BIO577: Media Preparation

During the quarter we will be utilizing bacterial hosts (Escherichia coli [E. coli] or
Agrobacterium tumefaciens) for replicating recombinant DNA plasmids we have
engineered. Bacteria need nutrients, a source of energy and certain environmental
conditions in order to grow and reproduce. In the environment, microbes have adapted to
the habitats most suitable for their needs, in the laboratory, however, these requirements
must be met by a culture medium. This is basically an aqueous solution to which all the
necessary nutrients have been added. Depending on the type and combination of
nutrients, different categories of media can be made.
Categories
Complex media are rich in nutrients, they contain water soluble extracts of plant or
animal tissue (e.g., enzymatically digested animal proteins such as peptone and
tryptone). Usually a sugar, often glucose is added to serve as the main carbon and energy
source. The combination of extracts and sugar creates a medium which is rich in
minerals and organic nutrients, but since the exact composition is unknown, the medium
is called complex.
Defined media are media composed of pure ingredients in carefully measured
concentrations dissolved in double distilled water i.e., the exact chemical composition of
the medium is known. Typically, they contain a simple sugar as the carbon and energy
source, an inorganic nitrogen source, various mineral salts and if necessary growth
factors (purified amino acids, vitamins, purines and pyrimidines).
Selective/differential media are media based on either of the two categories above
supplemented with growth-promoting or growth-inhibiting additives. The additives may
be species- or organism-selective (e.g., a specific substrate, or an inhibitor such as
cyclohexamide which inhibits all eukaryotic growth and is typically used to prevent
fungal growth in mixed cultures).

*We will be preparing four different types of media in todays lab. Each group will make
one of the four.

Liquid media
Liquid cultures of E. coli can generally be grown in LB (Luria-Bertani) medium. There
are a number of different LB broths, with different compositions, which are commonly
used. Different formulations contain different concentrations of NaCl and give rise to
varied yields of plasmid DNA. We will be using the recipe at the end of this handout to
obtain highest yields of plasmid DNA.

Preparation: To prepare 1 liter of LB medium, add 10 g NaCl, 10 g tryptone and 5 g

yeast extract to 950 ml deionized water and stir until dissolved. Adjust the pH to 7.0 with
5 M NaOH. Adjust the volume of the solution to 1 liter with deionized water. Sterilize by
autoclaving for 20 minutes.
Solid media
E. coli strains can generally be streaked and stored on LB plates containing 1.5% agar
and the appropriate antibiotic(s).

Preparation: Prepare LB medium according to the instructions for liquid media. Just
before autoclaving, add 15 grams agar per liter and mix. After autoclaving, swirl the
medium gently to distribute the melted agar evenly throughout the solution. Take care
that the hot liquid does not boil over when swirled. Pour plates in a laminar-flow hood or,
if no hood is available, on a cleaned bench surface next to a Bunsen burner. Use 3035
ml medium per standard 90-mm petri dish (~30 plates per liter of medium).

Dry plates either directly after solidification by removing the lids and standing the plates
in a laminar-flow hood for 1 hour. Alternatively, if you do not have access to a hood,
plates can be dried with the covers slightly open in a 37C incubator for 30 min, or left
upside down with lids on at room temperature for 23 days.
Store plates inverted at 4C.

Antibiotics
Bacterial strains carrying plasmids or genes with antibiotic selection markers should
always be cultured in liquid or on solid medium containing the selective agent. Lack of
antibiotic selection can lead to loss of the plasmid carrying the genetic marker and
potentially to selection of faster-growing mutants. Antibiotics and nutrients such as
amino acids are inactivated by the high temperatures of an autoclave. They should be
sterilized by filtration through a filter unit with a pore size of 0.2 m, and added to the
cooled, autoclaved medium from properly stored stock solutions.

Preparation: Prepare stock solutions of antibiotics separately from batches of liquid or

solid media, sterilize by filtration, aliquot, and store in the dark at 20C. Recommended
stock and working concentrations for commonly used antibiotics are shown in the table
below. Prepare solid media as outlined above. Cool autoclaved agar medium to below
50C (when you can hold it comfortably) before adding heat-sensitive antibiotics and
nutrients.

Table 1. Concentrations of commonly used antibiotics

Antibiotic Stock solutions Working concentration

Concentration Storage (dilution)

Ampicillin 50 mg/ml in water 20C 100 g/ml (1/500)

(sodium salt)
Chloramphenicol 34 mg/ml in ethanol 20C 170 g/ml (1/200)
Kanamycin 10 mg/ml in water 20C 50 g/ml (1/200)
Streptomycin 10 mg/ml in water 20C 50 g/ml (1/200)
Tetracycline HCl 5 mg/ml in ethanol 20C 50 g/ml (1/100)
Recipes for media:

Liquid LB solid LB +Kan a solid LB +Chlb solid YEP +Chlb

10 g tryptone 10 g tryptone 10 g tryptone 20 g peptone
5 g yeast extract 5 g yeast extract 5 g yeast extract 10 g yeast extract
10 g NaCl 10 g NaCl 10 g NaCl
dH2O to 950 ml dH2O to 950 ml dH2O to 950 ml
15 g agar 15 g agar 15 g agar

All recipes are for one liter total volume.

Liquid LB must be aliquoted out into capped tubes (5 ml for each tube). All solutions
must be autoclaved for 20 minutes. Once the solid media has cooled sufficiently, the
appropriate antibiotic must be added.
a
Kan is the abbreviation for kanamycin. The final concentration of kanamycin should be
50 g/ml. The main stock solution is at a concentration of 50 mg/ml, therefore we will
add 1 ml of the main stock to our cooled (but not solidified!) media.

bChl is the abbreviation for chloramphenicol. The final concentration of chloramphenicol

should be 30 g/ml. The main stock solution is at a concentration of 34 mg/ml, therefore
we will add 882 l of the main stock to our cooled media.