BISC209: Streaking for Isolation

Protocol for Streaking for Isolation

Nutrient agar plates can be streaked using a three-phase or a four phase pattern (illustrated in Figure A-1) and either an inoculating loop or a sterile swab.

Isolation streak technique: from broth, slant, or plate to plate

To streak a plate using an inoculating loop: YouTube Demo [1]

The handling of the plate can be accomplished in a number of ways, all of which attempt to minimize possible contamination by either keeping the lid over the plate or by keeping the plate upside down (see Figure A-2).

1. Sterilize the loop and allow it to cool. Obtain cells from the stock culture.

• if using a broth and a loop allow the film of liquid inside the loop to “break” in the tube so that only those cells adhering to the wet wire and not those in a film within the wire are transferred.
• if using a sterile cotton swab, remove the sterile cotton swab from its container; be sure not to touch the swab to anything. Insert the swab into the stock culture tube, roll the swab on the side of the container to remove excess liquid.
• if using a colony or growth from a slant, just touch the sterile loop to the colony or growth

2. Using the swab or the loop streak the first section of the plate using tight sweeping lines that stay within that section: (1/3 for a 3-phase pattern) or (1/4 for 4-phase) of the plate. It is fine to overlap your streaks in this section.

3. Sterilize the loop and allow it to cool in the air for 15 seconds. Touch the loop to an unused edge of the agar surface to cool it completely before continuing. Then pull the loop through one edge of the previous streaks one or two times to re-inoculate the loop. Now streak the second third of the plate, avoiding the first third (see Figure A-4).

4. Sterilize and cool the loop as in the last step. Pull the loop through one edge of the streaks in the second third of the plate to obtain inoculum. Now streak the last third of the plate.

To streak a plate using a sterile swab:

1. Remove the sterile cotton swab from its container; be sure not to touch the swab to anything. Insert the swab into the culture of and then roll the swab on the side of the container to remove excess liquid. Using the swab like a loop, streak the first section of the plate. Remove the sterile cotton swab from its container; be sure not to touch the swab to anything. Insert the swab into the culture of and then roll the swab on the side of the container to remove excess liquid. Using the swab like a loop, streak the first section of the plate.

2. Switch to the loop and proceed to streak the plate as described for streaking with a loop.

Figure A-4. Patterns used in streaking a plate to obtain isolated colonies. If an inoculating loop is used, it is sterilized between each phase.

FigureA-5: Two procedures for streaking a plate. (a) Streaking a plate while holding the lid ajar. Note that the lid shields the agar from airborne contamination: (b) streaking a plate while holding the bottom of the plate. Note that the agar surface faces downward, thereby minimizing contamination from the air.