Biotech 2nd Edition Sample Ch. 4 Biotechnology Sample Ch. 4 - Page 12

Prepare and autoclave all media in containers that are at least 2 times the volume of the prepared media volume. This allows room for boiling. Purpose To prepare LB agar and LB broth for growing E. coli cultures. Materials Balance, tabletop milligram Weigh boat, 3.5”×3.5” Lab scoops LB agar base Beakers, 400 mL Glass rods Magnetic stir bars Hot plate stirrer, 7”×7” Hot hands protector Media bottle, 250 mL Permanent lab marker pens LB broth base Beaker, 250 mL Media bottle, 125 mL Sterilizer/autoclave Autoclave tape Procedure Note: Recipes can be adjusted for any desired volume of media. Part I: Preparation of 125 mL of LB agar • 125 mL of agar is the maximum that can be prepared in the 250-mL media bottles. • Use Mass1/Volume1= Mass2/Volume2 to determine the proper amounts of media base to use. 1. Wash a 250-mL media bottle and black cap. Label it with sample name, date, and your initials. (This is for sterilizing the media.) 2. Wash a 400-mL glass beaker. (This is for mixing the media.) 3. Measure out the amount of LB agar base required for the volume of agar desired. Use the Mass1/Volume1= Mass2/Volume2 equation to calculate the mass needed for the desired volume. Record that mass (M2) in your notebook along with the calculations. Pour the LB agar base into the clean 400-mL beaker. 4. Very slowly, add 90 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 agar mixture, the LB agar base will eventually become suspended. Note: Some agar recipes require the addition of NaOH. If necessary, determine the amount of 1 M of NaOH to add to the suspended agar. 5. Add more water until a total volume of 125 mL of suspended agar is achieved. 6. Gently add a magnetic stirrer to the beaker by sliding it down the side of the beaker. Move the beaker of agar suspension onto a stirring, hot  plate. 7. Heat on medium high, gently stirring the entire time, until just before it boils. Do not let it boil. The agar suspension should become clear. 8. Using hot hand protectors (like the Bel-Art Hot Hand® Protector), carefully remove the beaker from the hot plate and pour the hot agar suspension into the labeled, clean, 250-mL Figure 4.6.  A pressure cooker can be used to media bottle. Very loosely cap the bottle