• It is necessary to separate individual bacterial cells from one another on an agar surface in order to produce pure colonies. A colony of bacteria is made up of millions of identical bacterial cells, each originating from one single cell.
• Students should streak gently across the agar surface to avoid tearing the agar. If the cells are embedded in the agar it will not be possible to spread them out.

You will need:

• A source plate with colonies of several different species of bacteria growing on it

• 1 sterile agar plate per student

• Sterile toothpicks

• Sharpie marker for labeling

• Incubator, and extra plates for streaking practice

What to do:

1. Label the bottom of the plate with the date, type of agar being used, source of bacteria, and your initials.

    

2. Begin by looking at the bacteria growing on the plate you have already swabbed. Look for a colony of bacteria that you believe you can touch with a toothpick without touching any other colonies.

    

3. Once a bacterial colony is chosen for study, making sure you have chosen one that is bright pink or red in color, smooth, and uniformly round in shape, you are ready to begin the streak plate method.
4. Remove a colony from the MacConkey agar plate using a sterile inoculation loop or toothpick.
5. Replace the lid.
6. Remove the lid of the sterile agar plate, and place it upside-down on the table top.
7. Then with a gentle wiping motion, a series of horizontal streaks spread the bacteria across the edge of the new LB agar plate. The streaks should appear as smudges on a mirror. If too much pressure is applied during streaking, the agar surface may be torn.
8. After the first set of streaks is applied, the plate is rotated slightly. Then, using a new sterile toothpick, make three parallel streaks coming away from the first streak. The last streak is applied by streaking through the end of the three small parallel streaks and then streaking back and forth over the remainder of the plate without crossing over.
9. The object is to spread the bacteria so thin that individual cells are isolated on the surface of the agar.
10. Each of the isolated cells will give rise to a colony of its descendants when the plate is incubated.

After incubating at 37^o C for 24 hours, you should have a genetically pure colony.

Questions to Answer:

1. After picking a colony with a toothpick, streaking it on an agar plate and incubating the plate overnight, a student observes that some of the colonies growing on the plate are white while others are pink. Choose the best explanation for the difference in color.

a. The colony chosen mutated overnight and produced a different type of bacteria.

b. The colony was not spread out properly on the agar plate.

c. The student must have touched more than one colony with the toothpick when choosing a colony for isolation.

d. The ingredients in the agar that cause pink pigmentation are not evenly distributed.

2. If the student where to take a toothpick and touch a white colony that was completely isolated from any other colonies and then streak a new agar plate with the toothpick, what type of bacteria would you expect to see after incubation?

a. White colonies of uniform size

b. Pink colonies of uniform size

c. White and pink colonies of uniform size

d. White colonies of different sizes

3. A student chooses a colony of bacteria and completes the procedure for streak plate isolation. Nothing grows on the streak plate. The student notices that the plate bacterial colonies were originally picked from contains tetracycline. The student also notices after further examination that the plate used to isolate colonies contained tetracycline and ampicillin. Using this information, what is the best conclusion the student can draw from this experiment?

a. The combination of tetracycline and ampicillin is deadly to this type of bacteria.

b. This type of bacteria is not resistant to tetracycline.

c. This type of bacteria is resistant to ampicillin.

d. This type of bacteria is sensitive to ampicillin.