Biotech 2nd Edition Sample Ch. 4 Biotechnology Sample Ch. 4 - Page 18

AB LB 1/06 7/2 oli ce lls isolated colony top of plate bottom of plate Figure 4.11.  Streaking for Isolated Colonies.  Use the triple-Z method of streaking, flaming between streakings. This method results in a significant decrease in the number of cells spread each time so that isolated colonies result in the last “Z.” 37ϒC cool it on a spot of uncontaminated agar, and collect a colony of E. coli from the stock plate. If using a sterile, disposable plastic loop, remove it from the sterile packet without touch the end loop or contaminating other loops in the packet, then collect a bacteria colony. 3. Streak the bacterial colony onto a sterile LB agar Petri plate (see Figure 4.11). a. Be careful to hold the top of the Petri plate over the bottom to minimize contamination. b. Streak the sample back and forth across the agar on the top one-eighth of the plate. This is the first set of “Z”s. c. Rotate the plate 90°. Flame-sterilize the metal inoculating loop. Cool on uncontaminated agar. If using the plastic loop, discard it into disinfectant and get another sterile loop. d. Streak the loop at a right angle through the previous streak. Go through the first streak only once to pick up the fewest bacteria cells possible. Make a “Z”-shaped streak. This is the second “Z.” e. Rotate the plate another 90°. Flame-sterilize the metal inoculating loop. Cool on uncontaminated agar. If using the plastic loop, discard it into disinfectant and get another sterile loop. f. Streak the loop again, at a right angle, through the second “Z” streak. Go through that streak only once to pick up the fewest bacteria cells possible. Make a third “Z.” Flamesterilize the inoculating loop. Place on laminar flow hood counter to cool. If using the plastic loop, discard it into disinfectant. g. Replace lid. Place your inoculated LB plate, upside down, in one of the incubation ovens designated by the supervisor. 4. Incubate the bacterial plate culture, upside down, for 24 to 36 hours at 37°C. 5. Before leaving the lab area, discard any biological waste in the biohazard bag, disinfect your workspace, and wash your hands. Data Analysis/Conclusion Evaluate your ability to isolate individual bacteria cells and grow them into isolated colonies. How many isolated colonies are present in the final “Z”? Describe how you might improve the technique next time. Are any of the colonies good candidates for starting a broth culture? Part II: Starting a Broth Culture Purpose To grow a broth culture to use as a source of cells for DNA isolation. DNA Isolation and Analysis 83