Exercise 3: Isolation Streak

Introduction

A culture that contains more than one species of microorganism is known as a mixed culture. Practically all patient specimens or environmental samples start as mixed cultures. Mixed cultures cannot be used for characterization or identification of microorganisms. Instead pure cultures containing a population of clones of the same microorganism species must be prepared. The process of converting mixed population into a pure culture is called isolation. The most common technique for isolating in organisms in pure culture is the isolation streak or streak plate.

In an isolation streak, the sample is dragged or “streaked” over the surface of a plated agar medium. Done properly the cell density decreases with each streak, eventually leading to individual cells being deposited separately on the agar surface. Individual cells that have been separated from the others will grow into distinct colonies consisting only of the original cell type. Because some colonies develop from pairs, chains, or small clusters of cells instead of from individual cells, the term colony-forming unit (CFU) is used to describe the origin of the colony.

Course Intended Outcomes

Demonstrate and apply basic aseptic techniques, standard lab safety procedures, and maintain aseptic environment in a microbiology laboratory.

Properly handle, prepare and maintain bacterial cultures, and isolate pure microbial species from a mixed culture.

Materials Used

Students will work individually for this exercise, but materials should be shared in small groups. Each group will need the following materials for this exercise.

• Nutrient Agar (NA) plate, 1 per student
• Bunsen burner
• Rubber tubing
• Striker
• Inoculating loop
• Tube rack

Additional materials and cultures will be assigned by your instructor.

Procedure, Day 1

There are multiple ways to correctly perform an isolation streak, but there are a few key steps that must be followed to get good results.

• • Only go to the culture tube ONCE, for streaking the first quadrant.
  • Sterilize your loop between quadrant streaks
  • Be sure that your 2^nd, 3^rd and 4^th quadrant streaks touch the previous quadrant 2 – 3 times or may not pick up any bacteria to isolate.
  • Keep the streaks for each quadrant moving in one direction. The more surface area that you use the better. It is the dragging along the media surface that separates the cells.

Technique:

1. Use a sterile loop to collect a small portion of the sample/specimen assigned.
2. Streak 1: Streak the specimen back and forth in one quadrant of the agar plate. Stay close to the plate’s edge and make long streaks. Keep a low angle with the loop to avoid cutting the agar. Flame the loop and then let it cool.
3. Streak 2: Rotate the plate nearly 90° and begin with the sterile loop dragging across the end of the first streak pattern. Intersect the first streak only two or three times. Flame the loop and then let it cool.
4. Streak 3: Rotate the plate nearly 90° and begin with the sterile loop dragging across the end of the second streak pattern. Intersect the second streak only two or three times. Flame the loop and then let it cool.
5. Streak 4: Rotate the plate nearly 90° and begin with the sterile loop dragging across the end of the third streak pattern. Extend into the middle of the plate. Flame the loop.
6. Refer to the illustrations in Figure 3-1 for acceptable streak patterns.

Sampling/Inoculation:

1. Your instructor will assign the cultures to be used for inoculation by your group.
   • Collect the required materials for the lab.
   • Place all tubes in a tube rack.
   • Never hold a culture tube by its cap.
2. Bacterial culture tubes are always to be shared with your table group.
3. Clearly label plate (on the bottom side) with your initials, organism name, and section day/time.

Procedure, Day 2

1. Collect your plates inoculated in the previous lab from the back bench.
2. Examine plates for isolated colonies. Well isolated colonies are typically found in quadrants 3 and 4 on a good streak plate.
3. Identify the characteristics of your isolated colonies: size, color, morphology.
4. Make note of any procedural errors or contamination on your plate.
5. Record your results in your lab notebook.

Lab Clean-up

Return bacteria cultures to the front bench.

Plates are disposable and should be thrown away in the biohazard container.

Disinfect your benchtop and wash your hands before leaving the lab.

Results and Interpretation

Illustrate the results of your plate streak in your lab notebook. It is also recommended that you take a picture of your results and save for future reference.

Critical Thinking Questions

On which “quadrant” did the isolated colonies appear? What can be done to improve your technique on your next attempt?

Describe the results that would occur if you accidentally “missed” contacting quadrant 1 before continuing your streak into quadrant 2?