Xylose Lysine Deoxycholate (XLD) Agar

Last updated on June 24th, 2021

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

Xylose lysine deoxycholate (XLD) agar is both a selective and differential medium for the isolation, cultivation and differentiation of gram-negative enteric microorganisms. This media is primarily used for isolation and differentiation of Salmonella and Shigella from both clinical and non-clinical specimens. XLD selectively promotes the growth of Salmonella and Shigella by inhibiting other enteric pathogens and differentiates Gram-negative enteric bacteria on the basis of xylose fermentation, lysine decarboxylation and the production of hydrogen sulphide from the sodium thiosulphate.  

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

Principle of XLD agar

The key ingredients of XLD agar are three sugars (xylose, lactose, and sucrose), lysine, and ferric ammonium citrate. Xylose is rapidly fermented by most Gram-negative enteric bacteria including Salmonella and causes acidification of the medium turning the phenol red indicator to 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.

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.

Composition of XLD

Ingredients in Per litre formulations

Lactose7.5gm
Sucrose7.5gm
Sodium Thiosulfate6.8gm
L-Lysine5.0gm
Sodium Chloride5.0gm
Xylose3.75gm
Yeast Extract3.0gm
Sodium Deoxycholate2.5gm
Ferric Ammonium Citrate0.8gm
Phenol Red0.08gm
Agar15.0gm

Preparation of media

  1. Suspend 55 g 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.

Note: 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, differentiation is based on it

Colony characteristics on XLD AgarBasis of reactionPossible pathogens
Red coloniesAlkaline reaction, non-fermentation of xylose/lactose/sucrose or fermentation of xylose followed by decarboxylation of LysineShigella spp Providencia spp Pseudomonas spp H2S non-producing Salmonella spp
Red colonies with black centreXylose positive, lysine decarboxylase positive, capable of producing H2S,thus black centered colonies in alkaline pHH2S producing Salmonella spp S. Typhi S. Typhimurium
Yellow opaque coloniesFerment xylose but not lactose and sucrose, lysine negative, gives acid pHE.coli, Klebsiella/Enterobacter Citrobacter, Serratia and Proteus spp
Yellow coloniesLactose or sucrose fermentation, lysine negative, gives acid pHPossible coliforms Sucrose positive Proteus spp

Quality control of XLD agar

XLD Cultures: Left: Red-pink black centered colonies of Salmonella typhimurium  Right: Red-pink Shigella colonies
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 testing and performance testing

  1. Sterility testing: Incubate un inoculated plates of XLD agar for 48 hours at 35-37°C and observe for 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.
OrganismGrowth and colony morphology
S.typhimurium ATCC 14028Good growth, Red colonies with black center
S.flexneri ATCC 12022Luxuriant growth, red colonies
Escherichia coli ATCC 25922Partially inhibited, Large, flat, yellow colonies
Enterococcus faecalis ATCC 29212Partial 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. 
Xylose Lysine deoxycholate (XLD) Cultures.
Xylose Lysine deoxycholate (XLD) Cultures.
Left: Yellow Escherichia coli colonies Right: Red-pink Salmonella colonies (Some Proteus species look identical)

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 alongside.
  • Nonenteric 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-centred colonies similar to Salmonella but can be distinguished from Salmonella colonies, the latter being large, with big black centres.
  • Prolonged incubation (more than 48 hour) may lead to false-positive results.
  • Salmonella Paratyphi, S. Choleraesius, S.Pullorum and S.Gallinarum produce red colonies without black centre, 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.
About Nisha Rijal 46 Articles
I am working as Microbiologist in National Public Health Laboratory (NPHL), government national reference laboratory under the Department of health services (DoHS), Nepal. Key areas of my work lies in Bacteriology, especially in Antimicrobial resistance.