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
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Chapter 4 Laboratory Manual