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Microbial Growth and Culturing

Culturing Techniques

Requirements for Growth

The same physical and chemical requirements for microbial growth in the environment must be accurately mimicked in the lab for successful culturing.

The same conditions required for microbial growth in their normal habitat are required when growing microorganisms (microbes) in the lab. Culturing is the process of growing and maintaining microbes in a laboratory setting. As some environmental conditions are impossible to recreate in the laboratory, not all microbes are capable of being cultured. Most microbes need to grow at between 20°C and 45°C, so an incubator—a piece of equipment that regulates conditions to support the growth of cells—is used to keep the bacteria at the proper temperature. Some incubators can also maintain specific atmospheric conditions that can support specific microbial survival. Anaerobic chambers are used to grow obligate anaerobes. Considerations must be made for microbes that come from extreme environments, such as psychrophiles or thermophiles, which grow in extremely cold or hot temperatures.

Microbes are grown in a medium, a substance containing nutrients that support the growth of cells. Media used for bacterial growth contain salts, nutrients, and sugars required for bacterial growth, as well as buffers that regulate the pH of the media. Different bacteria may require additional nutrients or a higher salt concentration; this can be adjusted in media formulations for particular microbes being grown. Media can be liquid or solid depending on the growth requirements of the microbe.

Physical Forms of Growth Media

Media used to grow microbes can be solid or liquid and are designed to provide the appropriate nutrients and moisture needed.

Microbes can be grown in liquid or solid media. Regardless of its physical form, the medium is designed to provide an environment that supports microbial growth. This environment includes appropriate nutrients and supporting conditions such as moisture and a surface for growth.

Liquid media are typically called broths or infusions. They are liquid solutions containing nutrients. Thioglycolate broth is a type of medium designed to test whether a bacterium is aerobic or anaerobic. Both aerobic and anaerobic microbes are able to grow in thioglycolate media, although they will localize differently within the tube containing the media. Aerobic microbes, which require oxygen for survival, will grow at the top of a tube of liquid media, while anaerobic microbes, which must avoid oxygen, will settle and grow at the bottom of the tube.

Solid media contain agar, a mixture of two sugars that produces a gelatin-like surface that provides a base for a variety of different growth media. Solid media are typically heated in order to melt the agar and are poured into a petri dish and allowed to cool and solidify before microbes are plated. Once microbes have been plated on agar in a petri dish, the petri dish is loosely covered and flipped upside down. Turning the petri dish upside down prevents the condensation that forms inside the petri dish from dripping onto the growing microbes and affecting their growth. The petri dishes are then kept in an incubator or other location that maintains optimal environmental conditions for their growth. Most bacteria and fungi can be grown on solid media, though algae culturing usually requires liquid media.

An intermediate form of growth media is semisolid media, which contain a lower percentage of agar than solid media. This allows for bacteria to penetrate into the agar and can be used to observe the ability of bacteria to move. It is also the best culture condition for microaerophiles, as they can grow into the agar at this consistency instead of only on the surface of the agar, allowing these bacteria to regulate the amount of oxygen they encounter.

Types of Growth Media

Microbes can be grown in liquid, semisolid, or solid media, depending on the traits of the microbe.

Types of Growth Media

Selective media are used to enhance growth of certain microbes, and differential media provide a visual cue to separate microbial types.

Some media are designed to support the growth of specific microbes while preventing others from growing, and these are referred to as selective media. By understanding the composition of selective media, scientists can understand certain information about the properties of the microbes that do or do not grow on them. Selective media are a useful tool in diagnostic medicine to identify microbes that may be causing a disease. An example of selective media is mannitol salt agar. Mannitol salt agar has a high salt content, which prevents most bacteria from growing; only Staphylococci, such as Staphylococcus aureus, can grow in high salt. There are media available to select for non-bacterial species also. For example, Sabouaud’s dextrose agar contains the sugar dextrose supplemented with short amino acid chains and is used to specifically grow certain species of fungi.

Another category of media that can differentiate between microbes is called differential media. Differential media are used to distinguish between closely related bacteria through visual observation of changes to the media used to culture the bacteria. An example of this is blood agar, which contains 5% sheep's blood and allows for the distinction of bacteria that have different capacities to kill red blood cells. Blood agar permits the growth of all bacteria because it does not contain any selective agent, such as an antibiotic. Instead, different kinds of bacteria can be identified visually by how much the red blood cells in the agar have been lysed, or disintegrated, around each bacterial colony.

Media can be both selective and differential. Mannitol salt agar is selective for staphylococci due to the high salt content. It is also differential due to the presence of mannitol, a type of sugar, and phenol red, a pH indicator. If the bacteria can ferment the mannitol, an acid is produced that changes the color of phenol red from red to yellow. Only Staphylococcus aureus ferments mannitol on this media, making it an important diagnostic tool in clinical settings.