Preparation of Luria Broth Media Solution and Luria Broth Media with Agar for Petri Plates

Dr. Megan Macnaughtan Research Group Procedures and Protocols

Jacob Myers

Friday, November 13, 2009

Preparation of Luria Broth Media Solution and Luria Broth Media with Agar for Petri Plates
I. Objective a. The objective of this procedure is to outline the steps used to prepare Luria Broth (LB) media to be used for bacterial culture growth in solution and bacterial colony growth on Petri plates. II. Overview a. A LB media solution is prepared using the following dry components: i. Yeast extract ii. Tryptone iii. Sodium chloride (NaCl). b. To prepare LB media with agar to be used for colony growth, the following component is to be added to the LB media: i. Agar. III. Procedure a. Luria Broth Media i. Weigh out each dry component of the solution as follows: 1. Yeast extract: 2. Tryptone: 3. NaCl: 5g 10 g 10 g

ii. Add each component to a 1 L Erlenmeyer flask containing 800 mL of water iii. Stir to dissolve components into solution. iv. Transfer the solution to a 1 L Volumetric flask and dilute to 1 L. v. Transfer the 1 L-LB media solution to a 1 L media storage bottle and label with appropriate information. vi. Autoclave the solution at(??conditions for autoclave sterilization??)to ensure that the LB is sterilized of all foreign matter and contaminants. vii. Store the solution in the refrigerator.

LSU Department of Chemistry

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Preparation of Luria Broth Media Solution and Luria Broth Media with Agar for Petri Plates
Dr. Megan Macnaughtan Research Group Procedures and Protocols

Jacob Myers b. Luria Broth Media with Agar i. Weigh out the dry component as follows: 1. Agar: 2.5 g

Friday, November 13, 2009

ii. Add the agar component to a 1 L Erlenmeyer flask containing 500 mL of LB media. iii. Autoclave the solution at(??conditions for autoclave sterilization and agar dissolution into media??)to ensure that the LB media with agar is sterilized of all foreign matter and contaminants AND that the agar dissolves into solution. iv. NOTE: The agar will NOT dissolve into solution until that LB media with agar is autoclaved. v. Once autoclaved, the LB media with agar can either be used immediately or stored either in the refrigerator or on the shelf. vi. NOTE: If the LB media with agar is not used immediately, allow the media to cool and place a cap on the storage bottle to ensure no contaminants get into the media. The media will solidify while being stored. To liquefy the media after it has solidified heat in an appropriate microwave for 30 seconds to 1 minute. **NOTE: Other components, such as M9 media, vitamins and minerals, or antibiotics, may be added proportionately to these LB media solutions depending on the growth conditions for the bacteria.**

LSU Department of Chemistry

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