Streaking Agar Plates

Goal: Take a source of microorganisms and spread the material over an agar plate to obtain single isolated colonies. The isolated colonies will allow you to observe specific characterisitcs such as colony size, shape and color. If special media are you used, you can also determine other traits such as hemolysis (if streaking on blood agar) or ability to ability to ferment the sugar lactose (if using MacConkey Agar).

Materials:

Gloves
Inoculating loop OR sterilized wooden sticks for streaking.
Permanent marker or grease pencil to label your plates beforehand.

Bunsen burner or sterilizing heater if intending to sterilize the inoculating loop between streaks.

Swab for collecting the primary inoculum, if intending to collect bacteria from an environmental source

Agar plate

Incubator, if incubating at a controlled temperature, such as 37°C. However, many common microbial species will grow on plates left at room temperature, through their growth may be slower than if the plates were incubated at 37°C.
Antibacterial soap to wash your hand.
5% bleach solution to clean your work area when finished.

Method:

Primary Streak
Secondary Streak

Primary Streak (shown in Yellow).

Hold the plate as though you are going to "paint" the surface of the agar . If you are right-handed, hold the plate in your left hand, and the inoculating loop in your right - as through you would a paint brush. Hold the plate in your right hand, and inoculating loop in your left if you are left-handed.

Dip your inoculating loop (sterile swab, or sterile stick as shown in the above picture) into a broth culture, or touch it to the material you want to spread - an isoated colony or swab an area for which you want to quantitate the microbial species present.

Go back and forth a number of times in a small area of the Petrie plate. The goal is to spread your material completely over this inital area of the plate.
Secondary Streak (shown in Blue).

Sterilze your inoculating loop, or use a fresh, sterile inoculating stick or swab. Squelch it in an unused area of the agar before steaking to cool it. If you were to use the original, unsterile loop, you will not be diluting the individual microbes you plates in the first streak.

Pick up the plate and rotate it 1/4 of a turn to your left (if right-handed), or to your right (if left handed).

Run the loop through the previous streak 2-3 times, then draw it along 1/3 of the remaining plate, as shown by the blue line in the above image.

It's OK if you gouge a little bit of the agar, but with practice you should be able to streak cleanly across the surface.

Tertiary Steak
Quarternary Streak

Tertiary Streak (Orange)

Rotate the plate another 1/4 turn and sterilize your inoculating loop or take a fresh, sterile stick or swab. Squelch a heated inoculated loop it in an unused area of the agar before steaking to cool it.

Run the loop through the previous, secondary streak 2-3 times, and draw the streak over a remaining 1/3 of the plate, as shown.
Quarternary Streak (Black)

Rotate the plate another 1/4 turn and sterilize the inoculating loop. Squelch a heated inoculated loop it in an unused area of the agar before steaking to cool it.

Run the loop through the previous tertiary streak 2 times and draw over the remaining free space in the plate, being careful not to contact the primary streak (yellow).

Incubate the plate as needed, and check 18-24 hours later for growth!


Tertiary Streak (Orange) Rotate the plate another 1/4 turn and sterilize your inoculating loop or take a fresh, sterile stick or swab. Squelch a heated inoculated loop it in an unused area of the agar before steaking to cool it. Run the loop through the previous, secondary streak 2-3 times, and draw the streak over a remaining 1/3 of the plate, as shown.	Quarternary Streak (Black) Rotate the plate another 1/4 turn and sterilize the inoculating loop. Squelch a heated inoculated loop it in an unused area of the agar before steaking to cool it. Run the loop through the previous tertiary streak 2 times and draw over the remaining free space in the plate, being careful not to contact the primary streak (yellow).