The media in general use for the isolation of various  Enterobacteriaceae  may be categorized  as differential and/or selective.  The differential character is usually demonstrated by the sugar lactose and some type of indicator system such as neutral red or brom thymol blue.  The selective nature is express by incorporating such materials as bile salts, crystal violet ,or brilliant green.  The indication of growth and the color of the colony gives the observer an idea what type of organism is present.
 

Hektoen Enteric Agar (HE)
 

 -is a differential, selective medium for the isolation and differentiation of Salmonella and Shigella from other gram negative enterics.
 

Proteose, Peptone	12 g	Sodium Chloride	5 g
Yeast Extract	3 g	Sodium Thiosulfate	5 g
Bile Salts No. 3	9 g	Ferric Ammonium Citrate	1.5 g
Lactose	12 g	Saccharose(Sucrose)	12 g
Brom Thymol Blue	0.065 g	Acid Fuchsin	0.1 g
Salicin	2 g	Agar	14 g

 This is an effective medium for the rapid differentiation between lactose-fermenting and nonlactose-fermenting organisms in fecal specimens.  It also detects H2S producers.  Besides various nutrients to support growth, the medium contains three carbohydrates (lactose, salicin, and sucrose) and two indicator dyes (bromothymol blue and acid fuchsin).  Rapid lactose fermenters produce bright yellow to salmon-pink colonies against a faint pink red to red background of the medium.  Nonlactose-fermenting colonies generally are blue green to green in color. Sodium thiosulfate and ferric ammonium sulfate are ingredients in the medium to detect H2S and such producers yield a black center in the colonies.  HE agar is clear and green in color prior to inoculation.
 

  Organism                                   Colony Appearance

 Shigella                                                    Green, moist, raised colonies

 Salmonella, Proteus                       Blue-green, with or without a black center

 Coliforms (rapid lactose           Salmon-pink to orange,surrounded by a zone of
  fermenters)                               bile precipitate

MacConkey Agar

  -a medium relatively selective for Enterobacteriaceae and differential for lactose + and lactose - organisms.
 

Peptone	1.7 g	Proteose Peptone	3 g
Lactose	1.0 g	Bile Salts	1.5 g
Sodium Chloride	5.0 g	Neutral Red	0.03 g
Crystal Violet	0.001 g	Agar	14 g

 

 The selective agents, bile salts and crystal violet, inhibit most gram-positive and other non enteric bacteria.  Colonies of lactose fermenters are distinguished by red colonies surrounded by a zone of precipitated bile acids; lactose non fermenters are “colorless.”  A moderate inoculum is used.

       Organism                                                  Colony Appearance

 Salmonella and Shigella                                            colorless

 Proteus                                                                      colorless

 Escherichiacoli                                                 pink to red w/bile ppt.
 

Salmonella-Shigella Agar (SS)

  -for selective isolation of Salmonella and some Shigella organisms.
 

Beef Extract	5 g	Proteose Peptone	5 g
Lactose	10 g	Bile Salts No. 3	8.5 g
Sodium Citrate	8.5 g	Sodium Thiosulfate	8.5 g
Ferric Citrate	1 g	Brilliant Green	0.00033 g
Neutral Red	0.025 g	Agar	14 g

 

  A higher concentration of bile salts than in MacConkey agar and the brilliant green make this medium more selective, so that many coliforms such as E. coli are inhibited, and a heavier inoculum of stool may be used.  Lactose fermenter colonies are reddish pink; colonies of lactose non fermenters are “colorless”; and some Proteus and Salmonella colonies may have black centers due to Fe2S3 formation.

           Organism                                                (Growth) Appearance

  Salmonella and Shigella                                    (good to fair)  colorless

  Proteus                                                                    (poor)  colorless

  Escherichiacoli and other coliforms            (inhibited if not-reddish pink)
 

XLD Agar (XLD)

  -selective and differential medium for differentiating the enteric pathogens     especially Shigella  from nonpathogenic bacteria.
 

Yeast Extract	3 g	Ferric Ammonium Citrate	0.8 g
L-Lysine	5 g	Sodium Thiosulfate	6.8 g
Xylose	3.75 g	Sodium Chloride	5 g
Lactose	7.5 g	Phenol Red	0.08 g
Saccharose (Sucrose)	7.5 g	Agar	15 g
Sodium Desoxycholate	2.5g

 

    XLD Agar is the xylose lysine agar base devised by Taylor to which the sodium desoxycholate, sodium thiosulfate and ferric ammonium citrate have been added to make a complete medium.  This selective and differential plating medium is designed for direct isolation of Shigella.

    Sodium desoxycholate in the formula provides inhibition of gram-positive bacteria.  It also markedly inhibits the swarming of Proteus strains.

     Differentiation of Shigella and Salmonella from nonpathogenic bacteria is accomplished by three reactions: (1) xylose fermentation, (2) lysine decarboxylation and (3) hydrogen sulfide production. Xylose as a carbohydrate source in the formula differentiates Shigella and Providencia, which ferment xylose slowly or not at all, from the other enterics, which ferment xylose rapidly.  Salmonella are further differentiated from nonpathogenic xylose fermenters by the lysine decarboxylase reaction.  As the organisms rapidly exhaust the xylose and decarboxylate the lysine, a reversion to alkaline conditions stimulates the Shigella reaction. Lactose and sucrose, added in excess, prevent the lysine-positive coliforms from similarly reverting. The production of hydrogen sulfide under alkaline conditions, this black precipitation is inhibited.
 

      Organism                                                        Colony Appearance

 Salmonella                                                               red, black centers

 Shigella, Providencia                                                             red

 Escherichia, Klebsiella,                                                       yellow
      Proteus, Enterobacter
 
 

KLIGLER IRON AGAR (KIA)
 

  -a screening medium for primary differentiation of lactose-negative enteric bacteria.
 

Beef Extract	3 g	Yeast Extract	3 g
Peptone	15 g	Proteose Peptone	5 g
Lactose	10 g	Dextrose (Glucose)	1 g
Ferrous Sulfate	0.2 g	Sodium Chloride	5 g
Sodium Thiosulfate	0.3 g	Phenol Red	0.024 g
Agar	12 g

 

      Lactose fermenters are indicated by a yellow slant and butt after 18 to 24 hours of incubation.  Lactose nonfermenters (but glucose fermenters) are characterized by a red slant and yellow butt.  Since the medium has 10 times more lactose than glucose, the organism cannot oxidize the large quantity of fermentation acids in the area of aerobic conditions  (Krebs cycle) (slant) within the incubation period.  On the other hand, a glucose fermenter (lactose nonfermenter) can easily oxidize the smaller amount of acid to neutral substances (H2CO3, NH4HCO3, etc.) and, therefore, causes a reversion of the slant to the original red color within 18 to 24 hours.  Remember the butt is anaerobic so that the Kreb’s cycle is inoperative.

  Gas formation is indicated by cracks, separations or bubbles in the agar.

  The formation of hydrogen sulfide is detected by a black coloration in the medium.  Since hydrogen sulfide is a soluble gas, an indicator such as FeSO4 must be present to allow the formation of the black indicator compound, Fe2S2.  The major sources of hydrogen sulfide are the decomposition of proteins, peptones,  or sulfur-containing amino acids such as cysteine.

  For convenience and standardization in recording KIA reactions, a + is used for an acid reaction observed on the slant and/or butt.  Gas is recorded as a circle around the + of the butt.  Presence or absence of H2S is recorded as + or -.  Therefore, a reading of +, O, + indicates an acid slant, an acid butt and gas production, and  H2S  production.