Kligler’s Iron Agar (KIA): Principle, Procedure and Results

Kligler’s Iron Agar (KIA) is used for the detection of carbohydrate fermentation. Reactions of KIA helps to include/exclude particular bacterial isolate in the family Enterobacteriaceae.  If an organism cannot ferment glucose, then in KIA, alkaline slant-alkaline-butt (no change) reaction is observed, this reaction alone is sufficient to exclude an isolate from the Enterobacteriaceae family. KIA is also used for the presumptive identification of Salmonella Shigella and other members of the Enterobacteriaceae family.

The composition of Kligler’s Iron Agar (KIA) is identical to Triple Sugar Iron Agar (TSI) except that 10 gm sucrose is added in TSI.

Composition of Kligler’s Iron Agar

  • Beef extract: 3 gm
  • Yeast extract: 3 gm
  • Peptone: 15 gm
  • Proteose peptone: 5 gm
  • Lactose 10 gm
  • Glucose: 1 gm
  • Ferrous sulfate: 0.2 gm
  • Sodium chloride: 5 gm
  • Sodium thiosulfate: 0.3 gm
  • Agar: 12 gm
  • Phenol red: 0.024 gm
  • Distilled water to equal 1 L
  • Final pH : 7.4

The incorporation of four protein derivatives- beef extract, yeast extract, peptone and proteose peptone makes KIA nutritionally very rich. The lack of inhibitors permits the growth of all but the most fastidious bacterial species (excluding the obligate anaerobe).

Glucose and lactose are carbohydrate source and are evenly distributed throughout both the slant and butt (deep) portion of the tube. However, lactose is present in a concentration of 10 times that of glucose. Unlike TSI, sucrose is absent in KIA.  Sodium thiosulfate is the source of the sulfur and ferrous sulfate acts as a hydrogen sulfide detector.

Phenol red indicator is yellow below a pH of 6.8. Because the pH of the un-inoculated medium is buffered at 7.4, relatively small quantities of acid production results in a visible color change.

Preparation of KIA

Kligler iron agar (KIA) is best prepared from ready to use dehydrated powder available from commercial suppliers of culture media. The medium is usually used at a concentration of 5.5 gm in every 100 ml distilled water (concentration may vary depending on the manufacturer).

  1. Prepare the medium as instructed by the manufacturer. When cooled to 50-55°C, mix well and dispense in 6 ml amounts in large size tubes (APPROX. 16-160 mm).
  2. Sterilize by autoclaving (with caps loosened) at 121°C for 15 minutes.
  3. Allow the molten agar to solidify in a slant position. This results in essentially two reaction chambers within the same tube.
    • The slant portion: It is exposed throughout its surface to atmospheric oxygen, is aerobic.
    • The butt or deep portion: It is protected from the air and is relatively anaerobic.
  4. Date the medium and give it a batch number and store in a cool dark place or at 2-8 °C.
Inoculation in KIA

Note: It is important when preparing the media that butt should be longer than the slope or at least equal, approximately 3 cm each so that this two-chamber effect is preserved.

Shelf-life: About 3 weeks or longer providing the tube caps are tightly screwed and there is no change in the appearance of the medium to suggest contamination, deterioration, or alteration of pH.

pH of the medium: This should be within the range of pH 7.2- 7.6 at room temperature.

Inoculation of KIA

Use a straight wire to inoculate KIA medium, first stabbing the butt and then streaking the slope in a zig-zag pattern. After inoculation, make sure the tube tops are left loose.

Principle/ working mechanism of KIA

Bacteria growing on KIA produces three general types of reactions.

  1. No change in the medium i.e. Alkaline Slant/Alkaline Butt (K/K): No carbohydrate fermentation. Presence of non-fermentative gram-negative bacilli (organisms that are unable to produce acids from the fermentation of glucose or lactose) e.g. Pseudomonas aeruginosa.
  2. Non-lactose fermenter i.e. Alkaline Slant/Acid Butt (K/A): Glucose is fermented but not lactose. This is characteristic of non–lactose-fermenting bacteria, such as Shigella species. Initially,  non-lactose fermenter does acidification of both the deep and the slant of the medium via the fermentation of the glucose but the slant reverts back to alkaline pH as alkaline amines are formed from the oxidative decarboxylation of peptides (derived from protein in the medium) near the surface.
  3. Lactose fermenter Acid Slant/Acid Butt (A/A): Complete permanent acidification of both the deep and the slant of the tube by lactose-fermenting bacteria. Glucose and lactose both are fermented. This is characteristic of lactose-fermenting coliforms, such as Escherichia coli and the Klebsiella–Enterobacter species.

Gas production: Space at the bottom of the tube and the split in the agar in the middle of the tube indicates gas (CO2 ) production by the organism.


H2S production:  Black deep (butt) indicates hydrogen sulfide (H2S) production. Sometimes the butt portion will be entirely black. In such a case, it is assumed that butt portion of the tube is acidic (yellow color is masked due to the H2S production). Non–lactose-fermenting, hydrogen sulfide-producing bacteria, such as Salmonella species, Citrobacter species, and Proteus species produce alkaline slant/acid (black) butt.

Detail Explanation of Principle:

Non-Lactose Fermenter

If the KIA tube is inoculated with a glucose-fermenting organism that cannot utilize lactose, only a relatively small quantity of acid is obtained from the fermentation of 0.1% concentration of glucose in the medium.

Initially, during the first 8 to 12 hours of incubation, even this amount of acid may be sufficient to convert both the deep and the slant color to yellow.

Within the next few hours, however, the glucose supply is completely exhausted, and the bacteria begin oxidative degradation of the amino acids within the slant portion of the tube where oxygen is present. This results in the release of amines that soon counteract the small quantities of acid present in the slant; by 18 to 24 hours, the entire slant reverts to an alkaline pH, and the color returns to red.

In the deep (anaerobic portion) of the tube, however, amino acid degradation is insufficient to counteract the acid formed, and the medium remains yellow.

Thus, the alkaline-slant–acid-butt reaction on KIA is an important indicator that the test organism is a non–lactose-fermenter.

Lactose Fermenter

If the KIA tube is inoculated with a lactose-fermenting organism, then, even though the glucose is completely used up after the first 8 to 12 hours, fermentation continues as the organism is able to use lactose (present in 10 times the concentration of glucose).

 Consequently, when the tube is examined at the end of 18 to 24 hours, acid production from fermentation of lactose is still occurring, and both the slant and the deep appear yellow, resulting in an acid-slant–acid deep reaction.

Results of Kligler Iron Agar (KIA)

Kligler’s iron agar (KIA) tubes with several reaction patterns
A: Acid/Acid, Gas; B: Acid/Acid, No gas; C: Alkaline/Acid; D: Alkaline /Acid, H2S+; E: Alkaline/Alkaline

Organism and their reaction in KIA

Name of the Organism Reactions
(Red= Alk, Yellow = Acid)
Gas H2S
Pseudomonas aeruginosa Alk/Alk
E.coli
AD Group
Acid/Acid
Alk/Acid
+
+

Shigella species (S.dysenteriae, S.flexneri, S.boydii, and S. sonnei) Alk/ Acid
Salmonella Paratyphi A Alk/ Acid +
Salmonella Paratyphi B Alk/ Acid + +
Salmonella Paratyphi C Alk/ Acid + +
Salmonella Typhi Alk/ Acid +
Other Salmonella serovars Alk/ Acid + (weak) d
Klebsiella pneumonia Acid/Acid ++
Klebsiella oxytoca Acid/Acid ++
Enterobacter aerogenes Acid/Acid ++
Enterobacter cloacae Acid/Acid ++
Citrobacter freundii Acid/Acid or Alk/Acid + +
Serriatia mercescens Alk/Acid +
Proteus vulgaris Alk/Acid +/- +
Proteus mirabilis Alk/Acid + +
Morganella morganii Alk/Acid +
Yersinia enterocolitica Alk/Acid

Alk: Alkaline; + 90% or more strains are positive; – 90% or more strains are negative; ++ strong positive reaction; d: different strains give different results.

Since Vibrio cholerae ferments glucose as well as sucrose, alkaline-slant/ acid deep is observed in Kligler’s Iron Agar but acid-slant/acid-butt reaction is seen on Triple Sugar Iron Agar (TSI). Gas is not formed and
H2S is not produced.

*Since V. cholerae ferments glucose as well as sucrose, alkaline-slant/ acid deep is observed in Kligler’s Iron Agar but acid-slant/acid-deep reaction is seen on Triple Sugar Iron Agar (TSI).

References and further reading

  • Color Atlas and Textbook of Diagnostic Microbiology, Koneman, 5th edition
  • Bailey & Scott’s Diagnostic Microbiology, Forbes, 11th edition
  • District Laboratory Practice in Tropical Countries Part-2. Monica Cheesbrough, 2nd edition
About tankeshwar 386 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion, I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.

3 Comments

  1. Good day, I love all your works, they have been really educative. Please can you post something about the Isolation of Saccharomyces cerevisiae? I am a masters student in microbiology working on it, and I have been having difficulties in assessing information. Your contributions would be highly appreciated.Thanks.

    • Dear Ayoade, I appreciate your feedback. I’m honoured to get your request to write about this particular topic but I’m so sorry, I have very little exposure/practical knowledge about the isolation of Saccharomyces cerevisiae. As I am working in the diagnostic Microbiology laboratory of a Hospital and teaching Medical students, my expertise is mainly about pathogenic microorganisms. I hope you can get this information from Food Microbiologist or any books in Food Microbiology. Wishing you all the best.

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