B. Transfer E.coli cells from glycerol stock to LB agar plates

Day 1 (cont.)

Bacterial stocks are typically stored in a glycerol solution in the -80°C freezer. To isolate a single bacterial colony, you will streak it onto an agar plate in a specific way.

  1. Label the bottom of an agar plate with the bacterial strain and the date.

Caution

The LB agar plate should contain no antibiotics because the E.coli do not contain a plasmid that carries an antibiotic resistance gene.

  1. Fill a small tub or dish with dry ice. Remove the vial containing the E.coli from the freezer and place it on the dry ice.
  2. Sterilize your inoculation loop by passing it at an angle through the flame of a Bunsen burner until it burns red.

Caution

Let the loop cool before you scrape your bacterial cells/ inoculum from the vial. If the loop is too hot, it will kill the cells.

Caution

Do not place the sterilized inoculum loop down on the bench or it will recontaminate it.

  1. Touch the sterile loop to the bacteria in the glycerol stock. Gently scrape a small amount of the inoculum from the vial with your loop.
streaking for single colony

Figure 2 How to streak bacteria onto an agar plate to achieve single colonies

  1. Gently spread the bacteria over a section of the plate, as shown in Figure 2, to create streak #1.
  2. Using a freshly sterilized loop, drag through streak #1 and spread the bacteria over a second section of the plate, to create streak # 2.
  3. Using a freshly sterilized loop, drag through streak #2 and spread the bacteria over the last section of the plate, to create streak # 3.
  4. Incubate the plate with the newly streaked bacteria overnight (12-18 hours) at 37°C.

Invert the plates, so that if water condensation occurs, the water will collect on the plastic lid, not on the agar.