Biotech 2nd Edition Sample Ch. 4 Biotechnology Sample Ch. 4 - Page 11

Laboratory 4e Making Media for Bacteria Cell Culture (Media Prep) Background In the next several activities, bacteria cells are grown and used as a source of DNA. Bacteria are grown on or in their food source, called media. If the medium is a solid, it is called agar; if it is liquid, it is called broth. The decision to use one type of medium or another depends on the kind of bacteria to be grown and the intended use. If single, isolated colonies are needed, cells must be grown on agar, usually in Petri plates. Depositing single, isolated cells on the agar surface results in individual colonies of cells. Each isolated cell grows and divides hundreds of times resulting in a colony of identical cells. All the cells in a colony are clones of each other. A plate culture is ideal for separating bacteria into individual colonies and isolated, pure, uncontaminated cultures. Once isolated colonies are grown, a single colony can be transferred to broth culture with the goal of maximizing cell growth and reproduction. In a broth culture, cells have better access to nutrients. They grow, produce molecules, and divide into new cells at a maximum rate. Broth cultures, sometimes as large as tens of thousands of liters, are used in manufacturing to obtain a maximum number of cells to make a maximum amount of protein product. E. coli bacteria will grow in many kinds of media, but they grow particularly well in Luria Bertani (LB) agar and broth. LB agar is an “all purpose” agar that supports the growth of many types of bacteria and fungi. Other nutrients may be added to the agar for different tests. Media base (dry mixture of media components) is available for purchase from supply houses. The recipe for making a 1-L batch of agar or broth is printed on most stock media-base containers. Using a simple ratio equation, Mass1/Volume1= Mass2/Volume2, you can determine the proper amounts of media for the volume of media needed: Media Prep Equation Mass2 Mass1 = Volume1 Volume2 M1 = the mass of media base to use in the original recipe’s volume (in grams) V1 = the volume of solvent (dH2O) in the original recipe’s volume (usually 1000 mL or 1 L) M2 = the mass of media base needed for the desired volume (in grams) V2 = the desired final volume of media (in mL or L) For example, suppose the recipe on a media bottle says to use 22 g of media base in 1 L of dH2O. For an experiment, only 300 mL of prepared media are needed. What amount of media base is needed to make the 300-mL volume? Convert 1 L to 1000 mL. 22g /1000 mL = M2/300 mL M2 = 22 g × 300 1000 M2 = 6.6 g of media dissolved in dH2O to a total volume of 300 mL 76 Chapter 4   Laboratory Manual