Lysine Iron Agar (LIA): Principle, Composition, Results, and Interpretation

Two enteric isolates both show K/A (alkaline slant, acid butt) on KIA — both grow on XLD agar as pink colonies without black centres. Both could be Shigella. LIA differentiates them: the first shows K/K with purple butt (lysine decarboxylation positive) — Salmonella (which decarboxylates lysine). The second shows R/A (red-purple slant, acid butt — lysine deamination) — Proteus or Providencia (which deaminates lysine, producing a different alkaline reaction on the slant with a distinctly different colour).

LIA is the medium that resolves the KIA ambiguity. Used in combination with KIA or TSI, it dramatically narrows the identification before any formal panel is run.

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

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.

Principle

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.

Figure: Breakdown of Lysine

In the anaerobic butt, organisms capable of glucose fermentation produce acid, resulting in yellow color. If the organism produces lysine decarboxylase, it removes CO2from 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.

The difference between decarboxylation and deamination — why it matters:

• Lysine decarboxylation (K/K result, purple butt): The organism removes the carboxyl group from lysine, producing cadaverine — a strongly alkaline amine. The entire butt turns purple. This is the Salmonella pattern.
• Lysine deamination (R/A result, red/burgundy slant): The organism removes the amino group from lysine under aerobic conditions on the slant, producing alpha-keto acid. The slant turns a distinctive red/burgundy colour — different from the standard alkaline pink/red. This is the Proteus/Providencia/Morganella pattern. These two reactions produce different colours on the slant and help separate organisms that look similar on KIA.

Procedure

Preparation of the medium

1. Suspend 33 grams of the medium in 1000 mL of demineralized water.
2. Heat to boiling with agitation to completely dissolve
3. Dispense into tubes and sterilize by autoclaving at 121°C for 15 minutes.
4. Cool in a slanted position to that deep butts are formed.

Inoculation of the medium

1. 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.
2. Streak the slant of the medium while removing the inoculating loop from the stab.
3. Cap the tube tightly and incubate at 35°C to 37°C in ambient air for 18 to 24 hours.

Results

Proteus spp. are capable of deaminating lysine in the presence of oxygen, resulting in a red color change on the slant of the medium.

Figure: A: Alkaline slant and alkaline butt (K/K)B: Alkaline slant/alkaline butt, H2S positive (K/K, H2S)C: Alkaline slant/acid butt (K/A)D: Red slant/acid butt (R/A)E: Uninoculated tube

LIA + KIA Combined Interpretation

Used together, LIA and KIA (or TSI) provide complementary information for enteric identification:

Key distinguishing use: LIA differentiates Salmonella (K/K — decarboxylation positive) from Citrobacter (K/A — decarboxylation negative, may show H₂S on KIA) — a common confusion in enteric workups.

Limitations of Lysine Iron Agar

1. 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.
2. LIA is not a substitute for TSI.

Quality Control

1. Alkaline slant and butt: H2S positive: Citrobacter freundii (ATCC8090)
2. Alkaline slant and butt: Escherichia coli (ATCC25922)
3. Alkaline slant and butt: H2S positive: Salmonella typhimurium (ATCC14028)
4. Red slant, acid butt: Proteus mirabilis (ATCC12453)

How to Remember: LIA

"LIA completes what KIA starts": KIA tells you carbohydrate fermentation and H₂S. LIA tells you what the organism does with the amino acid lysine. Together they answer four questions: Does it ferment glucose? Lactose? Produce H₂S? Decarboxylate or deaminate lysine?

The colour memory — "Purple = decarboxylation, Red = deamination":

• Purple butt = cadaverine produced = lysine DEcarboxylation (Salmonella, E. coli)
• Red/burgundy slant = alpha-keto acid = lysine DEamination (Proteus, Providencia)

The Salmonella pattern: K/A on KIA + K/K (purple) on LIA = Salmonella until proven otherwise.

Key Exam Facts in One Table

References

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4. Garcia LS. Clinical Microbiology Procedures Handbook. 4th ed. ASM Press; 2016.
5. Edwards PR, Fife MA. Lysine-iron agar in the detection of Arizona cultures. Appl Microbiol. 1961;9(5):478–480.
6. Difco Laboratories. Difco Manual: Dehydrated Culture Media and Reagents for Microbiology. 11th ed. Detroit: Difco Laboratories; 1998.