Kliger’s Iron Agar (KIA) - Biochemistry, Procedure, Result, Interpretation

Biochemistry of Kliger’s Iron Agar (KIA)

Microorganisms, usually of the Enterobacteriaceae family, first metabolize glucose in both the slant (aerobically as oxygen is present to serve as the final electron acceptor) and in the butt where an anaerobic condition is prevalent. After all the glucose has been converted to pyruvate, it is further metabolized through the aerobic Krebs cycle in the slant and through an anaerobic cycle in the butt.

As a result, acidic end products are released which changes the pH indicator present in the medium - phenol red - to change to yellow. Thus both slant and but of the Kliger’s Iron Agar (KIA) inoculated with glucose fermenters appear yellow. In cases of non-glucose fermenters, both the butt will remain red or even turn redder due to the release of alkaline end products.

After glucose is used up, the organisms may begin to metabolize ample lactose present in the Kliger’s Iron Agar (KIA). As the concentration of lactose is high, the test organism will continue to break them down, making continuous acid end products. This reaction is coined as (A/A) and the organism is a lactose fermenter.

If there is a production of gas, it will cause the media to break. In high gas producers, the entire media may have been raised in the tube.

In cases of the test organism not being able to use lactose, it must utilize the protein present in the media. The protein metabolism, which primarily occurs on the slant where oxygen is plentiful, produces by-products such as NH3 which is alkaline in nature. As a result, the phenol red indicator reverts back from yellow to red in color. Thus a non-lactose fermenter shows a red slant while the but remains yellow due to anaerobic glucose utilization. This reaction is termed alkaline/acid (Alk/Acid).

Moreover, glucose non-fermenters may metabolize peptone to produce alkaline end products and such reactions are termed alkaline/alkaline (alk/alk).

Kliger’s Iron Agar (KIA) can also detect hydrogen sulfide gas production. Some organisms are able to reduce amino acids with sulphur such as cysteine cystine, methionine while some can metabolize inorganic sulphur compounds including thiosulphate, sulphate, and sulphites. As a byproduct hydrogen sulfide gas (H2S) having a characteristic rotten egg smell is released.

The H2S gas if released can be detected by adding heavy metal salt with iron (FE2+) or lead (Pb2+) to the medium where sulphur substrates such as cysteine and sodium thiosulphate have been added. Even if the H2S gas is colorless, it reacts with metal salt (FeSO4) to produce an insoluble black FeS precipitate.

Biochemistry of Kliger’s Iron Agar (KIA)

Cysteine → (cysteine desulfurase) → α amino-acrylic acid + H2S

H2S + FeSO4 (Ferrous Sulphate) → FeS (Ferrous Sulphide) + H2SO4

Na2S2O3 → (thiosulphate reductase) → Na2SO3 + 2 H2S

Fe+++ + H2S → FeS (Ferrous Sulphide)

Procedure of Kliger’s Iron Agar (KIA)

The procedure of Kliger’s Iron Agar (KIA) includes:

1. Take a sterile Kliger’s Iron Agar (KIA) and inoculate the test organism by stabbing the but followed by streaking the slant in a zic-zac path.

2. Incubate the inoculated tube at 35-37°C for 18-24 hours.

3. Observe for changes in color in the slant and butt.

Result, Interpretation of Kliger’s Iron Agar (KIA)

A total of six types of reactions/results and interpretations are seen in the Kliger’s Iron Agar (KIA) test:

1. A/A, G+ = glucose fermentation with acid and gas production; lactose and/or sucrose are also fermented. Eg: E. coli, Klebsiella

   A/A, G+, H2S+= glucose fermentation with acid, gas, and H2S production; lactose and/or sucrose are also fermented. Eg: Citrobacter

2. Alk/A, G+ = glucose fermentation with acid, gas production; lactose and/or sucrose not fermented. Eg: Salmonella paratyphi A

   Alk/A, G+, H2S+ = glucose fermentation with acid, H2S, and gas production; lactose and/or sucrose not fermented. Eg: Salmonella paratyphi B, Salmonella paratyphi C, Proteus

3. Alk/A = glucose fermented with no gas production; lactose and/or sucrose not fermented. Eg: Shigella

4. Alk/NC = glucose, lactose, sucrose non fermenter. Eg: Pseudomonas

5. NC/NC = glucose, lactose, sucrose non fermenter. Eg: Alkaligenes, Acenetobacter

6. A/NC = glucose metabolized oxidatively

*NC = no change