XLD Agar (Xylose Lysine Desoxycholate Agar) ISO

The reactions are the degradation of the three fermentable carbohydrates: xylose, lactose and sucrose, with the production of acid, manifested in the color change from red to yellow. Sodium thiosulfate serves as a reactive substance with ferric ammonium citrate as an indicator of the formation of hydrogen sulfide under alkaline conditions. Lysine is included to enable the Salmonella group to be differentiated from the non-pathogens since, in its absence, salmonellae would quickly ferment the xylose, making it indistinguishable from non-pathogenic species. After the salmonellae exhaust the xylose present, the lysine is attacked through the enzyme lysine decarboxylase with a change to an alkaline pH, similar to the Shigella reaction. The bacteria that decarboxylate the L-lysine to cadaverine are identified by the presence of a purple-red color around the colonies due to the elevation of the pH. Phenol red is the pH indicator. Yeast extract is a source of vitamins, particularly of the B-group, essential for bacterial growth. Sodium chloride supplies essential electrolytes for transport and osmotic balance. Sodium desoxycholate is the selective agent and is thus inhibitory to Gram-positive microorganisms. Bacteriological agar is the solidifying agent.

Typical colonies of Salmonella on XLD agar have a black center and a lightly transparent zone of reddish color due to the color change of the indicator.

Salmonella H₂S-negative variants grown on XLD agar are pink with a darker pink center. Lactose-positive Salmonella grown on XLD agar are yellow with or without blackening.

Preparation

Suspend 54 grams of the medium in one liter of distilled water. Mix well and dissolve by heating with frequent agitation. Boil for one minute until complete dissolution. AVOID OVERHEATING. DO NOT AUTOCLAVE. Cool the medium according to the applicable normative and pour into Petri dishes as soon as it has cooled.

Preparation of large volumes, overheating and prolonged storage in a water bath are to be avoided. Precipitates may be formed but do not affect the performance of the culture media.

According to ISO 6579

For detection of Salmonella spp. in food, animal feed, animal faeces and environmental samples according to ISO 6579:

1. Pre-enrichment in non-selective liquid medium: inoculate the Buffered Peptone Water (LC-1402) with the sample or dilutions, and incubate at 34-38 °C for 18 ± 2 h.
2. Enrichment in/on selective media: inoculate, with the culture obtained in the pre-enrichment stage, the Rappaport Soy Broth (Vassiliadis) (LC-1174) or the Modified Semisolid Rappaport Vassiliadis Medium (MSRV) (LC-1376), and the MKTTN Broth (LC-1173). The Rappaport Soy Broth and the Modified Semisolid Rappaport Medium are incubated at 41.5 °C for 24 ± 3 h, and the MKTTN Broth at 34-38 °C for 24 ± 3 h.
3. Plating out on selective solid media: from the selective enriched cultures, inoculate two selective isolation agars: XLD agar and any other selective medium complementary to XLD agar (Salmonella Chromogenic Agar (LC-1122), Brilliant Green Agar (LC-1143), Bismuth Sulfite Agar (LC-1011), DCLS Agar (LC-1045), Desoxycholate Citrate Agar (LC-1067), Hektoen Enteric Agar (LC-1030), Salmonella Shigella Agar (LC-1064) and XLT4 Agar (LC-1159)). Incubate the XLD plates inverted at 34-38 °C for 24 ± 3 h. Incubate the second selective medium in accordance with the manufacturer's instructions.
4. Confirmation: subculture colonies of presumptive Salmonella and confirm their identity by biochemical and serological tests.

Note: according to Annex D of ISO 6579-1:2017, for the detection of enterica subspecies enterica serovars Typhi and Paratyphi, Selenite Cystine Broth (LC-1220) should be added as a selective enrichment medium and Bismuth Sulfite Agar (Wilson Blair) should be selected as a second selective medium (LC-1011).

According to ISO 19250

For detection of Salmonella spp. in water samples according to ISO 19250:

1. Pre-enrichment in non-selective medium: inoculate the Buffered Peptone Water (LC-1402) with the sample or dilutions, and incubate at 34-38 °C for 18 ± 2 h.
2. Enrichment in selective media: inoculate, with the culture obtained in the pre-enrichment stage, the Rappaport Soy Broth (Vassiliadis) (LC-1174) and the MKTTN Broth (LC-1173). The Rappaport Soy Broth is incubated at 41.5 ± 1 °C and the MKTTN Broth at 34-38 °C, both of them for 24 ± 3 hours.
3. Plating out on selective solid media: from the selective enriched cultures, inoculate two selective isolation agars: XLD agar and any other selective medium complementary to XLD agar (for instance, Brilliant Green Agar (LC-1143) or Bismuth Sulfite Agar (LC-1011)). Incubate the XLD plates inverted at 34-38 °C for 24 ± 3 hours. Incubate the second selective medium in accordance with the manufacturer's instructions.
4. Confirmation: subculture colonies of presumptive Salmonella and confirm their identity by biochemical and serological tests.

• International Standard UNE-EN-ISO 6579. Food Microbiology for human consumption and Animal Feed. Horizontal Method for the detection of Salmonella spp.
• ISO 19250 Water quality - Detection of Salmonella spp.
• ISO 6579 Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Salmonella spp. Amendment 1: Broader range of incubation temperatures, amendment to the status of Annex D, and correction of the composition of MSRV and SC.