Lysine Iron Agar (LIA) is a combination medium used for the differentiation of gram-negative bacilli (enterics) based on decarboxylation or deamination of lysine and the formation of hydrogen sulfide (H2S). In combination with Triple Sugar Iron (TSI) agar, LIA is used to identify members of Salmonella and Shigella.
Composition of Lysine Iron Agar
|Enzymatic digest of gelatin||5 g|
|yeast extract||3 g|
|Ferric ammonium citrate||0.5 g|
|Sodium thiosulfate||0.04 g|
|Bromocresol purple||0.02 g|
Lysine iron agar (LIA) contains peptone and yeast extract to support bacterial growth. The amino acid lysine is used for detecting deamination and decarboxylation reactions. A small amount of glucose (0.1%) is a fermentable carbohydrate.
Sodium thiosulfate is a source of reducible sulfur. Ferric ammonium citrate is included as a sulfur reduction indicator. The H2S-producing organism produces black color in the medium due to black precipitation of ferrous sulfide (FeS). Bromocresol purple, the pH indicator, is yellow at or below pH 5.2 and purple at or above pH 6.8.
Lysine iron agar contains an aerobic slant and an anaerobic butt. The test organism is stabbed in the butt and streaked on the slant in a fishtail streak. The tube is then tightly capped and incubated for 18 to 24 hours before reading the results.
In the anaerobic butt, organisms capable of glucose fermentation produce acid, resulting in yellow color. If the organism produces lysine decarboxylase, it removes CO2 from L-lysine and forms cadaverine, an alkaline product. Cadaverine neutralizes the organic acids formed by glucose fermentation, and the butt of the medium reverts to the original alkaline state (purple). The purple color throughout indicates lysine decarboxylation. The purple color in the slant with a yellow (acidic) butt indicates no lysine decarboxylation.
Deamination reactions require the presence of oxygen. Therefore, any evidence of deamination will be seen only in the slant. If the organism produces lysine deaminase, the resulting deamination reaction will produce compounds that react with the ferric ammonium citrate and a coenzyme, flavin mononucleotide (FMN), forming a burgundy (dark red color) on the slant. A red slant with a yellow (acidic) butt indicates lysine deamination.
Hydrogen sulfide (H2S) is produced in LIA by the anaerobic reduction of thiosulfate. Ferric ions in the medium react with the H2S to form a black precipitate in the butt.
Preparation of the medium
- Suspend 33 grams of the medium in 1000 mL of demineralized water.
- Heat to boiling with agitation to completely dissolve
- Dispense into tubes and sterilize by autoclaving at 121°C for 15 minutes.
- Cool in a slanted position to that deep butts are formed.
Inoculation of the medium
- With a straight inoculating needle, inoculate LIA by stabbing through the center of the medium to approximately within 3 mm of the bottom of the tube.
- Streak the slant of the medium while removing the inoculating loop from the stab.
- Cap the tube tightly and incubate at 35°C to 37°C in ambient air for 18 to 24 hours.
|Purple slant/purple butt||Alkaline slant/alkaline butt (K/K)||Lysine deaminase negative; Lysine decarboxylase positive|
|Purple slant/yellow butt||Alkaline slant/acid butt (K/A)||Lysine deaminase negative; Lysine decarboxylase negative; Glucose fermentation|
|Red slant/ yellow butt||Red slant/acid butt (R/A)||Lysine deaminase positive; Lysine decarboxylase negative; Glucose fermentation|
|Black precipitate||H2S production||Sulfur reduction|
Proteus spp. are capable of deaminating lysine in the presence of oxygen, resulting in a red color change on the slant of the medium.
Limitations of Lysine Iron Agar
- Proteus spp. that produces hydrogen sulfide but does not produce lysine decarboxylase will not blacken the LIA medium since the acid in the butt suppress H2S formation. Additional testing, such as triple sugar iron (TSI) agar, should be used as a follow-up identification method.
- LIA is not a substitute for TSI.
- Alkaline slant and butt: H2S positive: Citrobacter freundii (ATCC8090)
- Alkaline slant and butt: Escherichia coli (ATCC25922)
- Alkaline slant and butt: H2S positive: Salmonella typhimurium (ATCC14028)
- Red slant, acid butt: Proteus mirabilis (ATCC12453)
- Bailey & Scott’s Diagnostic Microbiology, Forbes, 11th edition
- Review of Medical Microbiology and Immunology, Lange Medical Books, 13th edition
- Koneman’s Color Atlas and Textbook of Diagnostic Microbiology (Color Atlas & Textbook of Diagnostic Microbiology), 7th edition