XL Agar Base

Intended Use

XL (Xylose Lysine) Agar Base is used for the isolation and differentiation of enteric pathogens and, when supplemented with appropriate additives, as a base for selective enteric media.
XLD Agar is the complete Xylose Lysine Desoxycholate Agar, a moderately selective medium recommended for isolation and differentiation of enteric pathogens, especially Shigella species.
XLD Agar meets United States Pharmacopeia (USP), European Pharmacopoeia (EP) and Japanese Pharmacopoeia (JP)^1-3 performance specifications, where applicable.

Summary and Explanation

A wide variety of media have been developed to aid in the selective isolation and differentiation of enteric pathogens. Due to the large numbers of different microbial species and strains with varying nutritional requirements and chemical resistance patterns, investigators have developed various formulae to meet general as well as specific needs relative to isolation and identification of the microorganisms.
XL Agar Base was developed by Taylor⁴ for the nonselective isolation and differentiation of gram-negative enteric bacilli. It is particularly recommended for obtaining counts of enteric organisms. This medium can be rendered moderately selective for enteric pathogens, particularly Shigella, by the addition of sodium desoxycholate (2.5 g/L) to make XLD Agar.⁴
XL Agar Base can be made selective for Salmonella by adding 1.25 mL/L of 1% aqueous brilliant green to the base prior to autoclaving. Its use is recommended for Salmonella isolation after selenite or tetrathionate enrichment in food analysis; both coliforms and Shigella are inhibited.⁴
XLD Agar was developed by Taylor in order to increase the efficiency of the isolation and identification of enteric pathogens, particularly Shigella.⁴ The pathogens are differentiated not only from the nonpathogenic lactose fermenters but also from many nonpathogens which do not ferment lactose or sucrose. Additionally, the medium was formulated to increase the frequency of growth of the more fastidious pathogens,⁴ which in other formulations have often failed to grow due to the inclusion of excessively toxic inhibitors. The results obtained in a number of clinical evaluations have supported the claim for the relatively high efficiency of XLD Agar in the primary isolation of Shigella and Salmonella.^5-9
XLD Agar is a selective and differential medium used for the isolation and differentiation of enteric pathogens from clinical specimens.^10-12 The value of XLD Agar in the clinical laboratory is that the medium is more supportive of fastidious enteric organisms such as Shigella.¹² XLD Agar is also recommended for the testing of food, dairy products and water in various industrial standard test methods.^13-17 General Chapter of the USP describes the test method for the isolation of Salmonella from nonsterile pharmaceutical products using XLD Agar as the solid culture medium.¹

Principles of the Procedure

Xylose is incorporated into the medium because it is fermented by practically all enterics except for the shigellae. This property enables the differentiation of Shigella species. Lysine is included to enable the Salmonella group to be differentiated from the nonpathogens. Without lysine, salmonellae rapidly would ferment the xylose and be indistinguishable from nonpathogenic species. After the salmonellae exhaust the supply of xylose, the lysine is attacked via the enzyme lysine decarboxylase, with reversion to an alkaline pH, which mimics the Shigella reaction. To prevent similar reversion by lysine-positive coliforms, lactose and sucrose (saccharose) are added to produce acid in excess.⁴ Degradation of xylose, lactose and sucrose generates acid products, which in the presence of the pH indicator phenol red, causes a color change in the medium from red to yellow.
To add to the differentiating ability of the formulation, an H₂S indicator system, consisting of sodium thiosulfate and ferric ammonium citrate, is included for the visualization of the hydrogen sulfide produced, resulting in the formation of colonies with black centers. The nonpathogenic H₂S producers do not decarboxylate lysine; therefore, the acid reaction produced by them prevents the blackening of the colonies.⁴ Sodium chloride maintains the osmotic balance. Yeast extract supplies B-complex vitamins which stimulate bacterial growth. Agar is the solidifying agent.
XLD Agar is both a selective and differential medium. It utilizes sodium desoxycholate as the selective agent and, therefore, it is inhibitory to gram-positive microorganisms.

User Quality Control

NOTE: Differences in the Identity Specifications and Cultural Response testing for media offered as both Difco™ and BBL™ brands may reflect differences in the development and testing of media for industrial and clinical applications, per the referenced publications.
Identity Specifications
Difco™ XLD Agar
Dehydrated Appearance: Pink, free-flowing, homogeneous.
Solution:                       5.5% solution, soluble in purified water upon
                                    boiling. Solution is red, very slightly to slightly opalescent.
Prepared Appearance:     Red, slightly opalescent.
Reaction of 5.5%
Solution at 25°C:           pH 7.4 ± 0.2
Cultural Response
Difco™ XLD Agar
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C for 18-24 hours. Incubate (*) cultures at 30-35°C for 18-48 hours and (**) culture at 35-37°C for 18-72 hours.

Identity Specifications
BBL™ XL Agar Base
Dehydrated Appearance: Fine, homogeneous, free of extraneous material.
Solution:                       4.5% solution, soluble in purified water
                                   upon boiling. Solution is dark medium to
                                   dark, red to rose-red, clear to slightly hazy.
Prepared Appearance:    Dark medium to dark, red to rose-red, clear
                                   to slightly hazy.
Reaction of 4.5%
Solution at 25°C:           pH 7.5 ± 0.2
BBL™ XLD Agar (prepared)
Appearance:                 Medium orange red to red, trace hazy.
Reaction at 25°C:          pH 7.4 ± 0.2
Cultural Response
BBL™ XL Agar Base
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C for 18-24 hours (up to 48 hours if necessary).

BBL™ XLD Agar (prepared)
Inoculate and incubate at 35 ± 2°C for 24 hours. Incubate (*) cultures at 30-35°C for 18-48 hours and (**) culture at 35-37°C for 18-48 hours.

Formula

XL Agar Base
Approximate Formula* Per Liter
Xylose.......................................................................... 3.5 g
L-Lysine....................................................................... 5.0 g
Lactose........................................................................ 7.5 g
Sucrose........................................................................ 7.5 g
Sodium Chloride............................................................ 5.0 g
Yeast Extract................................................................. 3.0 g
Phenol Red................................................................. 0.08 g
Agar........................................................................... 13.5 g
XLD Agar
Approximate Formula* Per Liter
Xylose.......................................................................... 3.5 g
L-Lysine....................................................................... 5.0 g
Lactose........................................................................ 7.5 g
Saccharose................................................................... 7.5 g
Sodium Chloride........................................................... 5.0 g
Yeast Extract................................................................ 3.0 g
Phenol Red................................................................. 0.08 g
Sodium Desoxycholate.................................................. 2.5 g
Ferric Ammonium Citrate............................................... 0.8 g
Sodium Thiosulfate....................................................... 6.8 g
Agar.......................................................................... 13.5 g
*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation from Dehydrated Product

XL Agar Base
1. Suspend 45 g of the powder in 1 L of purified water. Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder. Add brilliant green, if desired.
3. Autoclave at 118°C for 10 minutes. Cool to 55-60°C.
4. Add 20 mL of an aqueous solution containing 34% sodium thiosulfate and 4% ferric ammonium citrate. For XLD agar, add 25 mL of 10% aqueous sodium desoxycholate. Pour into plates.
5. Test samples of the finished product for performance using stable, typical control cultures.
XLD Agar
1. Suspend 55 g of the powder in 1 L of purified water. Mix thoroughly.
2. Heat with agitation just until the medium boils. DO NOT OVERHEAT. DO NOT AUTOCLAVE.
3. Cool to 45-50°C in a water bath and use immediately. Overheating causes precipitation.
4. Test samples of the finished product for performance using stable, typical control cultures.

Sample Collection and Handling

For clinical specimens, refer to laboratory procedures for details on specimen collection and handling.^10-12
For food, dairy or water samples, follow appropriate standard methods for details on sample collection and preparation according to sample type and geographic location.^13-17
For pharmaceutical samples, refer to the USP for details on sample collection and preparation for testing of nonsterile products.¹

Procedure

1. Collect water samples as described in recommended procedures. For clinical specimens, refer to appropriate standard references for details on testing protocol to obtain isolated colonies from specimens using XLD Agar.^10-12
For food, dairy and water samples, refer to appropriate standard references for details on test methods using XLD Agar.^13-17
For pharmaceutical samples, refer to USP General Chapter for details on the examination of nonsterile products and the isolation of Salmonella using XLD Agar.¹
A nonselective medium should also be streaked to increase the chance of recovery when the population of gram-negative organisms is low and to provide an indication of other organisms present in the specimen. Incubate plates, protected from light, at 35 ± 2°C for 18-24 hours. Colonies on XLD agar may require 48 hours incubation for full color development.

Expected Results

Degradation of xylose, lactose and sucrose generates acid products, causing a color change in the medium from red to yellow.
Hydrogen sulfide production under alkaline conditions causes colonies to develop black centers. This reaction is inhibited by the acid conditions that accompany carbohydrate fermentation.
Lysine decarboxylation in the absence of lactose and sucrose fermentation causes reversion to an alkaline condition and the color of the medium changes back to red.
Typical colonial morphology and reactions on XLD Agar are as follows:
E.coli .................................................. Large, flat, yellow; some strains may be inhibited
Enterobacter / Klebsiella ....................... Mucoid, yellow
Proteus............................................... Red to yellow; most strains have black centers
Salmonella ......................................... Red-yellow with black centers
Shigella, Salmonella H2S-negative ........ Red
Pseudomonas ..................................... Red
Gram-positive bacteria ......................... No growth to slight growth

Limitation of the Procedure

1. Red, false-positive colonies may occur with some Proteus and Pseudomonas species.
2. Incubation in excess of 48 hours may lead to false-positive results.
3. S. Paratyphi A, S. Choleraesuis, S. pullorum and S. gallinarum may form red colonies without black centers, thus resembling Shigella species.
4. Some Proteus strains will give black-centered colonies on XLD Agar.
*Store at 2-8° C.
†QC testing performed according to USP/EP/JP performance specifications.

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