Xylose Lysine Deoxycholate (XLD) Agar

Introduction of XLD Agar

Xylose Lysine Deoxycholate (XLD) agar is a selective growth medium used for the isolation of Salmonella and Shigella species from clinical samples and from food. XLD Agar was developed by Taylor for the differentiation, isolation, and identification of enteric pathogens, and to support the growth of more fastidious enteric organisms. XLD Agar was especially designed to allow the growth of Shigella species, and is a proven medium for the isolation of this organism. It has also been found to be an excellent medium for isolating Salmonella species as well. It has a pH of approximately 7.4, leaving it with a bright pink or red appearance due to the indicator phenol red. Sugar fermentation lowers the pH and the phenol red indicator registers this by changing to yellow. Most gut bacteria, including Salmonella, can ferment the sugar xylose to produce acid; Shigella colonies cannot do this and therefore remain red. After exhausting the xylose supply Salmonella colonies will decarboxylate lysine, increasing the pH once again to alkaline and mimicking the red Shigella colonies. Salmonellae metabolise thiosulfate to produce hydrogen sulfide, which leads to the formation of colonies with black centers and allows them to be differentiated from the similarly coloured Shigella colonies.

Composition  of XLD Agar

Ingredients Gms/Litre
Yeast extract 3.0
L- Lysine 5.0
Lactose 7.5
Sucrose 7.5
Xylose 3.5
Sodium chloride 5.0
Sodium deoxycholate 2.5
Sodium thiosulphate 6.8
Ferric ammonium citrate 0.8
Phenol red 0.08
Agar 15.0

Final pH (at 25°C) 7.4±0.2

Principle of XLD Agar

XLD Agar is both a selective and differential medium. The medium contains yeast extract, which provides nitrogen and vitamins required for growth. It utilizes sodium deoxycholate as the selective agent and, therefore, is inhibitory to gram-positive micro-organisms. Though the sugars xylose, lactose and sucrose provide sources of fermentable carbohydrates, xylose is mainly incorporated into the medium since it is not fermented by Shigella but practically by all enterics. This aids in differentiation of Shigella species. Sodium chloride maintains the osmotic balance of the medium. Lysine is included to differentiate the Salmonella group from the non-pathogens. Salmonella rapidly ferment xylose and exhaust the supply. Subsequently lysine is decarboxylated by the enzyme lysine decarboxylase to form amines with reversion to an alkaline pH that mimics the Shigella reaction. However, to prevent this reaction by lysine-positive coliforms, lactose and sucrose are added to produce acid in excess. Degradation of xylose, lactose and sucrose to acid causes phenol red indicator to change its colour to yellow. Bacteria that decarboxylate lysine to cadaverine can be recognized by the appearance of a red colouration around the colonies due to an increase in pH. These reactions can proceed simultaneously or successively, and this may cause the pH indicator to exhibit various shades of colour or it may change its colour from yellow to red on prolonged incubation. To add to the differentiating ability of the formulation, an H2S indicator system, consisting of sodium thiosulphate and ferric ammonium citrate, is included for the visualization of hydrogen sulphide produced, resulting in the formation of colonies with black centers. The non-pathogenic H2S producers do not decarboxylase lysine; therefore, the acid reaction produced by them prevents the blackening of the colonies.

Preparation of XLD Agar

  1. Suspend 56.68 grams in 1000 ml distilled water.
  2. Heat with frequent agitation until the medium boils.
  3. Transfer immediately to a water bath at 50°C.
  4. After cooling, pour into sterile Petri plates.
  5. It is advisable not to prepare large volumes that will require prolonged heating, thereby producing precipitate, thereby producing precipitate.

Results of XLD Agar

Salmonella Typhimurium on XLD agar.

Organisms Colony characteristics
Salmonella H2S positive Red colonies with black centers
Shigella spp. and Salmonella H2S negative Red  colonies
E.coli Large, flat, yellow colonies
Proteus spp. Red to Yellow colonies
Enterobacter / Klebsiella Mucoid, yellow colonies

Uses of XLD Agar

  1. XLD Agar is a selective differential medium for the isolation of Gram-negative enteric pathogens from fecal specimens and other clinical material.
  2. It is especially suitable for the isolation of Shigella and Salmonella species.
  3. Microbiological testing of foods, water and dairy products.

Limitations of XLD Agar

  1. Some Proteus strains may give red to yellow colouration with most colonies developing black centers, giving rise to false positive reactions.
  2. Non-enterics like Pseudomonas and Providencia may exhibit red colonies.
  3. S. Paratyphi A, S. Choleraesuis, S. Pullorum and S. Gallinarum may form red colonies without H2S, thus resembling Shigella
  4. Incubation in excess of 48 hours may lead to false-positive results.
  5. It is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.

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