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Introduction to Lab Ex. 18: Fermentation of Carbohydrates
Kligler Iron Agar (KIA)
Introduction to Lab Ex. 18 –
Fermentation Of Carbohydrates: Kligler Iron Agar (KIA)
Carbohydrates are good sources of energy for organisms.
The most efficient metabolic process to harvest the energy
from sugars is aerobic respiration.
In the absence of aerobic conditions, organisms may be able to
metabolize the sugars, though with minimal gain of ATP.
These processes are characterized by the end products.
The ability of cells to break down specific substrates is dependent
on their ability to produce appropriate enzymes.
Catabolic processes primarily include:
aerobic respiration, fermentation
Fermentation is the catabolism of sugars in the absence of oxygen
where the final electron acceptors is an organic molecule.
Fermentation typically results in the formation of organic acids and
alcohols that will accumulate in the medium.
This will result in the lowering of the pH of the medium.
With the inclusion of a pH indicator in the medium the lowering
of the pH can become a visual reaction.
Some bacteria characteristically produce gases during the fermentation
process, which are visible as cracks in the agar or
displacement of the agar in solid medium.
The ability to ferment specific sugars is dependant on the ability of the
bacterium to produce the specific enzymes required for the
transport and metabolism of that particular sugar.
Thus fermentation of various sugars can be used to characterize bacteria.
Kligler Iron agar is a combination medium.
While fermentation of various sugars can be tested to characterize
different bacteria, the ability to ferment glucose and lactose
are of special importance since these can be used to classify
bacteria to various groups.
Gram negative bacteria can be classified as lactose fermenters
and non-lactose fermenters.
To facilitate the testing of both sugars a combination medium is
used.
Kligler Iron agar also tests for the production of H2S by the
bacteria.
KIA contains the 2 sugars: glucose and lactose in a ratio of 1:10.
The medium is used in the form of slants.
The abundance of lactose in the medium compared to glucose allows
for differentiation of fermentation of one sugar against that
of both sugars.
Since lactose is a disaccharide (glucose and galactose) if lactose is
fermented then glucose must be fermented too.
Thus distinction is made between fermentation of one sugar (glucose)
or both sugars (glucose and lactose).
The reaction is made visible by the accumulation of various amounts
of acids altering the pH of the medium to varying extents
(either the slant alone – glucose alone fermented; or both slant
and the butt of the agar changing color – due to increased
amounts of acids produced by both sugars being fermented).
The production of gas in these reactions is made visible by cracks or
displacement of the agar in the medium.
Production of H2S is made visible by the addition of ferrous salts
that precipitate the H2S and form a black precipitate.
Possible reactions:
Alk – alkaline; A- acid; G- gas; - no reaction;
Butt alone yellow – Alk/A (Gas) – Glucose alone fermented,
lactose not fermented
Slant and Butt yellow – A/A (G) – Both glucose and lactose fermented
Slant pink no change in butt – Alk/- - no fermentation
Black coloration – H2S produced
Fermentation reactions have to be read within 18-24 hours
before sugar reversion can occur.
Tube deamination
amino acids
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glucose
lactose
H2S production
fermentation fermentation (black color)
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