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

Selective / Differential / Enriched media

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 organisms. 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 and 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)

Selective for Gram negative Organisms

MacConkey Agar (2% peptone, 1% lactose, 0.15% bile salts, 0.5% NaCl, 1.35% agar, 30 µg/ml neutral red, 1 µg/ml crystal violet) 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. Continue to streak plates until you clearly have 2 different lactose fermentation cultures.

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

Eosin–Methylene Blue (EMB) Agar (1% peptone, 0.2% dipotassium phosphate, 1.5% agar, 400 µg/ml eosin Y, 65 µg/ml methlene blue) 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 as well as agar and water. 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 negative bacteria are not dark blue or black. The growth of gram positive bacteria is generally inhibited on EMB agar.

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

Esherichia coli purple with black center/ green metallic sheen

Klebsiella pneumoniae dark centered colonies/ green metallic sheen

Enterobacter aerogenes pink colonies/ no metallic sheen

Proteus mirabilis colorless colonies

Salmonella typhimurium colorless colonies

Other selective media

Salmonella-Shigella (SS) Agar (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) 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.

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 spp.

Organism Colonial Appearance

Enterobacter aerogenes creamy pink colonies

Proteus mirabilis colorless colonies

Salmonella enteritidis colorless colonies

Shigella flexnerii colorless colonies

Bismuth Sulfite Agar (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) is a very 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.

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.

Sugar Fermentation Media

Carbohydrate fermentation tubes general recipe: Purple Broth base (1% peptone, 0.1% beef extract, 0.5% NaCl, 20 µg/ml bromcresol purple) plus 1% of the desired carbohydrate.

commonly used Fermentation MediaInoculate one tube for each organism: adonitol, dulcitol, glucose, inositol, lactose, mannitol, and sucrose. Incubate for 24 to 72 hours. The fermentation of carbohydrates by gram negative organisms gives rise to the production of pyruvic acid causing a change in color of the media from purple to yellow due to the reduction in pH.

A color change to yellow in the fermentation media indicates a positive test (+).

In positive tests only, gas production is evidenced by gas bubbles trapped in the smaller inner tube (Durham tube). If gas is present the result is recorded as +/gas.

A negative test (-) is evidenced by a purple to gray color and obvious growth* in the tube. Example, the lactose fermentation test contains 1% lactose dissolved in the Purple Broth base.

Phenol Red Agar with sugars mannitol or sucrose: 1% mannitol(or sucrose) is added to phenol red agar base (1% peptone, 0.1% beef extract, 0.5% NaCl, 1.5% agar and 25µl/ml phenol red) to test for the fermentation of mannitol by the bacteria. A positive test is a yellow color due to the formation of acidic products of mannitol fermentation. The indicator, phenol red, turns yellow when the pH falls below 6.8. Staphylococcus aureus usually ferments mannitol within 24 to 48 hours. An inoculum should be spread on the slant and then stabbed to the bottom of the butt, a region that remains fairly anaerobic. The inoculated slants should then be incubated at 37°C for 24-48 hours. Yellow color indicates a positive (+) test.