Quadrant Streaking

So- you've screened for antibiotic production and have a few bacteria that show promise. Let's isolate those colonies!

You'll Need:

• Your original Library Plate

• 1 new LB agar plate for each named colony

• Antibiotic Screen Plates

• Two inoculating loops

• Bunsen burner

• Beaker

1. Observe your Antibiotic Screen plates from your last lab. Which bacteria show some promise?

   Look for a ring of space (called a zone of inhibition) around Isolated colony

   your sample bacteria, or space where the ESKAPE didn't

   grow.

   Once you've identified some candidates, match them up from your library plate. The grid should be the same! Try to find ~6 colonies you want to isolate.

   

Zone of inhibition example

2. Label the new agar plates along the edges with your initials, the ID of the selected colony, and the date.
   

3. Flame both loops and place them loop-up in the beaker. Let them cool for 15-20 seconds. Tip: This speeds up the process because you can quickly alternate loops without waiting for a single one to cool each time.
   

4. Select the first colony you've chosen from your library plate and touch the loop to it, collecting a barely visible sample.
   

5. Starting on one edge of the plate, draw a tight squiggle with four long lines. Flame the loop and place it in the beaker, and select the next loop.

   Tip: The image below can be a helpful reference!

   
   

   

   
   

6. Don't touch your library plate again- the second quadrant will be inoculated by the first quadrant. Place the loop between the last two lines of the first quadrant and draw another tight squiggle with four lines.

   
   

   Tip: If you can’t see the previous quadrant, tilt the plate in the light to help you see the lines in the agar from before.

   
   

7. Repeat steps 6 and 7 once from the second quadrant, creating the third quadrant.

   
   

8. For the last quadrant, start the line from the last line of the previous quadrant as before. This time, pull the squiggle down into the middle of the plate in a straight line. Make sure your streak does not overlap with any other lines.

9. Incubate at 26°C for 24-48 hours.

   
   

   When you come back to the plates later, look for small, isolated dots of colonies along the last quadrant you made. These are likely to be your isolated colonies! Next, you'll take a closer look at your colonies through Gram staining. Good luck!