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Dehydrated Culture Media

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KLIGLER IRON AGAR

Code: CM0033

A medium for the identification of Enterobacteriaceae, based on double sugar fermentation and hydrogen sulphide production.

Typical Formula*

gm/litre

`Lab-Lemco’ powder

3.0

Yeast extract

3.0

Peptone

20.0

Sodium chloride

5.0

Lactose

10.0

Glucose

1.0

Ferric citrate

0.3

Sodium thiosulphate

0.3

Phenol red

0.05

Agar

12.0

pH 7.4 ± 0.2 @ 25°C

 
* Adjusted as required to meet performance standards

Directions
Suspend 55g in 1 litre of distilled water. Bring to the boil to dissolve completely. Mix well and distribute into containers. Sterilise by autoclaving at 121°C for 15 minutes. Allow to set as slopes with 1 inch butts.

Description
A differential medium for the identification of Enterobacteriaceae on a basis of double sugar fermentation and hydrogen sulphide production.

Oxoid Kligler Iron Agar based on the original medium1,2,3 combines the principles of Russell 4, double sugar agar, with ferric citrate as an indicator to detect hydrogen sulphide production. The medium is recommended for the identification of colonies picked off from plating media such as MacConkey Agar, Bismuth Sulphite Agar, or Desoxycholate Citrate Agar, etc.

Technique
Smear the surface of a Kligler Iron Agar slope and stab the butt with a colony picked off one of the solid media.
There are three reactions to record when interpreting a KIA tube.

1Carbohydrate utilisation:

(i) slant reaction

(ii) butt reaction

acidity: yellow colour

acidity: yellow colour

alkalinity: red colour

alkalinity: red colour

2 Gas production:

aerogenic

anaerogenic

bubbles or splitting of agar

no gas production

3 H2S production:
blackening in whole or part of butt:-
Record the slant reaction/the butt reaction/gas production/H2S production; in that order.
Red slant/yellow butt - glucose only fermented
Yellow slant/yellow butt - glucose + lactose fermented
Red slant/red butt - neither glucose nor lactose fermented

Reactions after 18 - 24 hours at 35°C

Organism

Slope

Butt

Gas

H2S

Shigella sonnei

Red

Yellow

-

-

Shigella dysenteriae

Red

Yellow

-

-

Salmonella typhi

Red

Yellow

-

+

Salmonella species

Red

Yellow

+

+

Enterobacter species

Red

Yellow

+

-

Klebsiella species

Yellow

Yellow

+

-

Escherichia coli

Yellow

Yellow

+

-

Proteus mirabilis

Red

Yellow

-

+

Morganella species

Red

Yellow

V

-

Citrobacter freundii

Yellow

Yellow

+

+

Yersinia species

Red

Yellow

V

-

V = variable, + = positive, - = negative.

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the prepared medium at 2-8°C.

Appearance
Dehydrated medium: Straw-orange coloured, free-flowing powder
Prepared medium: Red coloured gel

Quality control

Positive controls:
Expected results
 

Slope

Butt

Gas

H2S

Citrobacter freundii ATCC® 8090*

Yellow

Yellow

+

+

Shigella sonnei ATCC® 25931*

Red

Yellow

-

-

Alcaligenes feacalis ATCC® 19018
Red
Red
-
-
Negative control: 

Uninoculated medium.

No change

* This organism is available as a Culti-Loop®

Precautions
It is essential that Kligler Iron Agar is examined and reported at 18-24 hours. Early or late readings will give false results.
KIA will grow both oxidative and fermentative organisms. Confusion will result if care is not taken to distinguish between the two groups.
Always use a straight wire to inoculate the butt, to avoid splitting the agar with a loop.
Pure cultures are essential to avoid misinterpretation.
Do not use screw-capped tubes or bottles for KIA medium. It is essential that air is freely available to growth on the slant.

References
1. Kligler I. J. (1917) Am. J. Pub. Hlth 7. 1042-1044.
2. Kligler I. J. (1918) J. Exper. Med. 28. 319-322.
3. Bailey Sadie F. and Lacey G. R. (1927) J. Bact. 13. 182-189.
4. Russell F. F. (1911) J. Med. Res. 25. 217-229.

 
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