Inoculating And Plates

One of the most important techniques you will learn this semester is how to streak for isolation. As you might guess, the purpose of streaking for isolation is to produce isolated colonies of an organism on an agar plate. Streaks for isolation are useful when you need to separate organisms in a mixed culture, as you will when doing your mixed unknown, or when you need to study the colony morphology of an organism.

When streaking for isolation, you will begin by streaking a portion of your agar plate with an inoculum.  You will then streak successive areas of your plate in an attempt to dilute the original inoculum so that single colony forming units (CFUs) will give rise to isolated colonies.

The following are the steps for a streak for isolation:

1. Complete steps 1-13 on the Aseptic Transfers page. 

2. Lift the lid of the plate on one side, keeping the lid over the plate as much as possible. Watch how to work under the lid of a plate.

3. Streak your first quadrant by moving your loop back and forth across the section of the plate you have designated for your first quadrant. If you are inoculating from a broth, you will want to go back and forth approximately five times. If you are inoculating from solid growth, e.g. plate or slant, you will want to go back and forth only two to three times.

4. Flame your loop.

5. Streak your second quadrant by moving your loop back and forth between your first and second quadrants. You will want to move back and forth approximately three times.

6. Flame your loop.

7. Streak your third quadrant by moving your loop back and forth between your second and third quadrants. Move back and forth approximately three times.

8. Flame your loop.

9. Streak your last quadrant by going into the third quadrant approximately three times. Then, streak a “fishtail” pattern.

10. Tape your plate closed. If you have multiple plates, you can tape them together.

11. Incubate your plates upside down at the appropriate temperature.

There are other ways to inoculate a plate, including the spot streak and the streak for confluent growth (or lawn), both of which you will use this semester. However, the streak for isolation is by far the most important technique to learn when streaking plates and will be vital for your mixed unknown at the end of the semester. Always use a streak for isolation on your plates unless directed otherwise.

A spot streak involves drawing a single line of organism on a plate. You can place two to four organisms on a plate using this technique. Above are two examples of of possible spot streak patterns using three organisms.

During the semester, you will also use a streak for confluent growth. You can perform a streak for confluent growth with a cell spreader, a swab, or a loop. Below are the directions for using a swab or loop. If you use a swab, dispose of it properly!

1. Transfer inoculum onto the swab or loop you are using. 

2. Open one side of the lid of your plate; keep the lid as much over the plate as possible.

3. Placing your streaks as close together as possible, streak in one direction–horizontally, vertically, or diagonally, whichever is easiest for you.

4. Do not flame your loop or dispose of your swab. Continue using your tool as-is to complete your inoculation.

5. Turn your plate 45 degrees and complete the same streak pattern you did in step 3 above. 

6. Repeat steps 4 and 5.

7. Draw a line around the outer edge of the plate.

8. Tape your plate(s) and incubate at the correct temperature.

You can watch here how to streak a plate for confluent growth using an inoculating loop or a swab.