Lysine Iron Agar Test- Principle, Media, Procedure, Results, Uses

Lysine iron agar (LIA) test is a biochemical test that is used to differentiate gram-negative bacilli based on their ability to decarboxylate or deaminate lysine and to produce hydrogen sulfide (H2S). Lysine is an amino acid that can undergo two different metabolic pathways: decarboxylation and deamination. Decarboxylation is the removal of a carboxyl group from lysine, resulting in the formation of cadaverine, an alkaline compound. Deamination is the removal of an amino group from lysine, resulting in the formation of alpha-ketocarboxylic acid, which can react with iron to produce a reddish-brown compound. Hydrogen sulfide is a gas that can be produced by some bacteria from the reduction of sulfur-containing compounds, such as sodium thiosulfate. Hydrogen sulfide can react with iron to form a black precipitate of ferrous sulfide.

The LIA test can help to identify and differentiate various members of the Enterobacteriaceae family, such as Escherichia coli, Salmonella spp., Shigella spp., Proteus spp., and Citrobacter spp., as well as some other gram-negative bacilli, such as Morganella morganii and Providencia spp. The LIA test can also be used to distinguish between different serotypes of Salmonella enterica, such as Typhi, Paratyphi A, Paratyphi B, and Paratyphi C. The LIA test is simple, inexpensive, and easy to perform and interpret. It can provide valuable information for the diagnosis and treatment of infections caused by gram-negative bacilli.

Lysine iron agar (LIA) is a differential medium that can distinguish between gram-negative bacilli based on their ability to decarboxylate or deaminate lysine and to produce hydrogen sulfide (H2S).

The medium contains the following ingredients:

• Lysine: an amino acid that can be metabolized in two different ways by bacteria. Some bacteria can decarboxylate lysine, which means they remove a carboxyl group from the amino acid and produce an amine called cadaverine. This reaction is anaerobic and occurs in the butt of the medium. Other bacteria can deaminate lysine, which means they remove an amino group from the amino acid and produce an alpha-keto acid. This reaction is aerobic and occurs on the slant of the medium.
• Peptones: a source of nitrogen and carbon for bacterial growth.
• Glucose: a fermentable carbohydrate that can be used by bacteria to produce acid and gas. The amount of glucose in LIA is very small, so it is quickly exhausted by fermentation. This creates an acidic environment in the butt of the medium, which can be neutralized by lysine decarboxylation if the bacteria have this enzyme.
• Ferric ammonium citrate: a source of iron that can react with hydrogen sulfide gas to form a black precipitate of ferrous sulfide. This indicates the presence of H2S-producing bacteria in the medium.
• Sodium thiosulfate: a substrate for H2S production by bacterial enzymes. Some bacteria can reduce thiosulfate to sulfite and then to sulfide, which then combines with iron to form H2S gas.
• Bromcresol purple: a pH indicator that changes color depending on the acidity or alkalinity of the medium. It is yellow at or below pH 5.2 and purple at or above pH 6.8.

The medium has an aerobic slant and an anaerobic butt. The slant is exposed to oxygen, while the butt is protected from oxygen by the stab inoculation. The different reactions that can occur in LIA are summarized in the table below:

By observing the color changes and the presence or absence of gas and black precipitate in LIA, one can differentiate between various gram-negative bacilli based on their lysine metabolism and H2S production.

Lysine iron agar (LIA) is a combination medium that contains agar as the gelling agent and various nutrients and indicators to differentiate gram-negative bacilli based on their ability to decarboxylate or deaminate lysine and produce hydrogen sulfide (H2S). The composition of LIA per liter of demineralized water is as follows :

• Lysine: 10 g
• Pancreatic digest of gelatin: 5 g
• Yeast extract: 3 g
• Glucose: 1 g
• Ferric ammonium citrate: 0.5 g
• Sodium thiosulfate pentahydrate: 40 mg
• Bromocresol purple: 20 mg
• Agar: 13.5 g

The final pH of the medium is 6.7 +/- 0.2 at 25ºC.

Lysine is the amino acid that serves as the substrate for decarboxylation or deamination reactions. Pancreatic digest of gelatin and yeast extract provide nitrogen, carbon, vitamins, and minerals for bacterial growth. Glucose is a fermentable carbohydrate that produces acid in the anaerobic butt of the medium. Ferric ammonium citrate and sodium thiosulfate are the sources of iron and sulfur, respectively, for detecting H2S production. Bromocresol purple is the pH indicator that changes color from yellow (acidic) to purple (alkaline) depending on the lysine metabolism.

The medium has an aerobic slant and an anaerobic butt that allow different reactions to occur in different parts of the tube. The slant is inoculated by streaking with a straight inoculating needle, while the butt is inoculated by stabbing through the center of the medium to the bottom of the tube. The tubes are incubated aerobically with loose caps at 35-37ºC for 18-48 hours before reading the results.

To perform the lysine iron agar test, you will need the following materials:

• Lysine iron agar (LIA) slants
• Straight inoculating needle
• Pure culture of the test organism
• Incubator at 35-37°C
• Loose caps for the tubes

The steps of the test are as follows:

1. Allow the medium to reach room temperature before inoculating .
2. Collect the center of a well-isolated colony from a pure culture plate using a straight inoculating needle .
3. Inoculate the LIA slant by stabbing through the center of the medium to the bottom of the tube and then streaking the slant while withdrawing needle .
4. Cap the tube loosely and incubate aerobically at 35-37°C for 18-48 hours .
5. Examine the reaction of the medium by observing the color changes in the slant and the butt, and the presence of black precipitate or gas bubbles .

The results of the LIA test are based on the color changes of the slant and the butt of the medium, as well as the presence or absence of black precipitate and gas bubbles. The color changes are due to the pH indicator bromocresol purple, which is yellow at or below pH 5.2 and purple at or above pH 6.8. The following table summarizes the possible results and their interpretations:

Some examples of bacteria that can be identified by the LIA test are:

• Escherichia coli: Purple slant/yellow butt (K/A), negative for H2S and gas production
• Salmonella typhimurium: Purple slant/purple butt (K/K), positive for H2S and gas production
• Proteus vulgaris: Red slant/yellow butt (R/A), positive for H2S production
• Citrobacter freundii: Purple slant/purple butt (K/K), positive for gas production

• Lysine iron agar test is not sufficient for the complete identification of gram-negative bacilli. It is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for confirmation.
• It is important to stab the butt of the medium with a straight inoculating needle. Failure to stab the butt invalidates this test. The integrity of the agar must be maintained when stabbing.
• Caps must be loosened during this test or erroneous results will occur. Tight caps may prevent oxygen diffusion and affect the slant reaction.
• LIA is not as sensitive in detecting hydrogen sulfide (H2S) production as other iron-containing mediums, such as sulfide indole motility (SIM) medium and triple sugar iron agar (TSIA). H2S production may not be seen with organisms that do not produce lysine decarboxylase, such as Proteus spp., since acid in the butt may suppress H2S formation.
• Certain species or strains may give delayed reactions or completely fail to ferment glucose in the expected manner. However, in most cases if the organism fails to ferment glucose within 48 hours, and growth is definitely present, the organism is most likely not in the Enterobacteriaceae family.
• Slant reaction with Morganella morganii may be variable after 24 hours incubation and may require longer incubation.
• Gas production may be irregular or suppressed with organisms other than Citrobacter spp.
• Salmonella enterica serovar Paratyphi A does not produce lysine decarboxylase.

Lysine iron agar test is used to differentiate gram-negative bacilli based on decarboxylation or deamination of lysine and the formation of hydrogen sulfide (H2S). It is particularly useful for distinguishing different members of the Enterobacteriaceae family, such as Salmonella, Shigella, Escherichia, Proteus, Morganella, and Citrobacter . In combination with triple sugar iron (TSI) agar, LIA is used to identify Salmonella and Shigella species. LIA is also a sensitive medium for the detection of lactose-fermenting and non-lactose-fermenting salmonellae. LIA can help to confirm the results of other biochemical tests, such as indole, methyl red, Voges-Proskauer, and citrate tests.

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