Lysine Iron Agar

Intended Use

Lysine Iron Agar is used for the differentiation of enteric organisms based on their ability to decarboxylate or deaminate lysine and to form hydrogen sulfide.

Summary and Explanation

Edwards and Fife devised Lysine Iron Agar for the detection of Salmonella enterica subsp. arizonae (previously Arizona arizonae) cultures, especially those that ferment lactose rapidly.¹ This development followed closely the promulgation by Ewing and Edwards of a taxonomic scheme for the Enterobacteriaceae in which the principle division and groups within this family were defined and differentiation procedures described.² The various criteria for identification of cultures were summarized by Edwards and Ewing in their treatise on the Enterobacteriaceae.³ However, the taxonomy of the Enterobacteriaceae has changed dramatically in recent years.^4-6

Johnson et al. utilized Lysine Iron Agar and Kligler Iron Agar for primary differentiation of various groups of bacteria within the family Enterobacteriaceae and a combination of Lysine Iron Agar with Triple Sugar Iron Agar for identification of Salmonella, Shigella and Arizona group organisms from feces.⁷

Lysine Iron Agar aids in the differentiation of enteric bacilli on the basis of their ability to decarboxylate lysine, to deaminate lysine and to produce hydrogen sulfide. It is designed for use with other media (e.g., Triple Sugar Iron Agar) in appropriate identification schemes.

Principles of the Procedure

Dextrose serves as a source of fermentable carbohydrate. The pH indicator, bromcresol purple, is changed to a yellow color at or below pH 5.2 and is purple at or above pH 6.8.⁸ Ferric ammonium citrate and sodium thiosulfate are indicators of hydrogen sulfide formation. Lysine is the substrate for use in detecting the enzymes, lysine decarboxylase and lysine deaminase.

Cultures of enteric bacilli that produce hydrogen sulfide cause blackening of the medium due to the production of ferrous sulfides. Those that produce lysine decarboxylase produce an alkaline reaction (purple color) or neutral reaction in the butt of the medium. Organisms that deaminate the lysine cause the development of a red slant over an acid butt. Gas may be formed but its formation is often irregular or suppressed.

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™ Lysine Iron Agar
Dehydrated Appearance: Beige to greenish beige, free flowing, homogeneous.
Solution:                       3.45% solution, soluble in purified water upon
                                    boiling. Solution is reddish purple, slightly opalescent.
Prepared Appearance:     Purple, slightly opalescent.
Reaction of 3.45%
Solution at 25°C:            pH 6.7 ± 0.2

BBL™ Lysine Iron Agar
Dehydrated Appearance: Fine, homogeneous, free of extraneous material.
Solution:                       3.3% solution, soluble in purified water upon
                                    boiling. Solution is medium to dark, rose purple,
                                    clear to slightly hazy.
Prepared Appearance:     Medium to dark, rose purple, clear to slightly hazy.
Reaction of 3.3%
Solution at 25°C:            pH 6.7 ± 0.2

Cultural Response

Difco™ Lysine Iron Agar
Prepare the medium per label directions. Inoculate with fresh cultures and incubate at 35 ± 2°C for 18-48 hours.

Alkaline = red purple, no change in color
Acid = yellow
Red = lysine deaminase
+ H2S = black precipitate
– H2S = no black precipitate

BBL™ Lysine Iron Agar
Prepare the medium per label directions. Inoculate with fresh cultures and incubate at 35 ± 2°C for 24 hours.

A = Acid (yellow)
K = Alkaline (red purple, no change in color)
R = Red (lysine deaminase)
+ H2S = black precipitate
– H2S = no black precipitate

Formula

Difco™ Lysine Iron Agar
Approximate Formula* Per Liter
Peptone....................................................................... 5.0 g
Yeast Extract................................................................. 3.0 g
Dextrose...................................................................... 1.0 g
L-Lysine HCl................................................................ 10.0 g
Ferric Ammonium Citrate................................................ 0.5 g
Sodium Thiosulfate...................................................... 0.04 g
Bromcresol Purple........................................................ 0.02 g
Agar........................................................................... 15.0 g
BBL™ Lysine Iron Agar
Approximate Formula* Per Liter
Pancreatic Digest of Gelatin............................................ 5.0 g
Yeast Extract................................................................ 3.0 g
Dextrose...................................................................... 1.0 g
L-Lysine..................................................................... 10.0 g
Ferric Ammonium Citrate................................................ 0.5 g
Sodium Thiosulfate...................................................... 0.04 g
Bromcresol Purple........................................................ 0.02 g
Agar........................................................................... 13.5 g
*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation from Dehydrated Product

1. Suspend the powder in 1 L of purified water:
    Difco™ Lysine Iron Agar – 34.5 g;
    BBL™ Lysine Iron Agar – 33 g.
    Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
3. Autoclave at 121°C for 12 minutes.
4. Cool tubes in a slanted position to form slants with deep butts.
5. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

Using an inoculating needle, stab the butt twice then streak the slant with growth from a pure culture. Incubate tubes with loosened caps for 18-48 hours at 35 ± 2°C in an aerobic atmosphere. Difco™ & BBL™ Manual, 2nd Edition Triple Sugar Iron Agar slants should be inoculated in parallel unless results from this medium have already been obtained to distinguish coliforms from Shigella, for example.

Expected Results

Lysine decarboxylation is detected in the butt by an alkaline (purple) reaction. Lysine deamination is detected by a red slant. Hydrogen sulfide production is detected by the formation of a black precipitate. A negative reaction (purple slant and yellow butt) indicates fermentation of dextrose only.⁸ Hydrogen sulfide may not be detected in this medium by organisms that are negative for lysine decarboxylase activity since acid production in the butt may suppress its formation.⁸ For this reason H₂S-producing Proteus species do not blacken this medium.⁸

*Store at 2-8° C.

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