Kligler Iron Agar Test

In this kligler iron agar test post we have briefly explained about kligler iron agar test principle, objectives, requirements, kligler iron agar test procedure, uses and limitations.

Kligler Iron Agar Test

Remel Kligler Iron Agar (KIA) is a solid medium recommended for use in qualitative procedures for differentiation of enteric gram-negative bacilli on the basis of dextrose and lactose fermentation and hydrogen sulfide (H2S) production.

In 1918, Kligler described a medium for detection of H2S and differentiation of Salmonella spp. Bailey and Lacey further modified the medium by substituting phenol red indicator for Andrade indicator.

This medium became known as Kligler Iron Agar. It is recommended by Edwards and Ewing for determination of H2S production by enteric gram-negative bacilli. Gilardi has also recommended KIA for detection of H2S produced by some strains of Pseudomonas.


Casein and meat peptones supply nitrogenous compounds, amino acids, and vitamins necessary for bacterial growth. Sodium chloride is a source of essential electrolytes and maintains osmotic equilibrium. Lactose and dextrose are carbohydrate sources.  

Phenol red is an indicator of carbohydrate fermentation. Fermentation reactions are read on the slant and in the butt, indicated by a color change from red (alkaline) to yellow (acid). The dextrose concentration in kligler iron agar test is one-tenth the concentration of lactose. This serves to distinguish dextrose- only fermenting organisms from those which also ferment lactose.

The small amount of acid produced in the slant during dextrose fermentation oxidizes rapidly, causing the slant to revert to alkaline (red). The yellow acid reaction is maintained in the butt due to the absence of oxygen. Lactose fermenters result in yellow slants and butts because enough acid is produced in the slant by fermentation of both sugars to maintain an acid pH under aerobic conditions.

If the organism does not ferment dextrose, the slant and butt remain neutral (red). Ferric ammonium citrate is an indicator of H2S production. If H2S is produced from sodium thiosulfate, it reacts with ferric ammonium citrate to form a black precipitate (ferrous sulfate) in the medium. Gas production is indicated by bubbles, a splitting of the medium, or displacement of the medium.


Casein Peptone -10.0  gm, Sodium Thiosulfate – 0.3 gm, Lactose – 10.0 gm, Ferric Ammonium Citrate – 0.2 gm, Meat Peptone – 10.0 gm, Phenol Red – 25.0 mg, Sodium Chloride -5.0 gm, Agar -12.5 gm, Dextrose – 1.0  gm, Demineralized Water – 1000.0 ml. pH 7.4 ± 0.2 at 25°C


  1. Using a sterile straight inoculating needle, select an isolated colony from the culture plate.
  2. Remove tube cap, stab needle into the butt of the medium.
  3. Withdraw inoculating needle to the slant and streak back and forth up the slant surface.
  4. Replace cap loosely on the tube.
  5. Incubate aerobically overnight (18–24 hours) at 35 ± 2°C, observe and record reactions.
  6. Good growth must occur in the butt and slant, or equivocal reactions will result.


Kligler Iron Agar

Carbohydrate Fermentation

Slant Reaction

Positive Test : Yellow (acid)

Negative Test: Red (alkaline)

Butt Reaction

Positive Test: Yellow (acid)

Negative Test: Red (alkaline)

KIA Color Reaction

Red slant/ yellow butt: dextrose (+), lactose (-)

Yellow slant/ yellow butt: dextrose (+), lactose (+)

Red slant/ red butt: dextrose (-), lactose (-)

H2S Production

Positive Test: Black color throughout medium, a black ring at the juncture of the slant and butt, or a black precipitate in the butt

Negative Test: No black color development

Gas Production

Positive Test: Bubbles in the medium, cracking and displacement of medium, or separation of medium from side and bottom of tube.

Negative Test: No bubbles and no separation or displacement of the medium.

Kligler Iron Agar


1. Read and interpret kligler iron agar test reactions within an 18-24 hour incubation period. A reaction read at <18 hours may be falsely interpreted as negative because the carbohydrate fermented may not yet have produced enough acid to change the phenol red indicator. A reaction read at >24 hours may be incorrect due to peptone utilization which would result in an alkaline pH shift.5

2. H2S production in the kligler iron agar test butt may mask the acidity produced; however, if H2S is present an acid condition does exist, even if it is not observable.

3. Kligler iron agar test medium does not contain an inhibitor and many organism types may grow. Before inoculating KIA, be sure the organism is a catalase-positive, gram-negative bacillus.

4. To enhance the alkaline condition in the slant, a free exchange of air must be permitted. If kligler iron agar test tubes are tightly capped, an acid reaction caused solely by dextrose fermentation will also involve the slant. Therefore, tubes must have loosened caps during incubation.

5. The H2S indicator present in KIA is reported to be less sensitive than some methods, such as the lead acetate strip; therefore, some H2S- positive, gram-negative bacilli may not produce H2S in kligler iron agar test.

6. Before inoculation, a slight precipitate may be present on the slant. This will not effect the performance of the medium.

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