Kligler Iron Agar Test: Principle, Procedure, and Result
Kligler Iron Agar Test
Many of the biochemical tests are performed to detect the ability of the micro-organism to utilize the enzyme in the culture medium. Kligler iron test is also one of the biochemical tests, which employs a medium for the identification of the species of Enterobacteriaceae, based on the fermentation of the double sugar and the production of hydrogen sulphide.
Kligler Iron Agar Test was first determined by Kligler in the year 1918 to describe the medium for detecting the production of hydrogen sulphide and it also helps us to differentiate the species of Salmonella.
This was first modified by Bailey and Lacey by substituting the phenol red indicator instead of Andrade indicator in the medium.
The medium used in the test is commonly known as KIA. It is also recommended that determination of the Hydrogen sulphide depends on the production of the Enteric gram-negative bacilli and also in detecting the production of H2S by using some strains of Pseudomonas.
What is Kligler Iron Agar Test?
Kligler iron agar test is used for detecting the fermentation of carbohydrate in the medium.
Here the reaction of the KIA helps us to include or exclude a particular bacterial species to isolate it from the family of Enterobacteriaceae.
If the organisms cannot ferment the carbohydrate or glucose present in the medium, then alkaline Slant in alkaline butt reaction can be observed in the Klinger Iron Agar.
This reaction is sufficient for excluding and isolating the species belonging to the Enterobacteriaceae family.
Klinger Iron Agar is also helpful in identifying the species named Salmonella shigella along with the other members of the Enterobacteriaceae family.
Kligler Iron Agar Test Objective
The main aim of the test is to differentiate the organisms by demonstrating the medium with the hydrogen sulphide production and to determine the fermentation of dextrose and lactose.
Kligler Iron Agar Test Principle
The medium of Klinger iron consists of lactose and the glucose in addition to peptone, HM peptone B and the yeast extract, which helps us to enable the difference between the species of the enteric bacilli.
Generally, in this test, phenol red indicator is used as a pH indicator, which brings about a change in colour in the medium when the acid response is detected during the fermentation process of the sugars.
Fermentation of the dextrose results in the production of acid, which in turn changes the colour of the medium from red to yellow.
If there is only minimum amount of sugar present in the medium, the indicator remains red and there will not be any change in the colour.
On the other hand, when lactose is fermented, there will be large amount of acid production in the medium and it also avoids reoxidation and results in the yellow colour of the whole medium.
The combination of ferrous sulphate and the sodium thiosulphate enables us to detect the production of hydrogen Sulphide and it is evidenced by a black colour or through the formation of butt.
In some cases, it can also be determined by ring formation near or top of the butt.
The production of butts and yellow slants in the lactose fermentation is due to production of high amount of acid in the medium which induces the change in pH under the aerobic conditions.
Whereas, the tubes which does not show any change in colour indicates that there is no presence of glucose or lactose in the medium.
However, the gas production is detected by formation of the individual bibles or by splitting of the agar, due to the formation of the cracks in the butt of the medium.
Kligler Iron Agar Test Reagents
| Ingredients | Gram/Litre |
| Peptone | 15.0 |
| HM peptone | 3.0 |
| Yeast extract | 3.0 |
| Protease peptone | 5.0 |
| Lactose | 10.0 |
| Dextrose | 1.0 |
| Ferrous sulphate | 0.2 |
| Sodium chloride | 5.0 |
| Sodium thiosulphate | 0.3 |
| Phenol red | 0.02 |
| Agar | 15.0 |
Kligler Iron Agar Test Procedure
Initially, a well isolated colony from a solid culture medium is picked from the centre of the medium using an inoculating needle.
Here the medium used for identification of the colonies includes MacConkey Agar, Bismuth Sulphide Agar, or Deoxycholate Citrate Agar are used as the plating medias.
Further the stab is taken from the centre of the medium into the deep tube which has about 3 to 5mm of depth.
Then the inoculating medium is withdrawn and it is streaked against the surface of the slant.
The caps of the tubes are closed loosely before incubating so that there will be an aeration in the medium.
Then the tubes are incubated aerobically at a temperature of about 35ºC for about 18 to 48hours.
After incubation the tubes are observed for acid production which can be detected by change in colour or formation of butts.
Kligler Iron Agar Test Result
The results are interpreted by Carbohydrate fermentation, Kligler Iron Agar colour reactions and by Hydrogen sulphide production.
Carbohyderate Fermentation
Slant Reaction:
 Positive reaction- Yellow colour (acid)
 Negative reaction- Red colour (alkaline)
Butt reaction:
 Positive result _ Yellow (acid)
 Negative result – Red (alkaline)
Kligler Iron Agar Color Reactions
 Formation of red slant or yellow butt – Presence of dextrose and absence of lactose.
 Formation of yellow slant or yellow butt – Presence of dextrose and lactose
 Formation of red slant or red butt – Absence of dextrose and presence of lactose.
Production of Hydrogen Sulfide
 Positive Test: Here the positive result is determined but formation of black colour throughout the medium and a black ring at the juncture of the slant and butt. In some cases, it is also determined by formation of black precipitate in the butt.
 Negative Test: Here the negative test is indicated when there is no formation of black colour.
Gas Production
Positive Test: Formation of bubbles in the medium along with cracking or displacement of the medium. In some cases, it also leads to separation of the medium.
Negative Test: There will no bubbles and displacement or separation of the medium does not take place.
Kligler Iron Agar Test Citations
- The phenylalanine test on Kligler’s iron agar for the identification of Proteus. Nature . 1959 May 30;183(4674):1537-8.
- Rapid biochemical screening for Salmonella, Shigella, Yersinia, and Aeromonas isolates from stool specimens. J Clin Microbiol. 1994 Jun; 32(6): 1583–1585.
- Biochemical differentiation of the Enterobacteriaceae with the aid of lysine-iron-agar. Appl Microbiol . 1966 Mar;14(2):212-7.Â
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