Biotech 2nd Edition Sample Ch. 4 Biotechnology Sample Ch. 4 - Page 19

Procedure • Do all work in a sterile laminar flow hood or on a countertop. • Use all standard precautions with the Bunsen burner, (tying back hair, wearing goggles, etc.). • Dispose of any bacteria-contaminated products in autoclave bags and/or 10% bleach solution. 37ϒC 1. Obtain a Petri plate of LB agar containing isolated colonies, a 50-mL sterile, conical centrifuge tube, and a bottle of sterile LB broth. 2. Flame-sterilize (as demonstrated by the supervisor) the top of the LB broth bottle. 3. Using a sterile 10-mL pipet, transfer 10 mL of LB broth from the bottle to the 50-mL tube. 4. Flame-sterilize (as demonstrated by the supervisor) an inoculating loop, cool it on a spot of uncontaminated agar, and collect a colony of E. coli from the streaked plate. If using a sterile plastic loop, collect a colony of E. coli from the streaked plate. 5. Holding the tube at a 45° angle, add the colony to the broth, with a twist of the loop, to ensure that most of the colony gets into the broth (see Figure 4.12). Reflame the loop to remove any remaining bacteria. If using the plastic loop, discard it into disinfectant. 6. Incubate the broth culture in a shaking hot water bath or a shaking incubator oven at 37°C for 24 hours (see Figure 4.13). 7. Before leaving the lab, discard any biological waste in the biohazard bag, disinfect your workspace, and wash your hands. Note: After 24-hour incubation, remove 1 mL of culture and store (-20°C) in a sterile tightly capped 1.7 mL tube for Lab 4l. Figure 4.12.  Inoculating a Broth Culture.  Hold the tube “sideways” to decrease the chance of contaminants falling into the container. Figure 4.13.  In industry, large shaker flask ovens keep cells moving, aerated, and warm, so that they grow and divide at a maximum rate. Photo by author. Data Analysis/Conclusion Evaluate your broth culture. Does it look the way you think it should look? Is it cloudy and obviously full of bacteria cells? How do you know? How can you be sure that there are enough of the right kinds of cells? Propose a method for che