Use of Selective and Differential Media

There are many different types of media that are used in a microbiology laboratory. Generally speaking there are three types of media used; selective, differential, and supportive or nutritive.

Selective media are manufactured to support the growth of one type of microorganism while inhibiting the growth of another, in other words, it selectively grows one type of microorganism. It is not uncommon or this media to contain antimicrobials, dyes such as crystal violet, or even alcohol. Some of the more routine selective media types are EMB agar, mannitol salt agar, MAC, and a PEA agar.

Differential media is used to distinguish microorganisms from one another based on growth characteristics that are evident when growth is obtained. There are visible differences between microorganisms when growth is achieved. One fo the more common differential media used is the MacConkey agar. This differentiates between lactose fermenters and non-lactose fermenters.

Supportive media is used to support the growth of a wide range of microorganisms. They are typically non-selective because they want to achieve growth of a wide array of microorganisms. Some more common ones include the sheeps blood agar, as well as the chocolate agar. It has the added X and V factors to support the growth of Haemophilus influenzae.

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Triple Sugar Iron Agar (TSI)

The TSI is a multiple test medium. Its a slanted medium with a deep butt that is used to further investigate Gram-negative microorganisms. It differentiates the microbes by their ability to ferment glucose, lactose and/or sucrose with or without the production of gas and production of hydrogen sulfide.

The TSI medium contains three carbohydrates; 1.0% lactose, 1.0% sucrose and 0.1% glucose. Phenol red is added as a pH indicator. Ferric ammonium citrate and sodium thiosulfate are added as indicators for the production hydrogen sulfide.

There are multiple reactions that can be observed;

A reaction of alkaline/no change is denoted K/NC and it means that the organism can only catabolize peptones aerobically, hence only the slant exhibited a color change, usually a red/orange color. This means that no carbohydrates were utilized.

When the slant is alkaline after 18-24 hours of incubation it means that there was rapid depletion of glucose and there is a subsequent reliance on peptides for nutrients. This occurs because the concentration glucose is so low and therefore it is consumed quickly. Catabolism of peptones results in the release of ammonia (NH3) which yields an alkaline pH. The butt of the medium remains acid because the degradation of peptones occurs aerobically (i.e. in the slant).

Some organisms have the ability to ferment lactose and/or sucrose with glucose for their nutrients. This results in an acid slant and acid butt reaction denoted A/A and a color change of yellow/yellow will appear. Because the concentrations of lactose and sucrose are 10x the amount of glucose, therefore a large amount of acid is produced.

A TSI medium also is used to determine whether or not a microorganism can produce carbon dioxide and hydrogen gases from the fermentation of the carbohydrates present. Gas production is seen when a bubble forms, which splits the medium. A clear disc shaped area is seen within the medium.

Ferric ammonium citrate and sodium thiosulfate are both indicators that are added to view the presence of H2S hydrogen sulfide. A microorganism in an acidic environment acts on the sodium thiosulfate to produce H2S gas. H2S reacts with ferric ions to produce ions that produce ferrous sulfide which is an insoluble black precipitate.

It should be noted that the black precipitate of ferrous sulfide that indicates H2S production may mask an acidic condition in the butt of the tube. Since H2S is only produced under acidic conditions, when the butt of the tube is black, an acid butt exists as well even without the presence of the yellow color.

TSI Results K/K K/A A/A +g A/A +g,+H2S A/K +g, +H2S

-Caleb