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Culture Media for the Growth of Bacteria

©2000 Kenneth Todar, University of Wisconins-Madison

Culture Media for the Growth of Bacteria

For any bacterium to be propagated for any purpose it is necessary to provide the appropriate biochemical and biophysical environment. The biochemical (nutritional) environment is made available as a culture medium, and depending upon the special needs of particular bacteria (as well as particular investigators) a large variety and types of culture media have been developed with different purposes and uses. Culture media are employed in the isolation and maintenance of pure cultures of bacteria and are also used for identification of bacteria according to their biochemical and physiological properties.

The manner in which bacteria are cultivated, and the purpose of culture media, vary widely. Liquid media are used for growth of pure batch cultures while solidified media are used widely for the isolation of pure cultures, for estimating viable bacterial populations, and a variety of other purposes. The usual gelling agent for solid or semisolid medium is agar, a hydrocolloid derived from red algae. Agar is used because of its unique physical properties (it melts at 100 degrees and remains liquid until cooled to 40 degrees, the temperature at which it gels) and because it cannot be metabolized by most bacteria. Hence as a medium component it is relatively inert; it simply holds (gels) nutrients that are in aquaeous solution.

Culture media may be classified into several categories depending on their composition or use. A chemically-defined (synthetic) medium (Table 4a and 4b) is one in which the exact chemical composition is known. A complex (undefined) medium (Table 5a and 5b) is one in which the exact chemical constitution of the medium is not known. Defined media are usually composed of pure biochemicals off the shelf; complex media usually contain complex materials of biological origin such as blood or milk or yeast extract or beef extract, the exact chemical composition of which is obviously undetermined. A defined medium is a minimal medium (Table4a) if it provides only the exact nutrients (including any growth factors) needed by the organism for growth. The use of defined minimal media requires the investigator to know the exact nutritional requirements of the organisms in question. Chemically-defined media are of value in studying the minimal nutritional requirements of microorganisms, for enrichment cultures, and for a wide variety of physiological studies. Complex media usually provide the full range of growth factors that may be required by an organism so they may be more handily used to cultivate unknown bacteria or bacteria whose nutritional requirement are complex (i.e., organisms that require a lot of growth factors).

Most pathogenic bacteria of animals, which have adapted themselves to growth in animal tissues, require complex media for their growth. Blood, serum and tissue extracts are frequently added to culture media for the cultivation of pathogens. Even so, for a few fastidious pathogens such as Treponema pallidum, the agent of syphilis, and Mycobacterium leprae, the cause of leprosy, artificial culture media and conditions have not been established. This fact thwarts the the ability to do basic research on these pathogens and the diseases that they cause.

Other concepts employed in the construction of culture media are the principles of selection and enrichment. A selective medium is one which has a component(s) added to it which will inhibit or prevent the growth of certain types or species of bacteria and/or promote the growth of desired species. One can also adjust the physical conditions of a culture medium, such as pH and temperature, to render it selective for organisms that are able to grow under these certain conditions.

A culture medium may also be a differential medium if it allows the investigator to distinguish between different types of bacteria based on some observable trait in their pattern of growth on the medium. Thus a selective, differential medium for the isolation of Staphylococcus aureus, the most common bacterial pathogen of humans, contains a very high concentration of salt (which the staph will tolerate) that inhibits most other bacteria, mannitol as a source of fermentable sugar, and a pH indicator dye. From clinical specimens, only staph will grow. S. aureus is differentiated from S. epidermidis (a nonpathogenic component of the normal flora) on the basis of its ability to ferment mannitol. Mannitol-fermenting colonies (S. aureus)produce acid which reacts with the indicator dye forming a colored halo around the colonies; mannitol non-fermenters (S. epidermidis) use other non-fermentative substrates in the medium for growth and do not form a halo around their colonies.

An enrichment medium employs a slightly different twist. An enrichment medium (Table 5a and 5b) contains some component that permits the growth of specific types or species of bacteria, usually because they alone can utilize the component from their environment. However, an enrichment medium may have selective features. An enrichment medium for nonsymbiotic nitrogen-fixing bacteria omits a source of added nitrogen to the medium. The medium is inoculated with a potential source of these bacteria (e.g. a soil sample) and incubated in the atmosphere wherein the only source of nitrogen available is N2. A selective enrichment medium (Table 5b) for growth of the extreme halophile (Halococcus) contains nearly 25 percent salt [NaCl], which is required by the extreme halophile and which inhibits the growth of all other procaryotes.

Table 4a. Minimal medium for the growth of Bacillus megaterium. An example of a chemically-defined medium for growth of a heterotrophic bacterium.
ComponentAmountFunction of component
sucrose10.0 gC and energy source
K2HPO42.5 gpH buffer; P and K source
KH2PO42.5 gpH buffer; P and K source
(NH4)2HPO41.0 gpH buffer; N and P source
MgSO4 7H2O0.20 gS and Mg++ source
FeSO4 7H2O0.01 gFe++ source
MnSO4 H2O0.007 gMn++ Source
water985 ml
pH 7.0

Table 4b. Defined medium (also an enrichment medium)for the growth of Thiobacillus thiooxidans, a lithoautotrophic bacterium.
ComponentAmountFunction of component
NH4Cl0.52 gN source
KH2PO40.28 gP and K source
MgSO4 7H2O0.25 gS and Mg++ source
CaCl2 2H2O0.07 gCa++ source
Elemental Sulfur1.56 gEnergy source
C025%*C source
water1000 ml
pH 3.0

* Aerate medium intermittently with air containing 5% CO2.
Table 5a. Complex medium for the growth of fastidious bacteria.
ComponentAmountFunction of component
Beef extract1.5 gSource of vitamins and other growth factors
Yeast extract3.0 gSource of vitamins and other growth factors
Peptone6.0 gSource of amino acids, N, S, and P
Glucose1.0 gC and energy source
Agar15.0 gInert solidifying agent
water1000 ml
pH 6.6

Table 5b. Selective enrichment medium for growth of extreme halophiles.
ComponentAmountFunction of component
Casamino acids7.5 gSource of amino acids, N, S and P
Yeast extract10.0 gSource of growth factors
Trisodium citrate3.0 gC and energy source
KCl2.0 gK+ source
MgSO4 7 H2O20.0 gS and Mg++ source
FeCl20.023 gFe++ source
NaCl250 gNa+ source for halophiles and inhibitory to nonhalophiles
water1000 ml
pH 7.4

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