XLD Agar: Introduction, Composition, Principle, Preparation, Test Requirements, Procedure, Colony Characteristics, Modifications, Uses, and Limitations

Introduction of XLD Agar

XLD agar is a selective, differential, and indicator medium for the isolation of enteric pathogens. XLD stands for xylose lysine deoxycholate. It also supports the growth of more fastidious enteric organisms. XLD agar was developed by Taylor. It was specially designed to allow the growth of Shigella species and is a proven medium for the isolation of this organism. It has also been found to be an excellent medium for isolating Salmonella species as well. Salmonella and Shigella species colony characteristics on XLD agar –

Salmonella Typhi: Red colonies with a black center

S. Paratyphi: Only red colonies no black center

Principle of XLD Agar

Xylose lysine deoxycholate (XLD) agar is a selective, differential, and indicator medium. The selective agent is sodium deoxycholate, which inhibits the growth of gram-positive organisms. Xylose,  lysine, lactose, and sucrose act as both differential as well as carbohydrate source ingredients. Xylose is fermented by most enterics except for Shigella species, and these colonies appear red on this medium as a result. A second differential mechanism for Salmonella is employed by the addition of lysine. Lysine decarboxylation reverts the pH of the medium to an alkaline condition. To avoid this reversal to a Shigella reaction, lactose and sucrose are added in excess.

Medium is an indicator due to having two indicators, one pH indicator (phenol red) and another hydrogen sulfide indicator(ferric ammonium citrate). The addition of sodium thiosulfate and ferric ammonium citrate as a sulfur source and indicator, respectively, allows hydrogen sulfide (H₂S) forming organisms to produce colonies with black centers, under alkaline conditions. Organisms that ferment xylose, are lysine decarboxylase-negative and do not ferment lactose or sucrose cause an acid pH in the medium, and form yellow colonies e.g. Escherichia coli, Citrobacter, and Proteus species.

Composition of XLD Agar

Ingredients	Gm/Liter
Lactose	7.5
Sucrose	7.5
Sodium Thiosulfate	6.8
L-Lysine	5.0
Sodium Chloride	5.0
Xylose	3.75
Yeast Extract	3.0
Sodium Deoxycholate	2.5
Ferric Ammonium Citrate	0.8
Phenol Red	0.08
Agar	15.0
Deionized/distilled water	1000 ml

                               The final pH should be 7.4 +/- 0.2 at 25ºC.

Preparation of XLD Agar

1. Suspend  56.68 grams of dehydrated powder  XLD agar in 1000 ml distilled or deionized or purified water. Note: The amount of XLD agar varies from manufacturer to manufacturer e.g. Oxoid says  53 gm in 1 liter while Hardy Diagnostics 56.93 and Himedia 56.68.
2. Heat with frequent agitation until the medium boils.
3. Do not autoclave or overheat.
4. Transfer immediately to a water bath at 50°C.
5. After cooling, pour into sterile Petri plates.
6. It is advisable not to prepare large volumes that will require prolonged heating, thereby producing precipitate.

Storage and Shelf life of XLD Agar

• Store at 2-8ºC  and away from direct light.
• Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), or contamination.
• The product is light and temperature-sensitive; protects from light, excessive heat, moisture, and freezing.

Test Requirements for XLD Medium

• Type of specimen  -As may be clinical ( blood, feces) or non-clinical specimens like food samples and water.
• Inoculating loop
• Bunsen burner
• Incubator
• Control strains (Salmonella enterica ATCC 14028 and Escherichia coli ATCC 25922)

Test procedure (specimen/organism inoculation)

1. Allow the plates to warm at 37°C or to room temperature, and the agar surface to dry before inoculating.
2. Inoculate and streak the specimen as soon as possible after collection.
3. If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface.
4. Streak for isolation with a sterile loop.
5. Incubate plates aerobically at 35-37ºC. for 18-24 hours.
6. Examine colonial characteristics.

Colony Characteristics of various organisms in XLD Agar

• Salmonella: H₂S positive Red colonies with black centers
• Shigella species and Salmonella H₂S negative: Red  colonies
• E. coli: Large, flat, yellow colonies
• Proteus species: Red to Yellow colonies
• Enterobacter and  Klebsiella species:  Mucoid, yellow colonies
• Staphylococcus aureus: No growth

Shigella boydii in XLD agar

Salmonella Typhi (black colony) and Shigella growth on xylose lysine deoxycholate (XLD) agar

Shigella flexneri biochemical reactions and growth on SS agar, sorbitol MacConkey medium, and XLD agar

Modifications of XLD Agar

• XLD with Novobiocin (10.0 mg/mL novobiocin): XLD Agar with Novobiocin contains novobiocin, which is commonly used to inhibit the growth of Proteus species, and helps reduce the potential for false positives from this organism.
• Modified XLD (only 0.5 g/L sodium deoxycholate): Modified XLD Agar contains a reduced amount of sodium deoxycholate in order to permit the growth of a wider variety of enteric organisms normally inhibited on traditional XLD Agar.

Uses of XLD Agar

1. XLD medium is a selective, differential, and indicator medium for the isolation of Gram-negative enteric pathogens from fecal specimens as well as other clinical material.
2. It is also recommended medium for the isolation of  Salmonella and Shigella species.
3. It is also applicable for microbiological testing of foods, water, and dairy products.

Limitations of XLD Agar

1. Some Proteus strains may give red to yellow coloration with most colonies developing black centers, giving rise to false-positive reactions.
2.  Salmonella Paratyphi A, S. choleraesuis, S. pullorum, and S. gallinarum may form red colonies without H₂S, thus resembling Shigella species.
3. Some species of Salmonella may form red colonies without a black center, which resemble Shigella colonies. In addition, a few species of Shigella ferment lactose, and Salmonella that fail to decarboxylate lysine would not be detected on this medium.
4. Processing delays of over 2-3 hours of un-preserved stool specimens greatly jeopardize the recovery of many enteric pathogens, as these organisms are very susceptible to the acidic changes that occur with a temperature drop of the feces.
5. Red, false-positive colonies may occur with Proteus, Providencia, and Pseudomonas.
6. Incubation in excess of 48 hours may lead to false-positive results.
7. It is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.

Further Readings

1. https://catalog.hardydiagnostics.com/cp_prod/content/hugo/xldagar.htm
2. https://himedialabs.com/TD/M031.pdf
3. http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asppr=CM0469&c=UK&lang=EN
4. https://www.sciencedirect.com/science/article/pii/S0079635203801054
5. Cowan & Steel’s Manual for identification of Medical Bacteria. Editors: G.I. Barron & R.K. Felthani, 3rd ed 1993, Publisher Cambridge University Press.
6. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
7. Clinical Microbiology Procedure Handbook Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
8. Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr, and Sommers H.M.
9. Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
10. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
11.  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.