Kligler’s Iron Agar Test

Kligler’s Iron Agar Test is used for differential identification of gram-negative

Enterobacteriaceae family, based on fermentation of glucose, lactose and production of
hydrogen sulphide.

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.

3rd KIA Reaction : Alkaline Slant/Acid Butt (K/A)

 The base color changed into yellow, indicating that the specimen produces glucose.
 The slant color remains red, indicating that the specimen does not do fermentation of
lactose.
 There is split in agar, indicating that the specimen produces gas (CO2)
 It can be concluded that this bacteria is Non-Lactose Fermenter

4th KIA Reaction : Alkaline Slant/Alkaline Butt (K/K):

 The base and slant colors remain red, indicating that there is no carbohydrate
fermentation.
 There is split in agar, indicating that the specimen produces gas (CO2)
 It can be concluded that this bacteria cannot do carbohydrate fermentation