates LB agar plates+antibiotic
5g NaCl 5g NaCl 5g NaCl
5g tryptone 5g tryptone 5g tryptone
2.5g yeast extract 2.5g yeast extract 2.5g yeast extract
dH2O to 500mL dH2O to 500mL dH2O to 500mL
  7.5g agar 7.5g agar
    ampicillin*

Do not add the antibiotic until after autoclaving and cooling to 55 degrees Celsius.

  1. Place magnetic stirrer into the bottom of the flasks and stir to mix.

Note

The contents do not have to be completely in solution, but any powder left on the sides of the flask will caramelize on the glass during autoclaving.

  1. Cover the top of the flasks with aluminum foil and label with autoclave tape.
  2. Autoclave on the liquid setting for 20 minutes or according to your autoclave’s specifications.
  3. After removing the solution from the autoclave, allow the LB agar solution to cool to 55 degrees Celsius.

Note

This can be done by placing the flask in a 55 degree Celsius oven or water bath because this will hold the temperature and it can be left unattended for some time.

Note

The LB can cool to room temperature on the bench top.

  1. Add the appropriate amount of the desired antibiotic to one flask of LB and one flask of LB + agar. The other two flasks (containing LB and LB + agar) will not contain any antibiotic. Table 2 lists commonly used antibiotics. The table specifies what the concentration of the stock solution should be made at.

Table 2 Commonly used antibiotics and suggested stock and working concentrations

Antibiotic Stock solution concentration* Working solution concentration
Ampicillin 50 mg/mL in H2O 20ug/mL
Carbenicillin 50 mg/mL in H2O 20ug/mL
Chloramphenicol 34 mg/mL in ethanol 25 ug/mL
Kanamycin 10 mg/mL in H2O 10 ug/mL
Streptomycin 10 mg/mL 10 ug/mL
Tetracycline** 50 mg/mL in ethanol 10 ug/mL

How do I insert the key (the asterisks)

When pouring agar plates, keep your bench area sterile by working near the flame of a Bunsen burner.

  1. Label the bottom of each Petri dish with the date and the antibiotic added. If no antibiotic has been added, label each Petri dish with just the date.

Caution

Be sure to pour the LB + agar + antibiotic into the Petri dishes that are labeled with ampicillin, and the LB + agar into the dishes that are labeled with the date only.

  1. Pour approximately 20 mL of LB agar per 10cm polystyrene Petri dish.

Caution

Pour slowly from the flask into the center of the Petri dish. When the agar has spread to cover about two thirds of the dish stop pouring and the agar should spread to cover the entire plate. You may need to tilt the plate slightly to get the agar to spread out completely. If you pour in too much, the plate will be fine, but it will reduce the number of plates you can make per batch.

Caution

If bubbles are introduced during the pouring, these can be removed by quickly passing the flame of an inverted Bunsen burner over the surface of the plate. Be careful, if you leave the flame too long it will melt the Petri dish. Also, be careful not to burn yourself.

  1. Place the lids on the plates and allow them to cool for 30-60 minutes (until solidified), then invert the plates. Let the plates sit for several more hours (or overnight).