Lysogeny Broth (LB Broth) is a nutritionally rich liquid medium used extensively in microbiology for cultivating bacteria, especially Escherichia coli (E. coli). It provides all the necessary components for bacterial growth, including sources for carbon, nitrogen, and essential salts. LB Broth supports rapid and robust growth, making it a standard preparation in molecular biology applications.
Required Ingredients and Ratios
The standard LB Broth formulation, often called LB-Miller, requires three main powdered ingredients dissolved in water. For a 1-liter batch, the components are 10 grams of Tryptone, 5 grams of Yeast Extract, and 10 grams of Sodium Chloride (NaCl). These ratios provide a balanced supply of nutrients and maintain the correct osmotic environment.
Tryptone, an enzymatic digest of casein, serves as the primary source of amino acids and peptides, supplying the nitrogen and carbon needed for bacterial growth. Yeast Extract supplies a complex mixture of organic compounds, including B vitamins and trace elements, which act as cofactors. Sodium Chloride (common salt) maintains the osmotic balance outside the bacterial cells, preventing cell damage.
Combining Components and Adjusting pH
To begin, dissolve the powdered ingredients in a volume of distilled or deionized water slightly less than the final desired volume (e.g., 900 milliliters for a 1-liter batch). Using pure water prevents contaminants from interfering with bacterial growth. The mixture should be placed on a magnetic stirrer until all solids are completely solubilized, resulting in a clear, amber liquid.
Next, adjust the pH of the solution, which is often slightly acidic after mixing. The optimal pH range for most E. coli strains is between 7.0 and 7.5. A concentrated solution of sodium hydroxide (NaOH) is added dropwise while monitoring the pH with a meter until the target value is reached. Finally, add the remaining distilled water to bring the volume precisely up to 1 liter, and transfer the liquid to a flask for heat treatment.
Sterilization Protocols
Sterilization is necessary to prevent contamination from unwanted microbes that would compete with the desired bacteria. The standard method for sterilizing LB Broth is autoclaving, a process using saturated steam under high pressure. The medium, contained in a loosely capped vessel to allow pressure equalization, is exposed to 121°C at 15 pounds per square inch (PSI) for 15 to 20 minutes. This combination of heat and pressure effectively kills all living cells and bacterial endospores, ensuring sterility.
Pressure Cooker Alternative
A household pressure cooker can serve as an alternative for sterilizing smaller batches if a laboratory autoclave is unavailable. The cooker must achieve and hold 15 PSI, corresponding to the 121°C sterilization temperature. Heat the broth until the pressure gauge reaches 15 PSI, and hold this pressure for a minimum of 15 minutes. Allow the vessel to cool slowly until the internal pressure returns to zero before opening.
Storage and Quality Control
Once the sterilization cycle is complete and the container has cooled, the LB Broth is ready for storage. The medium should be stored in a sealed container, such as a bottle with a secured cap, at room temperature or refrigerated at 4°C. Proper storage ensures the broth remains sterile and retains its nutritional quality for several months.
Before storing, label the container clearly with the date of preparation and the medium concentration for accurate tracking. Quality control involves visually inspecting the liquid prior to use; it should remain perfectly clear. Any cloudiness or turbidity indicates microbial contamination, meaning the medium is no longer sterile and should not be used.