XLD Agar: Composition, Principle, Results, and Uses

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.

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.

Yeast extract provides sources of nitrogen, carbon, and vitamins required for organism growth. Sodium deoxycholate present in the medium inhibits the growth of Gram-positive organisms. Sodium chloride maintains the osmotic balance, and agar is the solidifying agent.

Ingredients in Per liter formulations:

Ingredients	Amount (Gram/Liter)
Lactose	7.5 gm
Sucrose	7.5 gm
Sodium thiosulfate	6.8 gm
L-Lysine	5.0 gm
Sodium chloride	5.0 gm
Xylose	3.75 gm
Yeast extract	3.0 gm
Sodium deoxycholate	2.5 gm
Ferric ammonium citrate	0.8 gm
Phenol red	0.08 gm
Agar	15.0 gm

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.

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.

Result Interpretation

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

Colony characteristics on XLD Agar	Basis of reaction	Possible pathogens
Red colonies	Alkaline reaction, non-fermentation of xylose/lactose/sucrose or fermentation of xylose followed by decarboxylation of lysine	Shigella spp, Providencia spp, Pseudomonas spp, and H2S non-producing Salmonella spp
Red colonies with a black center	Xylose positive, lysine decarboxylase positive, capable of producing H2S, thus black centered colonies in alkaline pH	H2S producing Salmonella spp S. Typhi S. Typhimurium
Yellow opaque colonies	Ferment xylose but not lactose and sucrose, lysine negative, gives acid pH	E.coli, Klebsiella/Enterobacter Citrobacter, Serratia and Proteus spp
Yellow colonies	Lactose or sucrose fermentation, lysine negative, gives acid pH	Possible coliforms Sucrose positive Proteus spp

Quality Control

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.

Organism	Growth and colony morphology
S.typhimurium ATCC 14028	Good growth, Red colonies with a black center
S.flexneri ATCC 12022	Luxuriant growth, red colonies
Escherichia coli ATCC 25922	Partially inhibited, Large, flat, yellow colonies
Enterococcus faecalis ATCC 29212	Partial to complete inhibition, clear pinpoint colonies

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. 

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

• Bailey & Scott’s Diagnostic Microbiology, Forbes, 11th edition
• Review of Medical Microbiology and Immunology, Lange Medical Books, 13th edition
• Koneman’s Color Atlas and Textbook of Diagnostic Microbiology (Color Atlas & Textbook of Diagnostic Microbiology), 7th edition