Xylose Lysine Deoxycholate (XLD) Agar: Principle, Uses, Composition & Preparation

By Prof Walter Jaoko

Introduction

What Is XLD Agar?

Xylose Lysine Deoxycholate agar (XLD agar) is a selective growth medium used for the isolation and differentiation of Enterobacter, especially Salmonella and Shigella species from food, environmental samples and clinical specimens.The agar was developed by Welton Taylor in 1965. 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.

XLD Agar relies on Xylose fermentation, lysine decarboxylation and production of hydrogen sulfide for the primary differentiation of shigellae and salmonellae from non-pathogenic bacteria. Xylose Lysine Deoxycholate (XLD) agar is not intended for use in the diagnosis of diseases or other hunan condition.

Principle

XLD Agar has been recommended for the identification of Enterobacteriaceae. XLD Agar was formulated by Taylor for the isolation and differentiation of enteric pathogens including Salmonella Typhi from other Salmonella species from foods, water and dairy products. The media formulation does not allow the overgrowth of other organisms over Salmonella and Shigella. The medium contains yeast extract, which provides nitrogen and vitamins required for growth. 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 Shigellae but practically by all enteric species. Sodium chloride maintains the osmotic balance of the medium. Lysine is included to differentiate the Salmonella group from the non-pathogens. Salmonellae 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. 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.Organisms which ferment xylose, are lysine decarboxylase-negative, and do not ferment lactose or sucrose cause an acid pH in the medium, and form yellow colonies e.g. Escherichia coli, Citrobacter and Proteus species.

Composition of XLD agar

Preparation of XLD Agar

• Suspend  56.68 grams dehydrated powder  XLD agar in 1000 ml distilled or deionized or purified water. Note: The amount of XLD agar varies manufacturer to manufacture e.g. Oxoid says  53 gm in 1 litre while Hardy Diagnostics 56.93 and Himedia 56.68.
• Heat with frequent agitation until the medium boils.
• Do not autoclave or over heat.
• Transfer immediately to a water bath at 50°C.
• After cooling, pour into sterile Petri plates.
• It is advisable not to prepare large volumes that will require prolonged heating, thereby producing precipitate.

Storage and Shelf life of XLD agar

• Store at 2-8ºC  and away from direct light.
• Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination.
• Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

Test procedure ( specimen/organism inoculation)

• Allow the plates to warm at 37°C or  to room temperature, and the agar surface to dry before inoculating.
• Inoculate and streak the specimen as soon as possible after collection.
• If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface.
• Streak for isolation with a sterile loop.
• Incubate plates aerobically at 35-37ºC. for 18-24 hours.
• Examine colonial characteristics.

Colony Characteristics of various organisms in XLD Agar

• Salmonella: H₂S positive Red colonies with black centers
• Shigella species and Salmonella H₂S negative: Red  colonies
• E. coli: Large, flat, yellow colonies
• Proteus species: Red to Yellow colonies
• Enterobacter and  Klebsiella species:  Mucoid, yellow colonies
• Staphylococcus aureus: No growth

Uses of XLD Agar

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

Limitations of XLD Agar

• Red, false-positive colonies may occur with some Proteus and Pseudomonas species.
• Incubation in excess of 48 hours may lead to false-positive results.
• S. paratyphi A, S. choleraesuis, S. pullorum and S. gallinarum may form red colonies without black centers, thus resembling Shigella species.
• Some Proteus strains will give black-centered colonies on XLD Agar.
• For identification, organisms must be in pure culture. Morphological, biochemical, and/or serological tests should be performed for final identification. Consult appropriate texts for detailed information and recommended procedures.
• A single medium is rarely adequate for detecting all organisms of potential significance in a specimen. Cultures of specimens grown on selective media should, therefore, be compared with specimens cultured on nonselective media to obtain additional information and help ensure recovery of potential pathogens.