XLD Agar: Composition, Principle, Colony Characteristics, and Uses

A family of four presents to the emergency department with 48 hours of bloody diarrhea after attending a wedding feast. Stool specimens are collected. In the laboratory, the technician has a specific challenge: distinguishing the causative pathogen (likely Salmonella or Shigella) from the millions of normal E. coli and other coliforms in each specimen. MacConkey agar will grow every gram negative enteric bacilli; blood agar will grow everything and tell you nothing about enteric pathogens specifically.

XLD agar was designed precisely for this moment. It uses a three-step biochemical sequence; xylose fermentation, lysine decarboxylation, and hydrogen sulfide production, to produce colony colors that are diagnostic on their own, before a single biochemical test is run.

Principle

Xylose lysine deoxycholate (XLD) agar is a selective and differential medium for the isolation, cultivation, and differentiation of gram-negative enteric microorganisms. This media primarily isolates and differentiates Salmonella and Shigella from clinical and non-clinical specimens.

Figure: Fig: Mixed culture of E coli and Salmonella in XLD agar

XLD agar selectively promotes the growth of Salmonella and Shigella by inhibiting other enteric pathogens and differentiates Gram-negative enteric bacteria based on xylose fermentation, lysine decarboxylation, and the production of hydrogen sulfide from the sodium thiosulphate.

XLD medium was formulated by Taylor for the isolation and differentiation of enteric pathogens.

Composition of XLD Agar

The key ingredients of XLD agar are three sugars (xylose, lactose, and sucrose), lysine, and ferric ammonium citrate.

Ingredients in Per liter formulations:

Final pH: 7.4 ± 0.2 — medium appears bright red at baseline

A note on reading XLD agar: Unlike most enteric media (which start pale or colorless), XLD agar is bright red to red-orange when freshly prepared, due to the phenol red indicator at its baseline alkaline pH (7.4). Organisms that produce acid turn colonies and surrounding medium yellow. Organisms that produce alkaline reactions or no fermentation maintain or restore the red color. This means yellow colonies on XLD indicate fermentation, and red colonies indicate alkalinity — the opposite of the yellow-means-danger interpretation students sometimes bring from other media.

Principle of XLD Agar

Xylose is rapidly fermented by most Gram-negative enteric bacteria, including Salmonella. This causes acidification of the medium, turning the phenol red indicator yellow. Since Shigella spp doesn’t utilize xylose, acidification does not occur, and red colonies are produced. This property aids in the differentiation of Shigella spp.

After the xylose supply is exhausted, Salmonella spp decarboxylates lysine, increasing the pH to alkaline condition, and also produces red colonies like Shigella spp. However, Salmonellae also metabolize thiosulfate to produce hydrogen sulfide, which leads to the formation of colonies with black centers and allows them to be differentiated from the similarly colored Shigella colonies.

Organisms that ferment lactose, sucrose, and xylose but are lysine decarboxylase negative cause an acid pH and produce yellow colonies.

The three-step sequence can be summarized as a decision tree:

The result: Salmonella ends up as red colonies with black centers because all three steps occur. Shigella ends up as red colonies without black centers because it skips step 1 but cannot do step 3. Coliforms (E. coli, Klebsiella) stay yellow because they ferment all three sugars but cannot reverse the acid pH.

Why this three-step system is better than single-sugar media: MacConkey and EMB use only lactose — both Salmonella and Shigella appear colorless and are indistinguishable. XLD adds two more reactions (lysine decarboxylation and H2S) that specifically differentiate Salmonella from Shigella with a single overnight incubation.

Preparation of Media

1. Suspend 55 gm of the medium in one liter of purified water.
2. Heat with frequent agitation until the medium reaches the boiling point.
3. Avoid overheating. Do not autoclave.
4. Transfer immediately to a water bath at 50°C.
5. After cooling, pour into sterile Petri plates.

It is advisable not to prepare large volumes that will require prolonged heating, thereby producing precipitate.

Colony Characteristics on XLD Agar

After incubation of the plates with test organisms, various colored colonies develop, and differentiation is based on it.

The two critical mimicry problems on XLD:

1. S. paratyphi A produces red colonies without black centers — indistinguishable from Shigella on XLD alone. Always confirm with TSI and serology.
2. Proteus spp. can produce small black-centered colonies resembling Salmonella. Urease test immediately differentiates them: Salmonella is urease-negative; Proteus is strongly urease-positive.

Quality Control

Figure: XLD Cultures: Left: Red-pink black centered colonies of Salmonella typhimurium Right: Red-pink Shigella colonies

The commercially available dehydrated media should be homogeneous, free-flowing, and light pink-beige in color. After preparation, the medium is bright red to reddish-orange, trace to slightly with neutral pH (7.20-7.60).

Quality testing of the prepared media plates should be done by performing sterility and performance testing.

1. Sterility testing: Incubate un-inoculated plates of XLD agar for 48 hours at 35-37°C and observe any growth. After 48 hours, the sterility test plate should remain clear. Discard the whole lot if any colonies are seen.
2. Performance testing: Inoculate known standard strains on XLD agar plates, incubate for 18- 24 hours at 35-37°C, and observe for growth and colony characteristics.

Uses of XLD Agar

1. For isolation and differentiation of Salmonella and Shigella spp from other enteric pathogens.
2. Isolation and screening of samples containing mixed flora suspected of harboring enteric pathogens, e.g., medical specimens or food products.
3. Detection of Salmonella in non-sterile pharmaceutical products (in accordance with EP, USP) and food, water dairy products, etc.

Figure: Xylose Lysine deoxycholate (XLD) Cultures. Left: Yellow Escherichia coli colonies Right: Red-pink Salmonella colonies (Some Proteus species look identical)

How to Remember

XLD = three ingredients doing three jobs:

• Xylose — first sugar fermented; separates Shigella (can't ferment) from everything else (can)
• Lysine — decarboxylated by Salmonella after xylose runs out; restores red colour; distinguishes Salmonella from coliforms
• Deoxycholate — inhibits Gram-positives

The three-step story as a clinical narrative:

Imagine Salmonella as a marathon runner passing three checkpoints: Checkpoint 1 (xylose): Salmonella ferments xylose → turns yellow → passes checkpoint 1 Checkpoint 2 (lysine): When xylose runs out, Salmonella decarboxylates lysine → turns alkaline → reverts to red → passes checkpoint 2 Checkpoint 3 (H2S): Salmonella reduces thiosulfate → H2S → black precipitate → passes checkpoint 3

Shigella can't pass Checkpoint 1 (no xylose fermentation) — stays red throughout, no black center. Coliforms pass Checkpoint 1 (ferment everything) — stay yellow, never revert.

The medium starts red — remember this: Yellow colonies on XLD = fermenters = coliforms. Red colonies on XLD = pathogens or non-fermenters. This is counterintuitive because on most media, color usually signals the presence of something (a positive result). On XLD, the red is the baseline — the absence of fermentation.

XLD in the enteric media trio:

• XLD: Best for Shigella recovery (especially S. dysenteriae); clearest Salmonella signature (red + black)
• DCA: Moderate selectivity; good for Salmonella; poorer for S. dysenteriae
• SS agar: Highest selectivity; best for suppressing coliforms; poorest for Shigella

If only one enteric medium: choose XLD. If two: XLD + MacConkey.

Limitations

• A single medium may not be enough to recover all pathogens, so a less selective medium such as MacConkey agar should also be used.
• Non-enteric pathogens such as Pseudomonas may grow in the medium producing red colonies like Shigella; therefore, further, identification is necessary.
• Proteus mirabilis may produce small black-centered colonies similar to Salmonella but can be distinguished from Salmonella colonies, the latter being large, with a big black center.
• Prolonged incubation (more than 48 hours) may lead to false-positive results.
• Salmonella Paratyphi, S. Choleraesius, S.Pullorum and S.Gallinarum produce red colonies without a black center, hence may appear like Shigella.
• Since it is a selective medium, further subculture onto a basal medium is essential before performing biochemical tests, serotyping, etc.

References

• Tille, P. M. (2017). Bailey and Scott's Diagnostic Microbiology (14th ed.). Elsevier.
• Taylor, W. I. (1965). Isolation of Shigellae. I. Xylose lysine agars; new media for isolation of enteric pathogens. American Journal of Clinical Pathology, 44(4), 471–475.
• Cheesbrough, M. (2006). District Laboratory Practice in Tropical Countries, Part 2 (2nd ed.). Cambridge University Press.