What is Xylose Lysine Deoxycholate (XLD) Agar?
- XLD Agar (Xylose Lysine Deoxycholate Agar) is a differential medium utilized for the isolation and separation of enteric gram-negative pathogens, specifically salmonella and shigella in clinical, environmental, and food samples.
- It relies on the fermentation of xylose, lactose, and sucrose, the decarboxylation of lysine, and the formation of hydrogen sulfide to differentiate shigella from salmonella.
- The American Pharmacopoeia (USP) and European Pharmacopoeia (EP) both prescribe XLD agar for the enumeration of bacteria.
- Xylose Lysine Deoxycholate agar (XLD agar) is used to isolate Salmonella and Shigella species from clinical samples and food. [1] [2] Welton Taylor created the agar in 1965. [3] Due to its pH of roughly 7.4, it appears bright pink or red due to the indicator phenol red. Sugar fermentation reduces the pH, as indicated by the yellowing of the phenol red indicator. The majority of gut bacteria, including Salmonella, can ferment the sugar xylose to create acid; however, Shigella colonies are incapable of doing so and so remain red. Salmonella colonies, upon depleting their xylose supply, will decarboxylate lysine, bringing the pH back to alkaline levels and resembling the red Shigella colonies. Salmonellae metabolize thiosulfate to create hydrogen sulfide, which results in the production of colonies with black centers and enables them to be distinguished from Shigella colonies of a similar hue.
- Some Enterobacteria, such as E. coli, will ferment the medium’s lactose to an amount that prevents pH reversion by decarboxylation and acidifies the medium, turning it yellow.
- Salmonella species: scarlet colonies with black cores in some cases. Because to the presence of Salmonella-type colonies, the agar itself will turn red.
- Shigella species: colonies colored red.
- Coliforms: colonies of yellow to orange hue.
- Colonies of Pseudomonas aeruginosa are pink, flat, and rough. Due to the color similarity, this form of colony is readily confused with Salmonella.
Composition of Xylose Lysine Deoxycholate (XLD) Agar
| Ingredients | Gms/Litre |
| Yeast extract | 3.0 |
| L- Lysine | 5.0 |
| Lactose | 7.5 |
| Sucrose | 7.5 |
| Xylose | 3.5 |
| Sodium chloride | 5.0 |
| Sodium deoxycholate | 2.5 |
| Sodium thiosulphate | 6.8 |
| Ferric ammonium citrate | 0.8 |
| Phenol red | 0.08 |
| Agar | 15.0 |
Final pH (at 25°C) 7.4±0.2
Principle of Xylose Lysine Deoxycholate (XLD) Agar
XLD Agar can be used as both a differential and selective medium. This medium also contains yeast extract which provides the necessary nutrients and vitamins for growth. The medium uses sodium deoxycholate to act as a selective agent, and is therefore ineffective against gram-positive microorganisms.
Although sucrose, lactose, and xylose are all fermentable carbohydrates, xylose is the main ingredient in the medium. This is because it is not fermented either by Shigella or by any enterics. This helps in the differentiation of Shigella species. The medium’s osmotic equilibrium is maintained by sodium chloride. Lysine is used to distinguish the Salmonella group and non-pathogens.
Salmonella quickly ferments xylose, exhausting the supply. The enzyme lysine-positive coliforms decarboxylate then decarboxylates lysine to form amines. These amines have a reversion to alkaline pH, which mimics the Shigella reaction. To prevent the reaction by lysine positive coliforms, lactose or sucrose are used to make excess acid. The yellow color of the phenol red indicator is caused by the degradation of sucrose, xylose and lactose to acid.
An increase in pH can cause a reddish hue around colonies to indicate that bacteria has decarboxylate Lysine to Cadaverine. These reactions can occur simultaneously or sequentially. The pH indicator may show different shades of colour, or may change from yellow to red with prolonged incubation. The formulation also includes an H2S indicator system that consists of ferric ammonium citrate and sodium thiosulphate.
This allows for visualization of hydrogen sulfuride, which results in colonies with black centers. Non-pathogenic H2S producers don’t decarboxylase Lysine and so the acid reaction they produce prevents blackening.
Type of specimen used in Xylose Lysine Deoxycholate (XLD) Agar
Samples from clinical trials such as
- Blood
- Faeces
- Food and dairy samples
- Water samples.
Purpose of Xylose Lysine Deoxycholate (XLD) Agar
This agar is recommended for the isolation and identification of Salmonella Typhi and other Salmonella species using clinical and nonclinical samples.
Preparation of Xylose Lysine Deoxycholate (XLD) Agar
- In 1000 ml of distilled water, you can suspend 56.68 grams
- Apply heat Keep stirring until the medium boils.
- Do not AUTOCLAVE or OVERHEAT.
- Transfer to a 50degC water bath immediately
- After cooling, pour into sterile Petri plates.
- It is best to not prepare large quantities that will need prolonged heating. This could lead to precipitate.
Medium in bottles Preparation: Melt the content of the bottle in a water bath at 100°C (loosing the cap partially detached) till thoroughly dissolved. Allow to cool immediately at 45-50°C. Mix well, avoiding foam formation, and then distribute in Petri dishes.
Physical properties of Xylose Lysine Deoxycholate (XLD) Agar
- Appearance: Light yellow to pink homogeneous free flowing powder.
- pH: 7.4 +0.2
- Gelling: Firm, comparable with 1.5% Agar gel
- Colour and Clarity of prepared medium: Red coloured clear to slightly opalescent gel forms in Petri plates
- Reaction: Reaction of 5.67% w/v aqueous solution at 25°C . pH : 7.4±0.2
Cultural Response: After incubation at 35-37degC, and at a specified amount of time the cultural response was observed. For bacteria growth on Soyabean Casein Digest Agar, the recovery rate is 100%.
Technique on XLD Agar
You can either plate the feces directly or use selective enrichment broths before streaking out. Salmonella enrichment can be done with Tetrathionate Broth (CM0029) or Selenite Broth (CM0395).
- Innoculate the dried, poured plates with a loopful inoculum, either from stool samples, rectal swabs, or a suitable enrichment broth.
- For 18-24 hours, incubate the plates at 35-37degC.
- Look out for common colonies.
Refer to the appropriate standards for testing food samples
Results and Colony Characteristics of Xylose Lysine Deoxycholate (XLD) Agar
From colonial appearances on XLD, a tentative grouping can be made of isolates. Three groups of Enterobacteriaceae are easily distinguished:
- Red zones with black centers: Salmonella, Arizona, and Edwardsiella;
- Red colonies and red centers: Shigella, Providencia and hydrogen sulphide negative salmonellae;
- Colonies with yellow haloes or yellow centers: the genera Escherichia, Enterobacter, Citrobacter, Kluyvera, Klebsiella, Hafnia, Serratia and Proteus, and the species Yersinia enterocolitica. These presumptive results must be confirmed by the usual biochemical tests.
| 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 H2S non-producing Salmonella spp |
| Red colonies with black centre | 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 |
Colony Characteristics of Different Microorganisms on Xylose Lysine Deoxycholate (XLD) Agar
| Organisms | Colony characteristics |
| Salmonella Typhimurium | red with black centres |
| Salmonella Abony NCTC | red with black centres |
| Escherichia coli ATCC 8739 | yellow |
| Escherichia coli NCTC 9002 | yellow |
| Escherichia coli ATCC 25922 (00013*) | yellow |
| Proteus vulgaris ATCC 13315 | grey with black centres |
| Salmonella Paratyphi A ATCC 9150 | red |
| Salmonella Paratyphi B ATCC 8759 | red with black centres |
| Salmonella Enteritidis ATCC 13076 (00030*) | red with black centres |
| Salmonella Typhi ATCC 6539 | red with black centres |
| Shigella dysenteriae ATCC 13313 | red |
| Shigella flexneri ATCC 12022 (00126*) | red |
| Shigella sonnei ATCC 25931 | red |
| Klebsiella aerogenes ATCC 13048 (00175*) | yellow |
| Enterobacter cloacae ATCC 13047 (00083*) | yellow |
| Staphylococcus aureus subsp. aureus ATCC 25923 (00034*) | inhibited |
| Enterococcus faecalis ATCC 29212 (00087*) | inhibited |
| Staphylococcus aureus subsp. aureus ATCC 6538 (00032*) | inhibited |
Quality Control for XLD Agar
Dehydrated media that is commercially available should be homogeneous, fluid, and light pink-beige. The medium should be bright red to reddish orange after being prepared. It should also slightly neutralize pH (7.20-7.60).
Perform sterility testing as well as performance testing to ensure that the media plates are in good condition.
- Sterility testing: Place un-inoculated plates on XLD agar incubated for 48 hours at 35 to 37degC. Watch for growth. The sterility test plates should be kept clear for 48 hours. If colonies are found, discard the entire lot.
- Performance testing: Innoculate standard strains on XLD plates. Let them incubate at 35-37°C for 18-24 hours, then observe the growth and colony characteristics.
| Positive control: | Expected Results (24 ± 3 hours at 37 °C) |
| Salmonella Typhimurium ATCC® 14028 * WDCM 00031 | Good growth; red colonies with black centre |
| Salmonella Enteritidis ATCC® 13076* WDCM 00030 | Good growth; red colonies with black centre |
| Negative control: | |
| Escherichia coli ATCC® 25922 * WDCM 00013 | Inhibited yellow colonies |
| Enterococcus faecalis ATCC® 29212* WDCM 00087 | No growth |
Uses of Xylose Lysine Deoxycholate (XLD) Agar
- XLD Agar can be used to isolate Gram-negative enteric Pathogens from feces and other clinical materials.
- It is particularly suitable for isolation of Salmonella and Shigella species.
- Microbiological testing of food, milk and water.
Storage and Shelf Life of Xylose Lysine Deoxycholate (XLD) Agar
Keep the prepared medium at 20-30°C and store between 10-30°C in tightly sealed containers. Make sure to use the product before the expiry date is printed on the label. To prevent product from clumping due to its hygroscopic nature, it should be stored dry after opening the package. Incorrect storage can lead to product lump formation. After use, store the product in a dry, ventilated place that is protected from extremes in temperature and ignition sources. Use the container before the expiry date printed on it.
Limitations of Xylose Lysine Deoxycholate (XLD) Agar
- Some Proteus strains can give off red or yellow coloration, with some colonies developing black centers. This could lead to false positive reactions.
- Red colonies may be seen in non-enteric species like Providencia and Pseudomonas.
- S. Paratyphi A and S. Choleraesuis may form red colonies, similar to Shigella.
- False-positive results may occur if the incubation time exceeds 48 hours.
- For complete identification, it is recommended that colonies grown from pure culture be subject to biochemical, immunological and molecular testing.
Xylose Lysine Deoxycholate (XLD) Agar
Xylose Lysine Deoxycholate agar (XLD) is a selective medium that allows for the isolation and growth of Salmonella and Shigella species using clinical samples or food. Taylor developed XLD Agar to aid in the differentiation, isolation and identification of enteric disease agents and support the growth more specialized enteric organisms. XLD Agar is a well-proven medium that allows the growth of Shigella species. It is also an excellent medium to isolate Salmonella species. It has a pH value of 7.4, giving it a bright pink to red color due to the indicator phenol.
The pH is lower when sugar fermentation occurs. The phenol red indicator changes to yellow in order to register this. Salmonella and most gut bacteria can ferment sugar xylose to make acid. Shigella colonies are unable to do this, so they remain red. Salmonella colonies can decarboxylate Lysine after exhausting their xylose supply. This will increase the pH to alkaline, mimicking red Shigella colonies. Salmonellae can metabolize thiosulfate and produce hydrogen sulfide. This allows them to differentiate from Shigella colonies that are similar in colour.
Principle of Xylose Lysine Deoxycholate (XLD) Agar
XLD Agar can be used as both a differential and selective medium. This medium also contains yeast extract which provides the necessary nutrients and vitamins for growth. The medium uses sodium deoxycholate to act as a selective agent, and is therefore ineffective against gram-positive microorganisms.
Although sucrose, lactose, and xylose are all fermentable carbohydrates, xylose is the main ingredient in the medium. This is because it is not fermented either by Shigella or by any enterics. This helps in the differentiation of Shigella species. The medium’s osmotic equilibrium is maintained by sodium chloride. Lysine is used to distinguish the Salmonella group and non-pathogens.
Salmonella quickly ferments xylose, exhausting the supply. The enzyme lysine-positive coliforms decarboxylate then decarboxylates lysine to form amines. These amines have a reversion to alkaline pH, which mimics the Shigella reaction. To prevent the reaction by lysine positive coliforms, lactose or sucrose are used to make excess acid. The yellow color of the phenol red indicator is caused by the degradation of sucrose, xylose and lactose to acid.
An increase in pH can cause a reddish hue around colonies to indicate that bacteria has decarboxylate Lysine to Cadaverine. These reactions can occur simultaneously or sequentially. The pH indicator may show different shades of colour, or may change from yellow to red with prolonged incubation. The formulation also includes an H2S indicator system that consists of ferric ammonium citrate and sodium thiosulphate.
This allows for visualization of hydrogen sulfuride, which results in colonies with black centers. Non-pathogenic H2S producers don’t decarboxylase Lysine and so the acid reaction they produce prevents blackening.
Type of specimen used in Xylose Lysine Deoxycholate (XLD) Agar
Samples from clinical trials such as
- Blood
- Faeces
- Food and dairy samples
- Water samples.
Purpose of Xylose Lysine Deoxycholate (XLD) Agar
This agar is recommended for the isolation and identification of Salmonella Typhi and other Salmonella species using clinical and nonclinical samples.
Preparation of Xylose Lysine Deoxycholate (XLD) Agar
- In 1000 ml of distilled water, you can suspend 56.68 grams
- Apply heat Keep stirring until the medium boils.
- Do not AUTOCLAVE or OVERHEAT.
- Transfer to a 50degC water bath immediately
- After cooling, pour into sterile Petri plates.
- It is best to not prepare large quantities that will need prolonged heating. This could lead to precipitate.
Medium in bottles Preparation: Melt the content of the bottle in a water bath at 100°C (loosing the cap partially detached) till thoroughly dissolved. Allow to cool immediately at 45-50°C. Mix well, avoiding foam formation, and then distribute in Petri dishes.
Physical properties of Xylose Lysine Deoxycholate (XLD) Agar
- Appearance: Light yellow to pink homogeneous free flowing powder.
- pH: 7.4 +0.2
- Gelling: Firm, comparable with 1.5% Agar gel
- Colour and Clarity of prepared medium: Red coloured clear to slightly opalescent gel forms in Petri plates
- Reaction: Reaction of 5.67% w/v aqueous solution at 25°C . pH : 7.4±0.2
Cultural Response: After incubation at 35-37degC, and at a specified amount of time the cultural response was observed. For bacteria growth on Soyabean Casein Digest Agar, the recovery rate is 100%.
Technique on XLD Agar
You can either plate the feces directly or use selective enrichment broths before streaking out. Salmonella enrichment can be done with Tetrathionate Broth (CM0029) or Selenite Broth (CM0395).
- Innoculate the dried, poured plates with a loopful inoculum, either from stool samples, rectal swabs, or a suitable enrichment broth.
- For 18-24 hours, incubate the plates at 35-37degC.
- Look out for common colonies.
Refer to the appropriate standards for testing food samples
Results and Colony Characteristics of Xylose Lysine Deoxycholate (XLD) Agar
From colonial appearances on XLD, a tentative grouping can be made of isolates. Three groups of Enterobacteriaceae are easily distinguished:
- Red zones with black centers: Salmonella, Arizona, and Edwardsiella;
- Red colonies and red centers: Shigella, Providencia and hydrogen sulphide negative salmonellae;
- Colonies with yellow haloes or yellow centers: the genera Escherichia, Enterobacter, Citrobacter, Kluyvera, Klebsiella, Hafnia, Serratia and Proteus, and the species Yersinia enterocolitica. These presumptive results must be confirmed by the usual biochemical tests.
Quality Control for XLD Agar
Dehydrated media that is commercially available should be homogeneous, fluid, and light pink-beige. The medium should be bright red to reddish orange after being prepared. It should also slightly neutralize pH (7.20-7.60).
Perform sterility testing as well as performance testing to ensure that the media plates are in good condition.
- Sterility testing: Place un-inoculated plates on XLD agar incubated for 48 hours at 35 to 37degC. Watch for growth. The sterility test plates should be kept clear for 48 hours. If colonies are found, discard the entire lot.
- Performance testing: Innoculate standard strains on XLD plates. Let them incubate at 35-37°C for 18-24 hours, then observe the growth and colony characteristics.
Uses of Xylose Lysine Deoxycholate (XLD) Agar
- XLD Agar can be used to isolate Gram-negative enteric Pathogens from feces and other clinical materials.
- It is particularly suitable for isolation of Salmonella and Shigella species.
- Microbiological testing of food, milk and water.
Storage and Shelf Life of Xylose Lysine Deoxycholate (XLD) Agar
Keep the prepared medium at 20-30°C and store between 10-30°C in tightly sealed containers. Make sure to use the product before the expiry date is printed on the label. To prevent product from clumping due to its hygroscopic nature, it should be stored dry after opening the package. Incorrect storage can lead to product lump formation. After use, store the product in a dry, ventilated place that is protected from extremes in temperature and ignition sources. Use the container before the expiry date printed on it.
Limitations of Xylose Lysine Deoxycholate (XLD) Agar
- Some Proteus strains can give off red or yellow coloration, with some colonies developing black centers. This could lead to false positive reactions.
- Red colonies may be seen in non-enteric species like Providencia and Pseudomonas.
- S. Paratyphi A and S. Choleraesuis may form red colonies, similar to Shigella.
- False-positive results may occur if the incubation time exceeds 48 hours.
- For complete identification, it is recommended that colonies grown from pure culture be subject to biochemical, immunological and molecular testing.
FAQ
What is the purpose of Xylose-Lysine-Deoxycholate?
Xylose Lysine Deoxycholate agar (XLD agar) is used to isolate Salmonella and Shigella species from clinical samples and food. Welton Taylor created the agar in 1965.
What is the principle of XLDA?
XLD agar preferentially increases the growth of Salmonella and Shigella by inhibiting other enteric pathogens, and differentiates Gram-negative enteric bacteria based on xylose fermentation, lysine decarboxylation, and hydrogen sulfide generation from sodium thiosulfate.
What is the composition of xylose lysine deoxycholate agar?
The components of XLD agar are yeast extract, sodium chloride, xylose, lactose, sucrose, l-lysine hydrochloride, sodium thiosulfate, iron (III) ammonium citrate, phenol red, sodium deoxycholate, agar, and distilled or deionized water.
Is Xylose-Lysine-Deoxycholate XLD Agar selective or differential?
XLD Agar, which stands for Xylose-Lysine-Deoxycholate, is a selective culture medium that can be used to isolate Salmonella and Shigella from food products or feces and other clinical samples. XLD Agar is a differential and selective medium. It includes yeast extract as a source of vitamins and nutrients.
