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Kligler’s Iron Agar Test 
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The Kligler’s Iron Agar test employs a medium for the identification of Enterobacteriaceae, based on double sugar fermentation and hydrogen sulphide production. 

Principle
Hydrogen sulfide determinations using Kligler Iron Agar should be limited to members of Enterobacteriaceae.


    Media:

    Composition of Kligler’s Iron Agar


    • Beef extract: 3 gm
    • Yeast extract: 3 gm
    • Peptone: 15 gm
    • Proteose peptone: 5 gm
    • Lactose 10 gm
    • Glucose: 1 gm
    • Ferrous sulfate: 0.2 gm
    • Sodium chloride: 5 gm
    • Sodium thiosulfate: 0.3 gm
    • Agar: 12 gm
    • Phenol red: 0.024 gm
    • Distilled water to equal 1 L
    • Final pH : 7.4
    Method
    1.    Stab the center of the medium into the deep of the tube to within 3-5mm from the bottom.
    2.    Withdraw the inoculating Loop and streak the surface of the slant.
    3.    Loosen closure on the tube before incubating.
    4.    Incubate aerobically at 35ºC. for 18-24 hours.
    5.    Read tubes for acid production of the slant/butt, gas, and hydrogen sulfide reactions.
    Expected Results
    Image result for Kligler’s Iron Agar Test
    1-uninoculated
    2-Glucose (+), Lactose(+),Hydrogen sulfide (-),Gas(-)
    3-Glucose (+), Lactose(+),Hydrogen sulfide (-),Gas(+)
    3-Glucose (+), Lactose(-),Hydrogen sulfide (-),Gas(-)
    4-Glucose (+), Lactose(-),Hydrogen sulfide (+),Gas(-)
    Carbohydrate Fermentation:
    • Red slant/ yellow butt – dextrose (+), lactose (-)
    • Yellow slant/ yellow butt – dextrose (+), lactose (+)
    • Red slant/ red butt – dextrose (-), lactose (-)
    Hydrogen Sulfide Production:
    • Positive Test – Black color
    • Negative Test – No black color 
    Gas Production:
    • Positive Test – Bubbles in the medium, cracking and displacement of the medium, or separation of the medium from the side and bottom of the tube
    • Negative Test – No bubbles and no separation or displacement of the medium.
    Uses
    • The test is recommended for the differential identification of gram-negative enteric bacilli from clinical and non clinical samples on the basis of the fermentation of dextrose, lactose and H2S production.
    • It is used as a differentiation medium for typhoid, dysentery and allied bacilli.
    • It differentiates Salmonella Typhi from other Salmonellae and also Salmonella Paratyphi A from Salmonella Scottmuelleri and Salmonella Enteritidis.
    • Kligler Iron Agar test differentiates lactose fermenters from the nonfermenters.
    Limitations
    • Failure to stab the butt invalidates this test.
    • Certain species or strains may give delayed reactions or completely fail to ferment the carbohydrate in the stated manner.
    • Results should be noted after 18-24 hours. Else it might result in erroneous results.
    • Pure cultures are essential when inoculating Kligler Iron Agar. If inoculated with a mixed culture, irregular observations may occur.
    • Hydrogen sulfide producing organisms may produce a black precipitate to such a degree that the reaction in the butt is completely masked. If hydrogen sulfide is produced, dextrose is fermented even if it is not observed.

    References
    www.time2026end.com
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