BISC209: Selective,and/or Differential, and/or Enriched media

Selective / Differential / Enrichment media
Selective media helps select for growth of certain organisms in a mixed population by using a ingredient that inhibits the growth of other microorganisms, but not the desired species or group. Enrichment media selects for certain microorganisms by including a nutrient that the desired microorganism or group can use and its competitors can not. (Sometimes enrichment media also limits alternate sources of nutrition). Differential media does not select for any particular group by inhibiting or enhancing their growth over competitors, but it does show a visible difference between or among groups of microorganisms. Some media can be both selective and differential.

Selective for Gram positive Organisms

Phenylethyl Alcohol Agar (PEA) PEA selects for the growth of Gram positive organisms by inhibiting the growth of Gram negative bacilli. The phenylethyl alcohol interfers with DNA synthesis in Gram negative organisms. This medium is particularly useful at inhibiting the overgrowth of Gram negative Proteus species that tend to swarm (they are highly motile) and, thus, make isolation of Gram positive organisms difficult in a mixed population .
Recipe: 10g tryptose, 3g Beef extract, 5g Sodium Chloride, 2.5g Phenylethyl alcohol, 15g Agar to 1 liter distilled or deionized water pH 7.1-7.5.

Mannitol Salt Agar (also see Targeted Enrichment media list)

Postive control organism: Staphylococcus epidermidis

Selective for Gram negative Organisms

Eosin–Methylene Blue (EMB) Agar is a differential medium for the detection of Gram negative enteric bacteria. The medium contains peptone, lactose, sucrose, dipotassium phosphate, eosin and methylene blue dyes. Eosin and methylene blue act as indicators to differentiate between Gram negative organisms that ferment lactose and those that do not ferment lactose. Most bacteria that ferment lactose form colonies on EMB agar that are dark blue to black with a metallic sheen due to precipitation of the dyes by the acid by-products of fermentation. Colonies produced by lactose non-fermentors are not dark blue or black. The growth of Gram positive bacteria is generally inhibited on EMB agar.

Recipe:10g peptone, 10g Lactose, 2g dipotassium phosphate, 0.4g eosin Y, 0.065 g methylene blue 15 g Agar. final pH 6.9-7.3

Table 2. Colonial appearance on EMB Agar after 18-24 hours at 35°C. A differential medium can be used to differentiate Gram negative enteric organisms based on the colony color.

Organism	Colonial Appearance
Escherichia coli	purple with black center/ green metallic sheen
Klebsiella pneumoniae	dark centered colonies/ green metallic sheen
Enterobacter aeorogenes	pink colonies/ no metallic sheen
Proteus mirabilis	colorless colonies
Salmonella typhimurium	colorless colonies

MacConkey Agar is a differential and selective medium that is used for the separation of lactose fermenting Gram negative enteric bacteria from Gram negative bacteria that do not ferment lactose. It is routinely used for the isolation of the pathogenic bacterial genera, Salmonella or Shigella, from clinical specimens, water samples and contaminated food.
MacConkey agar contains lactose, peptone, bile salts, sodium chloride, neutral red and crystal violet dyes as well as agar and water. Bile salts and crystal violet inhibit the growth of Gram positive organisms. The differential action of MacConkey agar is based on whether or not the organism ferments lactose (Table A-1). Colonies of lactose fermenting bacteria absorb the neutral red dye and thus, appear red in color. Bile salts may also be precipitated around a lactose-fermenting colony as a red halo. Colonies of bacteria that do not ferment lactose appear colorless and translucent. This differentiation is most obvious when discrete, single colonies are present on the MacConkey plate.
Recipe:2% peptone, 1% lactose, 0.15% bile salts, 0.5% NaCl, 1.35% agar, 30 µg/ml neutral red, 1 µg/ml crystal violet

Table A-1. Colonial appearance on MacConkey Agar after18-24 hours at 35°C. The growth of colonies on the plate indicate that the organism is most likely Gram negative and the color of the colony indicates that the organism is able to ferment lactose and/or form bile salts.

Organism	Colonial Appearance
Escherichia coli	pink to red colonies with bile salt precipitate
Enterobacter aerogenes	pink to red colonies
Proteus vulgaris	colorless colonies
Salmonella enteritidis	colorless colonies
Shigella dysenteriae	colorless colonies

Other selective media

Salmonella-Shigella (SS) Agar is a selective medium used for the detection and isolation of Salmonella and/or Shigella bacterial strains from clinical specimens, water and contaminated food products. Salmonella and Shigella, as well as other organisms that do not ferment lactose, form colorless opaque, translucent or transparent colonies on SS agar. Most lactose fermenting bacteria are inhibited, and the few that can grow form red, mucoid colonies, some of which may have black centers.
Recipe: 0.5% beef extract, 0.5% peptone, 1% lactose, 0.85% bile salts, 0.85% sodium citrate, 0.85% sodium thiosulfate, 0.1% ferric citrate, 1.35% agar, 333 µg/ml brilliant green, 25 µg/ml neutral red

Table 3. Colonial appearance on SS Agar after18-24 hours at 35°C. The color of the colonies of these Gram negative enteric organisms helps select for Salmonella and /or Shigella species.

Organism	Colonial Appearance
Enterobacter aerogenes	creamy pink colonies
Proteus mirabilis	colorless colonies
Salmonella enteridtidis	colorless colonies
Shigella flexnerii	colorless colonies

Bismuth Sulfite Agar is a highly selective medium for the isolation of Salmonella thyphi and other Salmonella species from clinical specimens, food products, water and sewage. Bismuth sulfite and brilliant green inhibit the growth of gram positive bacteria and many non-salmonellal members of the coliform group. H²S production occurs due to the presence of sulfur in the medium. H²S causes the iron in the media to precipitate causing colonies to turn black often with a metallic sheen. Recipe: 0.5% beef extract, 1% peptone, 0.55 dextrose, 0.4% disodium phosphate, 0.03% ferrous sulfate, o.8% bismuth sulfite indicator, 2% agar, 25 µg/ml brilliant green

Table 4. Colonial appearance on Bismuth Sulfite Agar after 18-24 hours, 35°C. This selective medium uses colony color to help identify species of Salmonella and some other coliform bacteria.

Organism	Colonial Appearance
Escherichia coli	brown-green colonies, if present
Enterobacter aerogenes	brown-green colonies, if present
Salmonella typhi	black colonies with metallic sheen
Salmonella enteritidis	black colonies with metallic sheen
Salmonella paratyphi	green colonies
Salmonella typhimurium	green colonies
Proteus morganii	green colonies
Shigella flexnerii	brown colonies, if present

Reference: Dehydrated Culture Media and Reagents for Microbiology. DIFCO Laboratories, Detroit, MI. 1984.

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