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


To differentiate the enteric gram-negative bacilli based on the production of hydrogen sulfide and fermentation of dextrose and lactose.

Principle of Kligler Iron Agar Test

Kligler Iron Agar (KIA) test is used in differentiating certain members of the Enterobacteriaceae by demonstrating hydrogen sulfide production and the fermentation of dextrose and lactose. Kligler agar used for the test comprises casein and meat peptones that supply nitrogenous compounds and amino acids, vitamins necessary for bacterial growth. The medium comprises two sources of carbohydrate, dextrose, and lactose and also contains phenol red as an indicator for carbohydrate fermentation. The use of two sugars helps to differentiate the organism on the basis of only dextrose fermenting from those which also ferment lactose. The dextrose concentration in the medium, however, is one-tenth of the concentration of lactose.

Non-lactose fermenters initially produce a yellow slant and butt as a result of dextrose fermentation. The concentration of dextrose is only one percent and, therefore, is rapidly exhausted. Once the dextrose is depleted in the medium, the reaction reverts to alkaline conditions demonstrated as red slant due to the oxidation of acids. Reversion does not occur in the butt of the medium where an acidic environment (yellow butt) is maintained. Lactose fermenting organisms produce yellow slants and butts. There is no reversion to red in the slant because enough acid is produced to maintain an acidic pH under aerobic conditions. Non-fermenters produce red slants and butts. H S production results in a blackening of the medium, either throughout the butt or in a ring formation near the top of the butt. Gas production is demonstrated by the presence of bubbles or cracks in the medium.

Media Used

Kligler Iron Agar

Proteose peptone5.0gm
Beef extract3.0gm
Yeast extract3.0gm
Sodium chloride5.0gm
Sodium thiosulfate0.3gm
Ferrous sulfate0.2gm
Phenol red0.024gm

Final pH 7.4 +/- 0.2 at 25ºC.

Procedure of Kligler Iron Agar Test

  1. With a straight inoculating needle, inoculate KIA by stabbing through the center of the medium to the bottom of the tube and then streaking the slant while withdrawing the needle.
  2. Incubate tubes aerobically with loose caps at 35-37ºC for 18-24 hours and examine the reaction of the medium.

Results of Kligler Iron Agar Test

Kligler Iron Agar Test

Image Source: Hardy Diagnostics

Carbohydrate fermentation

  • For slant and butt
    • Positive test: yellow (acid)
    • Negative test: red (alkaline)
  • For butt
    • Positive test: yellow
    • Negative test: red

Red slant/yellow butt: dextrose positive, lactose negative

Yellow slant/yellow butt: dextrose positive, lactose positive

Red slant/red butt: dextrose negative, lactose negative

H2S Production:

  • Positive Test: Black precipitate or color throughout the medium or at the junction between slant and butt  
  • Negative Test: No black color development

Gas production

  • Positive test: the presence of bubbles or cracks in the medium
  • Negative test: the absence of bubbles or cracks in the medium

Limitations of Kligler Iron Agar Test

  1. The medium doesn’t contain any inhibitor so many organism types may grow.
  2. Read and interpret the result within 18-24 hours time period. A reaction read at <18 hours may be falsely interpreted because the carbohydrate fermented may not have produced enough acid to change the phenol red indicator. A reaction read >24 hours may be interpreted incorrectly due to peptone utilization which would result in an alkaline pH shift.
  3. H2S production in the butt may mask the acidity produced.
  4. The H2S indicator present in KIA is considered less sensitive and therefore some H2S positive gram-negative bacilli may not produce H2S in KIA.
  5. The tubes must be loosely capped during incubation because, in tightly capped tubes, an acid reaction caused solely by dextrose fermentation will also involve the slant.

Quality control    

Escherichia coli ATCC 25922- yellow slant/yellow butt, H2S (-), gas (+)

Pseudomonas aeruginosa ATCC 27853- red slant/red butt, H2S (-), gas (-)

Salmonella enterica serovar Typhimurium ATCC 14028- red slant/yellow butt, H2S (+), gas (+)

About Author

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Sagar Aryal

Sagar Aryal is a microbiologist and a scientific blogger. He attended St. Xavier’s College, Maitighar, Kathmandu, Nepal, to complete his Master of Science in Microbiology. He worked as a Lecturer at St. Xavier’s College, Maitighar, Kathmandu, Nepal, from Feb 2015 to June 2019. After teaching microbiology for more than four years, he joined the Central Department of Microbiology, Tribhuvan University, to pursue his Ph.D. in collaboration with Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Saarbrucken, Germany. He is interested in research on actinobacteria, myxobacteria, and natural products. He has published more than 15 research articles and book chapters in international journals and well-renowned publishers.

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