Xylose Lysine Deoxycholate (XLD) Agar

By Nisha Rijal •  Updated: 05/21/22 •  5 min read

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 the isolation and differentiation of Salmonella and Shigella from both 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 on the basis of xylose fermentation, lysine decarboxylation, and the production of hydrogen sulphide from the sodium thiosulphate.  

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

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 Agar

Ingredients in Per liter formulations

Lactose7.5 gm
Sucrose7.5 gm
Sodium thiosulfate6.8 gm
L-Lysine5.0 gm
Sodium chloride5.0 gm
Xylose3.75 gm
Yeast extract3.0 gm
Sodium deoxycholate2.5 gm
Ferric ammonium citrate0.8 gm
Phenol red0.08 gm
Agar15.0 gm

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, 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, and H2S non-producing Salmonella spp
Red colonies with a black centerXylose 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

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 a 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

Nisha Rijal

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.

Keep Reading