Selective media can be used to  inhibit unwanted bacterial and thereby differentiate bacterial types. Media for isolating intestinal pathogens (MacConkey agar, Hektoen enteric (HE) agar) contain bile salts as a selective agent. Other agents that have selective properties are dyes, such as methylene blue and crysal violet and antimicrobila drugs. 

Selective media should be contrasted with differentail media which do not inhibit the growth of any particular microorganisms but are designed to siplay visible differences in how they grow. Differentiation shows up as variations in colony size or color (e.g., formations of gas bubbles and precipitates or media color changes). A single medium can be both selective and differential. MacConkey agar, for example is both slective and differentail due to its ability to suppress the growth of some organisms while producing a visual distinction among the ones that do grow. 

Selective Media for Isolation of Gram-negative Rods

The following types of media are useful for isolation and differentiation of Gram-negative organisms such as members of the family Enterobacteriaceae (gut bacteria).

MacConkey Agar: contains lactose, bile salts, neutral red and crystal violet. Bile salts and crystal violet inhibit growth of Gram-positve bacteria. Neutral red dye is a pH indicator that is colorless above a pH of 6.8 and red at a pH less than 6.5. Acid accumulating from lactose fermentation turns the dye red. Lactose fermenters turn a shade of red on MacConkey Agar, whereas lactose nonfermenters retain their normal color or the color of the medium. 

(1) Pour or no growth: organism is inhibited by crystal violet and/or bile so is Gram-positive.

(2) Good growth: organism is probably Gram-negative.

(3) Pink to red growth with or without bile precipitate: organism produces acid from lactose fermentation and is probably coliform

(4) Growth is colorless: organism does not ferment lactose is probably noncoliform. 

Eosin Methylene Blue (EMB) Agar: is commonly used for the test for the presence of coliforms in environmental samples. It is a complex (chemically undefined), selective, and differential medium. It contains peptone, lactose, sucrose and the dyes eosin Y and methylene blue. The dyes inhibit the growth of Gram-positive organisms and they react with vigorous lactose fermenters and (in the acidic environment) turn the growth dark purple or black. This dark growth is typical of Escherichia coliand is usually accompanied by a green metallic sheen. Other less aggressive lactose fermenters such as Enterobacter or Klebsiella species produce colonies that can range from pink to dark purple on the medium. 

(1) Poor growth or no growth: Organism is inhibited by eosin and methylene blue so is Gram positive.

(2) Good growth: organism is not inhibited by eosin and methylene blue os is gram-negative.

(3) Growth is pink and mucoid: organism ferments lactose with little acid production and is possible coliform.

(4) Dark purple to black, with or without green metallic sheen: organism ferments lactose and/or sucrose with acid production and is probable coliform.

(5) Growth is olorless (no pink, purple, or metallic sheen): organism does nto ferment lactose or sucrose and is probably noncoliform. 

 Other Types of Differential Media

HE Agar: differentiates Salmonella and Shigella from each other and form other enterics based on their ability to overcome the inhibitory effects of bile, reduce sulfur to H2S and ferment lactose, sucrose or salicin.

The test is based on the ability to ferment lactose, sucrose, or salicin and to reduce sulfur to hydrogen sulfide gas (H2S). Sodium thiosulfate is included as the source of oxidized sulfur. Ferric ammonium citrate is included as a source of oxidized iron to react with any sulfur that becomes reduced (H2S) to form the black precipitate ferrous sulfide (FeS). Bile salts are included to prevent or inhibit growth of Gram positive organisms. The bile salts also have a moderate inhibitory effect on enterics, so relatively high concentrations of animal tissue and yeast extract are included to offset this situation. Bromthymol blue and acid fuchsin dyes are added to indicate pH changes. Differentation is possible as a reslt of the various colors produced in the colonies and in the agar. 

Enterics that produce acid from fermentaiton will produce yellow to salmmon pink colonies. Neither Salmonella nor Shigella species ferment any of the sugars but do break down the animal tissue which raises the pH and gives the colonies a blue-green color. Salmonella species also reduce sulfur to H2S, so the solonies formed also contain FeS which makes them partially or completely black. 

XLD Agar: favors growth of Salmonella, Shigella or Providencia based on its ability to overcome the inhibitory effects of desoxycholate and differentiates them based on their ability to reduce sulfur to H2S, decarboxylate the amino acid lysine and ferment xylose or lactose.

Thioglycollate Broth: is designed to promote growth of a wide variety of fastidious microorganisms. Sodium thioglycollate in the media consumes oxygen and permits the growth of anaerobes. Oxygen removed during autoclaving will diffuse back into the meidum as the tubes cool. This produces a gradient of concentration from full aerobic at the top to anaerobic at the bottom. Thus fresh media will appear clear to straw colored with a pink region at the top. 

Experiment: Label 4 tubes having thioglycollate broth medium as 1. E coli (this is a facultative anaerobe) , B. subtilis (stribe aerobe so should see growth at top but nored because uses all the O2, 3. S. Aurues (facultative anaerobe (will see growth everywhere) and 4. control (expect to be just like the fresh medial with pink at top). 

Protocol Strategies:

(1) look at streaked colonies.

(2) obtain 3 plaes, TP, EMB and MAC. Draw line down middle to separate the plate. Label one side “1” and the other “2”

(3) Using sterile techniques, touch one colony and single streak on side 1. Repeat for the other 2 plates.  Then choose a different colony and repeat on side 2.