Bacteriology 102:
Kligler Iron Agar Reactions

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KIA Reactions
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Adapted from this page of our Differential Media Site, and serving as a prelude to how to
"program" such a medium to look for certain physiological types of enteric bacteria
(such as those presented here and here).

Formulation of Kligler Iron Agar
Source of amino acids which may be deaminated (alkaline rx.) Peptone
Proteose peptone
Beef extract
Yeast extract
Amino acid added to note its decarboxylation (alkaline rx.) None
Fermentable sugar(s) (acid rx.) Lactose (1%)
Glucose (0.1%)
pH indicator Phenol red:
  net acid = yellow
  net alkaline = red
Source from which H2S may be produced Sodium thiosulfate
Indicator of H2S production Ferrous sulfate

In the table on the right, note the relative amounts of the two sugars, lactose and glucose. By the degree of acid produced from fermentation, differentiation can be made between non-fermenters, glucose-fermenters (which produce a relatively small amount of acid) and those which ferment both glucose and lactose (producing a relatively large amount of acid which diffuses througout the medium and easily overneutralizes the aerobic deamination reaction in the slant).

Organisms which produce hydrogen sulfide from the reduction of thiosulfate are easily detected. The H2S reacts with the iron in the medium to produce ferrous sulfide, a black precipitate.

The medium is inoculated with the needle, first stabbing down the center to the bottom of the tube and then streaking up the slant. Incubation is for one day at 37°C. The various combinations of reactions are explained and illustrated below. (Tube "C" is the uninoculated control tube which shows an orange (neutral) reaction throughout.)

corresponding tube
no. above
1 2 3 4* 5
deamination of
amino acids
(aerobic alkaline rx.)
+ + + + +

(minor acid rx.)
+ + + +

(major acid rx.)
+ +
H2S production
(black color)
+ +**
typical examples Pseudomonas
(a non-enteric)
E. coli,
coliform strains of
Citrobacter that are H2S+,
H2S+ E. coli,
lactose+ Salmonella

Tube 4: Much gas is often seen for this tube, evidenced by cracks in the medium. Also, lactose fermenters which are methyl red-negative may show a "reversion" toward an alkaline reaction as neutral products are formed from some of the acid. This appears as shown in Tube 4A where a slight reddening of the slant occurs as the alkaline deamination reaction becomes no longer over-neutralized by acid from fermentation. How might such a tube appear after two or more days of incubation? (Regarding the methyl red test, recall the activities of enterics in MR-VP Broth, illustrated here.)

**  Tube 5: Enough acid can be produced to cause the black iron sulfide precipitate to break down and not be seen. In this case, the tube will look like no. 4.

Another way of looking at reactions in KIA is shown below. In modifying this medium to detect other fermentation reactions, one could substitute another sugar for glucose and also one or more other sugars for lactose. (One can assume that glucose is fermented if any other sugar is fermented.)

• Bacteriology 102 Home Page.
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Page last modified on 11/11/09 at 3:15 PM, CDT.
John Lindquist, Department of Bacteriology,
University of Wisconsin – Madison