Biotech 2nd Edition Sample Ch. 4 Biotechnology Sample Ch. 4 - Page 13

9. Place the media bottle into a pressure cooker or autoclave, along with the rest of the media to be sterilized (see Figure 4.6). Bolt down the pressure cooker’s top or autoclave’s door as directed by the supervisor, or according to the manufacturer’s directions. 10. Heat until the pressure gauge reads 15 pounds of pressure per square inch (psi). Caution: Follow all pressure cooker or autoclave instructions. 11. Keep the bottles at 15 to 20 psi for 15 to 20 minutes. Cool the agar to 65°C (just barely cool enough to hold bottles). Continue to step 12 if there is time to pour plates. If there is not enough time to pour plates, let the agar cool and solidify. It can be reheated in a microwave at 50% power for about 4 minutes to completely liquefy. Cool to 65°C before pouring. 12. Pour the liquid agar into Petri plates one-half full, in a laminar flow hood or disinfected lab tabletop, as directed by your instructor. Data Analysis/Conclusion Evaluate your ability to prepare 125 mL of LB agar. Consider the final volume, color, mix, solidity, and labeling of the sample. Describe which of these tasks you performed well, and which you could improve on and how. Procedure Part II: Preparation of 50 mL of LB Broth • 50 mL of broth is an appropriate volume for a 125-mL media bottle. • Use the Mass1/Volume1= Mass2/Volume2 to determine the proper amounts of media base to use. 1. Wash a 125-mL media bottle and black cap. Label it. (This is for sterilizing the media.) 2. Wash a 250-mL glass beaker. (This is for mixing the media.) 3. Measure out the amount of LB broth base required for the volume of broth desired. Record that mass (M2) in your notebook along with your calculations. Place the LB broth base into the clean 250-mL beaker. 4. Very slowly add 35 mL of distilled water, stirring as it is added. The water should, at first, make a thick paste. As you add more water and stir the broth mixture, the LB broth base will eventually become dissolved. LB broth base goes into solution much more easily than agar base. Note: Some broth recipes require the addition of NaOH. If necessary, determine the amount of 1 M of NaOH to add to the liquid broth. 5. Add more water, stirring, until a total volume of 50 mL of suspended broth is achieved. 6. If the broth base is not completely dissolved, move the beaker of broth suspension onto a stirring, hot plate. Gently add a magnetic stirrer to the beaker by sliding it down the side of the beaker. Heat on medium high until the broth base has dissolved, gently stirring the entire time. Do not let it boil. Using bottle holders, remove the beaker from the hot plate. 7. Pour the hot broth suspension into the labeled, clean, 125-mL media bottle. Very loosely cap the bottle with the black cap. Add a small piece of autoclave tape to the bottle. 8. Place the media bottle into a pressure cooker or autoclave, along with the rest of the class’s bottles. Bolt down the pressure cooker’s top or the autoclave’s door as directed by the instructor or the manufacturer’s directions. 9. Heat until the pressure gauge reads 15 psi. Caution: Follow all pressure cooker or autoclave instructions. 10. Keep the bottles at 15 psi for 15 to 20 minutes. Cool the broth to room temperature before using. Data Analysis/Conclusion Evaluate your ability to prepare 50 mL of LB broth. Consider the final volume, color, mix, and labeling of sample. Describe which of these you performed well, and which you could improve on and how. 78 Chapter 4   Laboratory Manual