Kligler Iron Agar

Intended Use

Kligler Iron Agar is used for the differentiation of members of the Enterobacteriaceae on the basis of their ability to ferment dextrose and lactose and to liberate sulfides.

Summary and Explanation

In 1911, Russell described a new double sugar tube medium for the isolation of typhoid bacilli from urine and feces.¹ Six years later, Kligler developed a simple lead acetate medium for the differentiation of the typhoid-paratyphoid group.² Subsequently, Kligler evaluated culture media used in the isolation and differentiation of typhoid, dysentery and allied bacilli and endorsed Russell’s medium.³ Bailey and Lacey substituted phenol red for the Andrade indicator previously used as a pH indicator.⁴
The current formulation of Kligler Iron Agar combines features of Kligler’s lead acetate medium with those of Russell’s double sugar agar.

Principles of the Procedure

Kligler Iron Agar, in addition to casein and meat peptones, contains lactose and dextrose which enable the differentiation of species of enteric bacilli due to color changes of the phenol red pH indicator in response to the acid produced during the fermentation of these sugars. The dextrose concentration is only 10% of the lactose concentration. The combination of ferric ammonium citrate and sodium thiosulfate enables the detection of hydrogen sulfide production.
Lactose nonfermenters (e.g., Salmonella and Shigella) initially produce a yellow slant due to acid produced by the fermentation of the small amount of dextrose. When the dextrose supply is exhausted in the aerobic environment of the slant, the reaction reverts to alkaline (red slant) due to oxidation of the acids. The reversion does not occur in the anaerobic environment in the butt, which remains acid (yellow butt). Lactose fermenters produce yellow slants and butts because enough acid is produced in the slant to maintain an acid pH under aerobic conditions. Organisms incapable of fermenting either carbohydrate produce red slants and butts.
Hydrogen sulfide production is evidenced by a black color either throughout the butt, or in a ring formation near the top of the butt. Gas production (aerogenic reaction) is detected as individual bubbles or by splitting or displacement of the agar.

User Quality Control

Identity Specifications
Kligler Iron Agar
Dehydrated Appearance: Fine, homogeneous, free of extraneous material.
Solution:                        5.2% solution, soluble in purified water upon boiling.
                                     Solution is medium to dark, orange to red, with or
                                     without a tint of brown, clear to slightly hazy.
Prepared Appearance:     Medium to dark, orange to red, with or without a tint
                                     of brown, clear to slightly hazy.
Reaction of 5.2%
Solution at 25°C:            pH 7.4 ± 0.2
Cultural Response
Kligler Iron Agar
Prepare the medium per label directions. Stab inoculate with fresh cultures and incubate at 35 ± 2°C for 24 hours.

Formula

Kligler Iron Agar
Approximate Formula* Per Liter
Pancreatic Digest of Casein........................................ 10.0 g
Peptic Digest of Animal Tissue.................................... 10.0 g
Lactose...................................................................... 10.0 g
Dextrose...................................................................... 1.0 g
Sodium Chloride.......................................................... 5.0 g
Ferric Ammonium Citrate............................................. 0.5 g
Sodium Thiosulfate...................................................... 0.5 g
Agar.......................................................................... 15.0 g
Phenol Red................................................................ 25.0 mg
*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation from Dehydrated Product

1. Suspend 52 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.
3. Dispense and autoclave at 121°C for 15 minutes.
4. Cool in a slanted position such that deep butts are formed. For best results, the medium should be used on the date of preparation or melted and resolidified before use.
5. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

To inoculate, carefully touch the center of an isolated colony on an enteric plated medium with a cool, sterile needle, stab into the medium in the butt of the tube, and then streak back and forth along the surface of the slant. Several colonies from each primary plate should be studied separately, since mixed infections may occur. Incubate tubes with loosened caps for 18-24 hours at 35 ± 2°C in an aerobic atmosphere.
To enhance the alkaline condition in the slant, free exchange of air must be permitted through the use of a loose closure. If the tube is tightly closed, an acid reaction (caused solely by dextrose fermentation) will also involve the slant.

Expected Results

After incubation, record the reaction in the slant and butt, noting gas formation and hydrogen sulfide production. Typical reactions produced by members of the Enterobacteriaceae (majority of the species in the particular genus) are presented in the following table.⁵

 *Store at 2-8° C.

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