STREAK PLATE TECHNIQUE FOR ISOLATION

STUDENT INSTRUCTIONS

Read these entire instructions thoroughly and watch a demonstration before you attempt the procedure yourself. Each student choose either E. coli or S. epi. Prepare a previously poured agar plate by labeling with name, date, and organism. Use a grease pencil to make "map" for a streaking pattern. Mark sections from behind the agar plate. (Do not remove the top until sample is ready for streaking). There are many methods for streaking. The most effective method depends on the concentration of bacteria within a particular inoculum as well as each individual technician’s dexterity. A typical pattern will be demonstrated which works with the samples used in this teaching lab.

Although we are practicing with pure cultures, the streak plate technique is designed to isolate species from mixed cultures. As the bacteria in a mixed sample are spread apart from each other and allowed to grow into colonies separated across the agar surface, an isolated colony can then be used as a source of inoculum for a pure culture. We will try this with an unknown mixture from an environmental culture.

PROCEDURE

Using aseptic technique, remove a loopful of bacteria from a pure culture.
Lift the lid of the Petri dish out of the way, but keep it angled over the dish.
Place the loop gently on the agar surface in section 1. (Never put enough pressure to dig into the agar surface!)
Gently sweep the loop back and forth across the agar surface, spreading the sample out in section 1.
Heat-sterilize the loop.
Allowing the loop to cool, touch it to a sterile part of the agar in section 2.
Sweep the loop across the agar surface from section 2 into section 1, then back to 2, then back to 1, etc. Take care not to double back over any streaks in section 2. Stop when you run out of area in section 2, and lift the loop.
Heat-sterilize the loop.
Allowing the loop to cool, turn the dish so that section 3 is accessible.
Place the loop in a sterile section of section 3, then gently stroke the agar surface, returning several times into section 2.
Do not double back over any streaks in section 3. Continue on in section 3 even though there is not space available for access to section 2. Above all, do not re-enter section 1.
Re-cover the dish, heat-sterilize the loop, invert, and incubate.

The goal of the above procedure is to dilute the original sample by heat- sterilizing. As you streak out the bacteria in section 1, they are separated from each other. By carefully streaking back into 1 from 2, you pull back into 2 from 1 only a small amount. As you streak from 2 to 3, you are pulling out even fewer. Your isolated colonies after incubation should be found in section 3. This is where single bacterium landed and multiplied into a clone of identical cells: a colony.

Invert the plates in the rack provided. Incubate the rack of plates in a minimum of 48 hours; retrieve and observe.