Sterilize the inoculating loop by placing it in the flame of the Bunsen burner. Pass the lower half of the loop through the flame until it glows red.

Allow the loop to cool. You can touch it to the sterile agar on the plate to make sure it has cooled down. Do not place the loop on the table or let it contact anything other than sterile agar or the desired culture now that it has been sterilized.

Dip the loop into the E. coli culture and then remove it.

Open the agar plate and gently glide the loop back and forth across the surface of one section of the agar. Take care to not scratch through the agar with the loop. The agar provides the nutrients the E. coli need to grow.

Place the loop in the Bunsen burner flame again to sterilize it. Once the loop rod cools, touch it to a sterile section of the plate to make sure it is cool, then glide the loop through your first streak to make a second streak. This second streak is a diluted version of the first streak. Repeat this sterilization and gliding process until several sections of the agar plate have been glided over. The reason you do this is so you can later pick from single, clonal colonies. This will help make sure you have at least one section that has individual colonies-- not too concentrated (which will have too much growth, and not allow you to pick from a single colony) and not too dilute (which would give no colonies).

Sterilize the loop in the flame one last time before putting it aside in the work area.

Put the top back onto the agar plate. Turn the plate upside down and place it into the incubator set to 37 degrees Celsius (98.6 degrees Fahrenheit). This ideal incubating temperature simulates the temperature of the human body where E. coli resides. Within 24 to 48 hours, visible colonies of E. coli bacteria will appear in the agar plate.