Xylose Lysine Deoxycholate (XLD) Agar: Principle, Uses, Composition, Preparation, and Colony Characteristics

Table of Content:

• Principle of XLD Agar
• Uses of XLD Agar
• Composition of XLD Agar
• Preparation of XLD Agar
• Colony Characteristics of XLD Agar
• Quality Control for XLD Agar
• Limitations of XLD Agar

A specific medium for the isolation of Salmonella and Shigella spp. from clinical specimens and food samples is Xylose Lysine Deoxycholate (XLD) Agar. Taylor created XLD Agar in the beginning to isolate and identify Shigella from stool samples. 

In addition to being distinguished from non-pathogenic lactose fermenters, numerous non-pathogens that do not ferment lactose or sucrose are also distinguished from pathogens. 

Additionally, the medium was formulated to increase the frequency of growth of the more fastidious pathogens, which in other formulations have often failed to grow due to the inclusion of excessively toxic inhibitors.

The claim for the comparatively high effectiveness of XLD Agar in the primary isolation of Shigella and Salmonella has been substantiated by the findings of a number of clinical tests. It is advised to test foods, dairy products, and water using XLD Agar, which is a component of the USP microbiological limit test for determining if Salmonella is present or absent in specimens.

Principle of XLD Agar

Both a selective and differential medium, XLD Agar is. It includes yeast extract, which is a source of vitamins and minerals. Gram-positive microorganisms are inhibited by it because sodium deoxycholate is used as the selective agent. Since Shigella is the only enteric that does not ferment xylose, xylose is introduced into the medium because this characteristic allows for the differentiation of Shigella species. Without lysine, salmonellae would quickly ferment the xylose and become indistinguishable from non-pathogenic species, making it difficult to identify the Salmonella group from non-pathogens. When the xylose supply is depleted by the salmonellae, the lysine is attacked by the enzyme lysine decarboxylase, and the pH returns to an alkaline state, simulating the Shigella reaction.

Lactose and sucrose are added to create too much acid in order to stop lysine positive coliforms from reverting in a similar way. The colour of phenol red indicator turns yellow with xylose, lactose, and sucrose conversion to acid. Bacteria that decarboxylate lysine to cadaverine can be recognized by the appearance of a red coloration around the colonies due to an increase in pH. The pH indicator may display a range of colour hues as a result of these simultaneous or subsequent reactions, or it may undergo a colour shift from yellow to red after an extended incubation.

An H2S indicator system, made of sodium thiosulfate and ferric ammonium citrate, is added to the formulation to increase its capacity to distinguish between various substances. This system allows for the visualisation of the hydrogen sulphide generated, which forms colonies with black centres. The acid reaction caused by non-pathogenic H2S producers inhibits the blackening of the colonies, which only occurs at neutral or alkaline pH, since they do not decarboxylate lysine.

Uses of XLD Agar

1. For the isolation of Gram-negative enteric pathogens from faecal specimens and other clinical material, XLD Agar is a selective differential medium.
2. Shigella and Salmonella species may be isolated using it very well.
3. Microbiological testing of foods, water and dairy products.

Composition of XLD Agar

Ingredients per liter of deionized water (Hardy Diagnostics XLD Agar)

Composition
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

Final pH 7.4 +/- 0.2 at 25 degrees C.

Preparation of XLD Agar

1. Suspend 55 grams of dehydrated medium in 1000 ml purified or distilled water.

2. Frequently stir the medium while heating it until it boils.

NOTE: AVOID AUTOCLAVE.

3. Transfer right away to a 50°C water bath.

4. Pour into sterilised Petri plates after chilling.

NOTE: It is advisable not to prepare large volumes, which will require prolonged heating and may produce precipitate.

Colony Characteristics of XLD Agar

Acid compounds produced during the degradation of xylose, lactose, and sucrose change the medium's colour from red to yellow. 

Colonies with alkaline conditions produce hydrogen sulphide, which results in the development of black centres. The acidic environment that results from fermentation of carbohydrates inhibits this process. 

In the absence of lactose and sucrose fermentation, lysine decarboxylation results in a return to an alkaline state, which changes the medium's colour back to red. 

Typical colonial morphology on XLD Agar are as follows:

• Salmonella typhi – Red Colonies, Black Centers
• Salmonella choleraesuis – Red Colonies
• Shigella sonnei – Red Colonies
• Shigella flexneri – Red Colonies
• Escherichia coli – Large, Flat, Yellow Colonies; some strains may be inhibited
• Proteus vulgaris – Yellow Colonies
• Enterobacter/ Klebsiella – Mucoid, Yellow Colonies
• Pseudomonas aeruginosa – Pink, Flat, Rough Colonies
• Gram-positive bacteria – No growth to slight growth

Quality Control for XLD Agar

Test Organisms	Incubation			Results
	Time	Temperature	Atmosphere
Salmonella enterica ATCC ® 14028	24hr	35 oC	Aerobic	Growth; red colonies with black centers
Shigella flexneri ATCC ® 12022	24hr	35 oC	Aerobic	Growth; red to pink colonies
Enterococcus faecalis ATCC ® 29212	24hr	35 oC	Aerobic	Partial to complete inhibition; clear, pinpoint colonies
Escherichia coli ATCC ® 25922	24hr	35 oC	Aerobic	Partial to complete inhibition; yellow to yellow red colonies

Limitations of XLD Agar

1. Some Proteus and Pseudomonas species have the potential to produce red, false-positive colonies.
2. More than 48 hours of incubation may produce false-positive findings.
3. Shigella species are resembled by S. paratyphi A, S. choleraesuis, S. pullorum, and S. gallinarum because they have the ability to produce red colonies without black centres.
4. On XLD Agar, some Proteus strains will produce colonies with black centres.
5. Organisms need to be in pure culture in order to be identified. For definitive identification, morphological, biochemical, and/or serological tests should be run. 
6. Rarely is a single medium sufficient to identify every organism in a specimen that could be significant. To gain further information and assure the recovery of possible pathogens, cultures of specimens produced on selective medium should be compared with specimens grown on nonselective media.