PREPARATION OF A CULTURE MEDIUM

The study of microorganisms in the laboratory generally requires growing organisms on a nutrient medium. Culture
media must meet the nutritional requirements of the organism you wish to culture. Minimal nutritional considerations
include the macronutrients; carbon, nitrogen, and phosphorus, and various other minerals, such as iron and
magnesium, in lesser amounts. In addition, most microbiological media incorporate buffers, weak acids or bases that
resist changes in pH. Of course, all living organisms require water.

Many different types and formulations of media are used for growing bacteria and fungi in pure culture. There are also
media specific for the selection and differentiation of a vast variety organisms. For our routine laboratory work, a
complex medium (exact composition is not known) prepared from plant, fungal and/or animal extracts is appropriate to
meet the nutritional needs of most of the organisms we study. Many different types of media are available commercially
as pre-formulated dry powders to be reconstituted in the laboratory. Appendix C at the end of this manual describes all
media used in General Microbiology.

A broth medium is one in which the ingredients are simply dissolved in water; a solid medium is usually prepared with
agar, a seaweed polysaccharide that acts as a gelling agent. Agar is a superior gelling agent in microbiological media
because of its unique physical properties. Once gelled, agar remains a solid up to 90°C (gelatin melts at 35°C). Solid
agar does not melt until it is heated to 100°C; liquid agar does not solidify until it is cooled to about 42°C allowing liquid
agar to be poured into Petri plates at a temperature relatively easy to handle. For most microorganisms, agar is an
ingredient which they cannot use as a
nutrient.

MATERIALS
Dehydrated trypticase soy broth (BBL)
Dehydrated agar
Graduated cylinder (100ml)
Balance and weighing paper
Flask (250 ml)
Pipette and tips
Test tubes & caps
Autoclave
PROCEDURE
1. Work by bench-side. Each group will prepare 100 ml of the medium. This will be enough for making ~14 slants (7 ml
per slant).
2. Measure 100 ml distilled water. Weigh out the dehydrated medium according to directions on the label but
recalculated for the volume you are preparing. Pour the weighed media into the 250 mL flask. In addition to the medium,
add 1.5% w/v dehydrated agar. Pour the water in the cylinder into the flask, Try to wash down the neck of the flask
where powder may be sticking. Swirl the flask to suspend the ingredients; the TSB will dissolve rather quickly; but the
agar will not dissolve until the medium is boiled in the microwave.
3. Heat the medium in a microwave oven - boil to dissolve the agar; use occasional light agitation. Watch it carefully.
Take great care as the agar can boil over. After the agar has completely dissolved, cool the medium in a water bath to
about 60°C.
4. To prepare agar slants, dispense about 7 ml of the melted cooled agar into clean tubes, using a Brinkman or
Eppendorf pipette . Cap the filled tubes with plastic caps.
5. Autoclave the medium 121°C, 15 psi for 15 min. (Autoclaving and slanting will be done for you after class.)