Kligler-Hajna

The medium of Kligler-Hajna is a medium allowing the simultaneous search for:

the use of the Lactose;
the fermentation of the Glucose;
production of H₂S;
production of Gas;
the Lysin décarboxylase.
the B-galactosidase for the bacteria Lactose - (test ONPG).

Composition

- Peptone ......................... 15 G - Beef extract ............... 3 G - Extracts from yeast ............... 3 G - peptic Peptone of meat ...... 5 G - Glucose ......................... 1 G - Lactose ......................... 10 G - phenol Red ................. 25 Mg - Sodium chloride .............. 5 G - Ferrous Sulfate .............. 0,2 G - Thiosulfate of sodium ........... 0,3 G - Agar-agar ....................... 11 G

pH = 7,5

Peptones are rich in Lysine. The medium is conditioned out of tubes with a slope and a base.

Sowing

the slope must be abundantly sown (tight scratches).
the base is sown simple puncture.

Incubation: 37 °C during 18 with 24:00 (not to exceed this time) after having to unscrew the stopper partially what allows the gaseous exchange.

Reading, interpretation

Use of glucids

To be able to use the Lactose, a bacterium must have two Enzyme S (diagram opposite):

a β-galactoside Perméase (1);
a β-galactosidase (2).

It follows from there a series of reactions of degradation of glucose (3) leading to the energy production E.

Glucose inhibits the synthesis of these two enzymes. It is what one calls the effect Glucose. Indeed, the use of glucids follows the law of Diauxie, the use of the Lactose will take place only after exhaustion of glucose. However, glucose is in small quantity compared to lactose and the slope is sown abundantly.

The slope and the base must be interpreted independently.

Interpretation of the slope

Initially, the bacteria use the Glucose like source of carbon and energy (because of the glucose effect). This use is accompanied by the production of organic acids from where the turn of the red to the yellow of the medium (figure 1A).
In the second time, because of fast development (in aerobiosis) and of the weak concentration in glucose in less than 12 midnight, the totality of glucose is degraded, there is disappearance glucose effect. Then, two possibilities:

If the bacterium is β-galactoside perméase + AND β-galactosidase +: the bacteria use lactose with production of organic acids: the turn with the yellow is confirmed (figure 1B).
If the bacterium is β-galactoside perméase + and β-galactosidase - OR β-galactoside perméase - and β-galactosidase -: the bacteria cannot use lactose. The bacteria use then as source of carbon and energy peptones: the metabolism protic releases from the products basic (ammonia, amines…) from where a recoloration with the red of the slope (figure 1).

As follows:

red Slope : bacterium Lactose - (figure 1A);
yellow Slope : bacterium Lactose + (figure 1B).

Interpretation of the base

Initially, the bacteria ferment glucose with important production of organic acids (because of the glucose effect) (figure 1D).
As on the slope, in less than 12 midnight, all glucose is fermented. However, if the bacteria are lactose -, the use of peptones with an alkalization does not allow the revirage indicator of pH, the base will remain yellow in 24:00 (figure 1D).
If the bacterium is not able to ferment glucose, it does not have there production of aciedes organic, the base remains red (figure 1C).

As follows:

red Base : bacterium Glucose - (figure 1C);
yellow Base : bacterium Glucose + (figure 1D).

Production of gas

The production of gas during the use of glucids is highlighted by the separation of the gélose and/or the bubbles in the gélose.

Production of hydrogen sulfide

It takes place starting from the ion Thiosulfate:

S_2O_3^ {2} + 10:00 ^+ + 8e^- \ longrightarrow 2:00 _2S + 3:00 _2O

The hydrogen sulfide reacts with the ions iron III (Fe³⁺) of iron citrate to form black an iron sulfide precipitate. As follows:

H₂S Bacteria +: black precipitate;
H₂S Bacteria -: no black precipitate;

Presence of a lysin décarboxylase

The Lysine décarboxylase (LDC) décarboxyle the Lysine presents in the medium in Cadavérine (very non-polar amine in basic medium) soluble in the Chloroforme. The Ninhydrine makes it possible to highlight the Cadavérine.

To pour 1 ml of Potash (1 mol/L) on the slope of the medium of Kligler.
To pour 1 ml of Chloroform and to let elutriate.
To take the chloroformic phase (in bottom) and to pour in a tube with hémolyse out of glass.
To add some drops of Ninhydrine (in chloroformic solution with 1 g/L) in the tube to hémolyse.
To incubate a few minutes with the bain-marie with 37 °C.
Reading:

Bacteria LDC +: coloring violet (presence of cadaverine);
Bacteria LDC -: the solution remains colorless.

Remark : the amino-acids do not interfere on the reaction because, in basic medium, they are polar thus insoluble in a non-polar solvent like the Chloroforme.

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