Isolation Techniques and Selective Media-- Part 3

Medium that detect fecal contamination in water and dairy products. It is also used to isolate and identify the presence of enteric lactose fermenters (coliforms), its original use was to isolate and identify Salmonella typhi, a lactose nonfermenter (noncoliform).

• Eosin Methylene Blue Agar

• Endo Agar

• Hektoen Enteric Agar

• MacConkey Agar

Streaked Endo Agar illustrating

a metallic sheen resulting from large amounts of acid

produced during lactose fermentation.

• Escherichia coli

• Clostridium coli

• Balantidium coli

• Entamoeba coli

Endo Agar

• contains color indicators sodium sulfite and basic fuchsin (which also double as Gram-positive inhibitors).

• Lactose is included as a fermentable carbohydrate.

• Escherichia coli and Klebsiella pneumoniae produce large

  amounts of acid, which gives the colonies

  a metallic sheen

• Lactose nonfermenters produce colorless to

  slightly pink growth

Eosin Methylene Blue Agar

• Some lactose fermenters, such as Escherichia coli

  and Klebsiella pneumoniae produce large

  amounts of acid, which gives the colonies

  a metallic sheen

• undefined medium designed to isolate Salmonella and Shigella species from other enterics based on the

  ability to ferment lactose, sucrose, or salicin, and to reduce sulfur to hydrogen sulfide gas (H2S).

• Both

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Eosin Methylene Blue Agar

• Agar is used for isolation of fecal coliforms.

• can be streaked for isolation or

  used in the Membrane Filter Technique

• contains peptone, lactose, sucrose,

  
  

  and the dyes eosin Y and methylene blue.

• The dyes inhibit growth of Gram-positive organ-

  isms and, under acidic conditions, also produce a dark purple

  
  

  complex usually accompanied by a green metallic sheen.

• It is has fixatives and dyes that promote the growth of E.coli

EOSIN METHYLENE BLUE AGAR : (clockwise from top) Escherichia coli, Enterobacter aerogenes, Salmonella typhimurium, and Enterococcus faecalis

• green

  metallic sheen of E. coli

• pink coloration with slight darkening shows E. aerogenes.

• E. coli produces colonies with dark

  centers.

• Some E. coli strains appear black

  without the green sheen

• pink coloration with slight darkening shows Salmonella typhimurium

Hektoen Enteric Agar

• used to isolate and differentiate Salmonella and Shigella species from other Gram-negative enteric organisms based on the ability to ferment lactose, sucrose, or salicin, and to reduce sulfur to hydrogen sulfide gas (H2S).

• undefined medium

• thiosulfate is included as a source of sulfur.

• Ferric ammonium citrate

  is added to react with H2S and form a black precipitate. Bile salts are

  included to inhibit most Gram-positive cocci.

• cocci. Bromthymol blue and

  acid fuchsin dyes are added as color indicators.

HEKTOEN ENTERIC AGAR:

(clockwise from top), Escherichia coli, Proteus

mirabilis, Shigella flexneri, and Enterococcus faecalis.

• E. coli produces yellow color because acid is an end-

  product of its fermentation of lactose.

• P. mirabilis does not ferment lactose but does produce a black precipitate from the reaction between ferric ammonium citrate in the medium and H2S from sulfur reduction.

• Shigella is also a lactose nonfermenter and is blue-green; it is not a sulfur reducer. E. faecalis is inhibited by the bile salts.

• Shigella is also a lactose fermenter and is blue-green; it is a sulfur reducer. E. faecalis is not inhibited by the bile salts.

• HEKTOEN ENTERIC AGAR with Salmonella

  typhimurium and Escherichia coli. The black Salmonella colonies due to sulfur reduction and

  the yellow E. coli colonies due to lactose fermen-

  tation with acid end-products.

• MAC CONKEY AGAR with Salmonella

  typhimurium and Escherichia coli. The black E.coli colonies due to sulfur reduction and the yellow Salmonella colonies due to lactose fermen-

  tation with acid end-products.

MacConkey Agar

• used to isolate and differentiate members of the

  Enterobacteriaceae based on the ability to ferment lactose.

• the standard medium include MacConkey Agar w/o CV (without crystal

  violet)

• control swarming bacteria (such as Proteus) that interfere with other

  results.

• Bile salts and crystal violet inhibit

  growth of Gram-positive bacteria.

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