Culturing: Media Selection and Inoculation Technique

Exercise 5

Introduction:

Many different species of bacteria look similar under the microscope and also have the same staining results (ex. Gram stain). To be able to differentiate between the different species, one can look at the metabolic differences (fermentation), as well as the environmental condition differences (temperature, pH, oxygen requirements). Being able to manipulate these conditions in a controlled environment can help to correctly identify the exact bacteria. Different media can be used to culture and identify bacteria.

Some bacteria require specific nutrients and conditions, while others can make due with whatever the environment has available. Some bacteria lack the enzymes to break down a complex carbon source, while others can break it down easily. Some bacteria require oxygen while others can not tolerate it. Some bacteria can live in harsh environments such as salt, while others will not grow in the harsh environment.

There are four different media types; complex, defined, selective, and differential.

Complex media is made of partially digested chemical compounds from organic substances such as yeast, meats, dairy products, tissues, and vegetable materials. The amount of each compound can not be known due to the differences between the organic compounds and the amount of digestion that has occurred. The amounts are not of value for the media. This type of media is important when culturing a mixed or diverse sample. Trypticase Soy Agar (TSA) is a complex media.

Defined media (synthetic) is a media that contains a known amount of each chemical it contains. This media provides (special) conditions for culturing fastidious organisms by providing their specific growth factors.

Selective media is used to isolate specific groups of bacteria. Usually the media is based on a known environmental condition range that they tolerate and most groups can not. An example is Manitol Salts Agar (MSA). This media contains salts that most organisms can’t tolerate due to their osmolity ranges. Microbes that normally exist under these conditions are able to grow. MSA is usually used for Staphylococcal species. Differential media provides chemical compounds that bacteria metabolize differently. The difference is observed by a colony or media color change. This media is often used to differentiate between groups of organisms with the same morphology and biochemical resemblances. Eosin Methylene Blue (EMB) is an example that can be used to differentiate between various Gram (-) rods of the intestinal tract. Escherichia coli and other enteric bacteria are able to ferment. Lactose fermentation results in a colony color change ranging from pink to purple.

Determination of oxygen requirements is a test to determine whether a microbe is an obligate aerobe, anaerobe, or facultative. The culture is put in