Updated 28 December 04
Please review the background information culturing microorganisms in Chapter 3 of your text.
Any medium for the cultivation of bacteria must provide certain basic nutritional requirements, which include (1) a carbon source that may also serve as an energy source; (2) water; (3) a nitrogen source; (4) a phosphate source; and (5) various mineral nutrients, such as iron and magnesium. Some bacteria are capable of growth on a medium consisting of a single carbon source, such as the carbohydrate glucose; a simple nitrogen source, such as ammonium salts; and inorganic salts, such as phosphates. This kind of medium is termed defined or synthetic because its exact chemical composition is known. For routine laboratory work, however, complex media are employed where the basic nutrients are provided by complex nutrients, such as plant and animal extracts in which the exact composition is not known. For example, beef extract and peptones (hydrolyzed protein) are the basic ingredients of nutrient agar. These materials supply a variety of carbon sources, nitrogen compounds in the form of amino acids, and a mixture of cofactors, such as vitamins. This basic medium can be further enriched to support the growth of more fastidious types of bacteria by the addition of carbohydrate sources, yeast extract, and materials such as plasma or blood, which provide a variety of complex nutritional factors. A broth medium is one in which the components are simply dissolved in water. The addition of agar-agar (a complex carbohydrate extracted from seaweed) results in a solid medium. Agar is an ideal solidifying agent for microbiological media because of its melting properties and because it has no nutritive value for the vast majority of bacteria. Solid agar melts at about100°C; liquid agar solidifies at about 42°C.
Because microbes are ubiquitously distributed in the environment, during the preparation of any culture medium, bacteria are introduced from many sources such as glassware, dry medium components, air, and so on. These bacteria would eventually grow and flourish if the medium were not sterilized, that is, if these unwanted microbes were not destroyed.
Sterilization procedures eliminate all viable microorganisms from a specified region. Culture dishes, test tubes, flasks, pipettes, transfer loops, and media must be free of viable microorganisms before they can be used for establishing pure cultures of microorganisms. The culture vessels must be sealed or capped with sterile plugs to prevent contamination. There are various ways of sterilizing the liquids, containers, and instruments used in pure culture procedures; these include exposure to elevated temperatures or radiation levels to kill microorganisms and filtration to remove microorganisms from solution.
Media preparation for the microbiology laboratory involves the use of an autoclave for sterilization, which permits exposure to high temperatures for a specified period of time. Generally, a temperature of 121°C (achieved by using steam at 15 lb/sq in) for 15 minutes is used to heat-sterilize bacteriological media. Much of the time spent in preparation for the bacteriology laboratory involves preparing the media for growing bacteria; that is, mixing and sterilizing the growth media in suitable sterile culture vessels.
In this exercise you will prepare a complex medium. You will mix the proper constituents to support the growth of microorganisms and sterilize the medium. You will also begin a microbiological investigation of an "environment."
We will make some broth and some agar media according to the directions below.
Note: Cultures no longer needed are placed in a "Kill" container. Please keep glass tubes and dishes separate from plasticware containing old cultures. We reuse the glass but not the plastic.