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, varies 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^oC and remains liquid until cooled to 40^oC, 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.

Types of Culture Media

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 (Table 4a) 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, known or unknown). Complex media are usually used for cultivation of bacterial pathogens and other fastidious bacteria.

Figure 2. Legionella pneumophila. Direct fluorescent antibody (DFA) stain of a patient respiratory tract specimen. © Gloria J. Delisle and Lewis Tomalty. Queens University, Kingston, Ontario, Canada. Licensed for use by ASM Microbe Library http://www.microbelibrary.org. In spite of its natural occurrence in water cooling towers and air conditioners, Legionella is a fastidious bacterium grown in the laboratory, which led to the long lag in identification of the first outbreak of Legionnaire's disease in Philadelphia in 1977. Had fluorescent antibody to the bacterium been available at that time, diagnosis could have been made as quickly as the time to prepare and view this slide.

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 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.

Component	Amount	Function of component
sucrose	10.0 g	C and energy source
K2HPO4	2.5 g	pH buffer; P and K source
KH2PO4	2.5 g	pH buffer; P and K source
(NH4)2HPO4	1.0 g	pH buffer; N and P source
MgSO4 7H2O	0.20 g	S and Mg++ source
FeSO4 7H2O	0.01 g	Fe++ source
MnSO4 7H2O	0.007 g	Mn++ Source
water	985 ml
pH 7.0