Lab Exercise 5: Pure culture techniques OBJECTIVES 1. Perform a streak-plate to separate the cells of a mixed culture so that discrete colonies can be isolated. 2. Perform a pour-plate (loop) dilution to separate cells of a mixed culture and compare growth characteristics beneath and on the surface of the agar. INTRODUCTION As you learned in Lab 4, microbes exist everywhere, and very rarely do they occur as a single species. Robert Koch, known as the father of medical microbiology, was one of the first to recognize that isolating a microbe (in his case, bacterium) away from other microbes was crucial for his own argument that microbes cause disease, as well as understanding characteristics of the microbe itself. His studies on Bacillus anthracis contributed to many of the laboratory techniques we still use today, including the method for isolating pure cultures of bacteria. The most commonly used method in the laboratory for isolating microbes is the streak plate, and to a lesser extent, the pour plate. Both methods rely on dilution of bacterial cells in a sample to the point at which a single cell can divide giving rise to a single pure colony. The pure colony is essentially a clone of cells that all are identical copies of the original cell and can be used for further study. Lab Exercises I. Streak plate method Table supplies 1 mixed culture of Serratia marcescens, Escherichia coli, and Chromobacterium violaceum Individual supplies 1 environmental body broth (from Lab Exercise 4) 2 nutrient agar plates Inoculating loop Protocol: 1. Prepare lab bench by removing extraneous items and cleaning surface with table disinfectant. 2. Label the bottom surface of your sterile agar plates. 3. Using the T-method (illustrated below) or quadrant method, outline the sections on the bottom of the agar plate. 4. Obtain mix culture and shake gently to suspend organisms. 5. Flame the loop until it is red-hot and let cool.
6. Remove cap of mix culture and flame the mouth of the tube (do NOT place cap down on the table). 7. Insert the loop into the tube and remove a loopful of broth with bacterial cultures. 8. Lightly flame the mouth of the culture tube again. 9. Return the cap to the tube and set in your tube rack. 10. Streak the plate, following either the T-method or quadrant streak shown below. Do not gouge into the medium with the loop and keep the lid over the plate as much as possible. 11. Flame the loop before setting it down. 12. Repeat the above protocol (# 4-11) using your body broth from Lab 4 and a new agar plate. Step 1:Do the initial inoculation then FLAME the loop, let cool about 15 seconds. Step 2: Use the sterile cooled loop and draw over the agar surface in the first section, flame and cool.
Step 3: Use the sterile cooled loop and draw of the agar surface in the second section. Note that you do NOT go back to the broth and you overlap with the first section a few times. Flame the loop and allow to cool. Step 4. Use the sterile cooled loop and draw of the agar surface in the third section. Again do NOT go back to the broth and overlap with the second section only. Now flame loop. Now place the plate in incubator. 13. Incubate the plates inverted at 30 C for 2 days. (NOTE: we will have to set up a separate 30 C incubator for this experiment. Follow instructions on where to put these plates). See animation at: http://www.sumanasinc.com/webcontent/animations/content/streakplate.html II. Pour plate method Table supplies Mixed culture of S. marcescens, E. coli, and C. violaceum Team supplies 3 nutrient agar pours 3 empty Petri dishes Inoculating loop
1. Label the bottom of three sterile Petri plates I, II, and III. 2. Remove 3 tubes of nutrient agar from the 50 C water bath in the back of the room. (NOTE: you must work quickly at this point or the nutrient agar will solidify in the tube). 3. Using aseptic technique, take one loopful from the mixed culture and add to the nutrient agar in tube I. 4. Mix the tube by rolling vigorously between the palms of both hands. DO NOT shake the tubes as the caps are not secure and medium will splash out. 5. Immediately, and using aseptic technique, take a loopful of the agar from tube I and add it to tube II. 6. As soon as this is done, you may pour the contents of tube I into the plate labeled plate I, being sure to pour it into the bottom of the plate. 7. Repeat steps 4-6, transferring a loopful of tube II to tube III, and then pouring the contents into plates II and III. 8. Allow media to solidify and then incubate the plates inverted at 30 C for 2 days. (NOTE: we will have to set up a separate 30 C incubator for this experiment. Follow instructions on where to put these plates). DATA AND OBSERVATIONS 1. Evaluation of streak plates: Show within the circle the distribution of the colonies on your streak plate. Did you get clear isolation of purple Chromobacterium violaceum, red Serratia marcescens, and white E. coli? If not, you maybe need to practice the technique again by subculturing the colonies (your instructor might want you to do this regardless). To subculture, use your wire loop to pick an isolated colony that you will use as inoculum for another streak plate.
2. Evaluation of pour plates: Show the distribution of colonies on the pour plate II or III. Indicate which colonies are growing on the medium and which are growing within it. DISCUSSION 1. Compare your results from the streak-plate and pour-plate methods. Which method achieved the best separation of species? 2. Which specie(s) are able to grow both on the surface and within the medium? In which location are the colonies larger? Why? 3. In regards to bacterial growth on solid media, define the term colony. 4. Explain how dilution is a common approach employed by any pure culture technique. 5. What advantage does the streak-plate method have over the pour-plate method? 6. Before inoculating and pouring molten nutrient agar into a plate, why must the agar first be cooled to 50 C? 7. Provide two reasons why plates should be inverted during incubation