Microorganisms need nutrients, a source of energy, and certain environmental conditions in order to grow and reproduce. In the natural environment, microbes have adapted to the habitats most suitable for their needs. In the laboratory, however, these requirements must be met by culture media. A culture medium is 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.
Selective and differential media are media based on either complex or defined media 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 (artidione), which inhibits all eukaryotic growth and is typically used to prevent fungal growth in mixed cultures.
Complex media are rich in nutrients that contain water-soluble extracts of plant or animal tissue (e.g., enzymatically digested animal proteins such as peptone and tryptone). Usually, some sugar, often glucose, is added to serve as the main carbon and energy source. The combination of extracts and sugar creates a medium that 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 and dissolved in double distilled water (i.e., the exact chemical composition of the medium is known). Typically, they contain a simple sugar as the source of carbon and energy, a source of inorganic nitrogen, various mineral salts, and, if necessary, growth factors (purified amino acids, vitamins, purines, and pyrimidines).
The mixture of necessary nutrients can be used as a liquid medium, or a solidifying agent can be added. "Agar agar" is a natural polysaccharide produced by marine algae and is the most commonly used solidifying agent added to media (end concentration usually 1.5 % w/v). If hydrolysis of the agar is suspected, a silica gel is used as a replacement solidifying agent.
Complex media contain often protein hydrolysate which are are excellent natural sources of amino acids, peptides and proteins in growth media. It is the most important source for nitrogenous nutrients. They are most often obtained by enzymatic digestion or acid hydrolysis of natural products, such as animal tissues, milk, plants or microbial cultures. The number of available protein hydrolsate, also called peptones, is enormous and can promote and sustain the growth of most common organisms. For the enzymatic digestion often papain, pepsin, trypsin or a mixture of enzymes of the pancreatic juice are taken. Below is a list of often used expressions and the definitions.
Before inoculation, it is important to maintain the sterile condition of the microbiological media and all materials it encounters. During any subsequent handling of the bacterial cultures, unwanted or contaminant organisms must be excluded by employing aseptic techniques.
Sterilization implies the destruction of all microorganisms including spores, this is accomplished using heat, chemicals, radiation, and filtration.
Heat sterilization causes denatures and coagulates vital proteins. It is simple to use, effective, easy to control, and cost-effective. There are various forms of heat sterilization.
Chemical sterilization is usually employed for delicate equipment such as optical instruments and electrical devices, which would otherwise be damaged by heat. Due to the toxicity of the chemicals used, this is not the most popular form of sterilization. Chemicals employed include gaseous ethylene oxide, which alkylates amino, sulfhydryl, carboxyl, and hydroxyl groups of microbial cell compounds; formaldehyde, used as a fumigant; and hydrogen peroxide vapor used in aseptic packaging.
Radiation is applied for heat-sensitive materials and for environmental samples such as soil and sediment where structural changes caused by heat need to be avoided. Two forms of radiation are used:
Filtration sterilization operates through the exclusion rather than the destruction of microorganisms. It is safe for the user and is employed for sensitive liquids and gases. Three types of filters are currently in use:
Table of the most commonly used media supplements, methods of sterilization, and solubility.
All stock solutions of amino acids can be autoclaved at 120 °C for 20 mins.
When undertaking research where medium composition plays an important role, (e.g. toxicity studies), care must be taken to observe the various interactions which can take place between media components.
* Can be avoided by addition of a HEPES buffer to reduce phosphate.
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