Kligler Iron Agar principles,composition and slants interpretation

Kligler iron slant MLTGEEKS

Kligler Iron Agar abbreviated KIA is a growth media recommended for the differential identification of gram-negative enteric bacilli from clinical and non clinical samples based on the fermentation  sugars that is dextrose, lactose and the production of Hydrogen sulphide

Those bacteria that ferment any of the three sugars in the medium will obviously produce byproducts. These byproducts are usually acids, which result in a change of color of the red pH-sensitive dye (phenol red) to a yellow color.

Some bacteria utilize thiosulfate anion as a terminal electron acceptor, reducing it to sulfide. If this occurs, the newly formed hydrogen sulfide (H2S) reacts with ferrous sulfate in the medium to form ferrous sulfide, which is visible as a black precipitate.

Bacteria that are non-lactose fermenter and ferments glucose, initially causes a yellow slant/yellow bottom (acid/acid reaction) after 8 hours but then converts to a red slant/yellow bottom after 24 hours (alkali/acid reaction).

Principle of Kligler iron Agar

  • Casein peptone and meat or proteose peptones supply nitrogenous compounds, amino acids, and vitamins necessary for bacterial growth.
  • Sodium chloride is a source of essential electrolytes and maintains osmotic equilibrium.
  • In addition to peptone, HM peptone B (Beef extract) and yeast extract, contains

Lactose and glucose (dextrose), which enables the differentiation of species of these enteric bacilli based on fermentation.

  • Phenol red is serves as the pH indicator, It exhibits a colour change in response to acid produced during the process of fermentation of sugars.
  • The combination of ferrous sulphate and sodium thiosulphate enables the detection of hydrogen sulfide production, which is evidenced by a black color either throughout the butt, or in a ring formation near the top of the butt.
  • Lactose fermenters produce yellow slants and butts because of lactose fermentation.The high amount of acids thus produced helps to maintain an acidic pH under aerobic conditions.
  • As for lactose non-fermenters like Salmonella and Shigella,they initially produce a yellow slant due to acid produced by the fermentation of the small amount of glucose (dextrose). When the glucose (dextrose) supply is exhausted in the aerobic environment of the slant, the reaction reverts to alkaline (red slant) due to oxidation of the acids produced. The reversion does not occur in the anaerobic environment of the butt, which therefore remains acidic (yellow butt).
  • Tubes showing original colour of the medium indicates the fermentation of neither glucose (dextrose) nor lactose.
  • Gas production (aerogenic reaction) is detected as individual bubbles or by splitting or displacement of the agar by the formation of cracks in the butt of the medium.
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Composition of Kligler iron agar

The following ingredients are used for the composition Kligler Iron Agar

IngredientsGrams/Litres
Casein Peptone15.000
HM Peptone B (Beef extract)3.000
Yeast extract3.000
Proteose peptone 5.000
Lactose10.000
Dextrose1.000
Ferrous sulphate0.200
Sodium chloride5.000
Sodium thiosulphate0.300
Phenol red0.024
Agar15.000
Final pH at temperature  25°C7.4±0.2

How to Prepare Kligler iron agar

The preparation of Kligler iron agar depends on each laboratory workload or the numbers of culture plates available. Anyway, note that a standard Petri dish is equivalent to 20ml when completely poured.

  • Start by suspending 57.52 grams in 1000 ml distilled water.
  • Now heat to boiling to dissolve the medium completely.
  • Then,mix well and distribute into tubes.
  • Don’t forget to sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
  • Allow the tubes to cool in slanted position to form slopes with  butts of about 1 inch.
  • Note that best reactions are obtained on freshly prepared medium and do not use screw capped tubes or bottles.

NB: It is also advisable to incubate a petri dish or tubes for 24 hour for quality control in order to avoid wastage of resources and ease early detection of contamination after the preparation.

Appearance of Medium after preparation

It’s appears red in coloured, clear to slightly opalescent gel forms in tubes as slants

Kligler Iron slant
Kligler Iron slant

Type of Inoculums

pure isolate from

  • Clinical samples
  • Food samples
  • Dairy samples
  • water samples

Tube reading and interpretation of Kligler iron slants

Incubation should be done at 35-37°C for 18-48 hours before reading

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OrganismsGas productionH2S ProductionSlantButt
Escherichia coli positive reactionnegative reaction, no blackening of mediumacidic reaction, yellowing of the mediumacidic reaction, yellowing of the medium
Klebsiella aerogenespositive reactionnegative reaction, no blackening of mediumacidic reaction, yellowing of the mediumacidic reaction, yellowing of the medium
Citrobacter freundiipositive reactionpositive reaction, blackening of mediumacidic reaction, yellowing of the mediumacidic reaction, yellowing of the medium
Proteus vulgarisnegative reactionpositive reaction, blackening of mediumalkaline reaction, red colour of the mediumacidic reaction, yellowing of the medium
Klebsiella pneumoniaepositive reactionnegative reaction,no blackening of medium acidic reaction, yellowing of the mediumacidic reaction, yellowing of the medium
Salmonella Paratyphi Apositive reactionnegative reaction,no blackening of mediumalkaline reaction, red colour of the mediumacidic reaction, yellowing of the medium
Salmonella Schottmuelleripositive reactionpositive reaction, blackening of medium alkaline reaction, red colour of the mediumacidic reaction, yellowing of the medium
Salmonella Typhinegative reactionpositive reaction, blackening of medium alkaline reaction, red colour of the medium acidic reaction, yellowing of the medium
Salmonella Enteritidispositive reactionpositive reaction, blackening of mediumalkaline reaction, red colour of the mediumacidic reaction, yellowing of the medium
Shigella flexnerinegative reactionnegative reaction,no blackening of medium alkaline reaction, red colour of the mediumacidic reaction, yellowing of the medium
Pseudomonas aeruginosanegative reactionnegative reaction, blackening of mediumalkaline reaction, red colour of the mediumalkaline reaction,red colour of the medium
Yersinia enterocoliticavariable reactionnegative reaction,no blackening of mediumalkaline reaction,red colour of the mediumacidic reaction, yellowing of the medium
Enterobacter cloacaepositive reactionnegative reaction,no blackening of mediumacidic reaction, yellowing of the mediumacidic reaction, yellowing of the medium
Kliger iron slant growth
Kliger iron slants growth

Some limitation of Kligler iron agar slant

  • The results obtained should be noted after 18-24 hours. Else it might result in erroneous results.
  • A straight wire loop should be used for inoculation of the specimen (Stabbing)
  • Pure isolates or culture should be used to avoid erroneous results.

Reference

  • Russell F. F., 1911, J. Med. Res., 25:217.
  • Kligler I. J., 1917, Am. J. Publ. Health, 7:1041.
  • Kligler I. J., 1918, J. Exp. Med., 28:319
  • Bailey S. F. and Lacey G. R., 1927, J. Bacteriol., 13:183.
  • Ewing, 1986, Edwards and Ewing’s Identification of the Enterobacteriaceae, 4th Ed., Elsevier Science Publishing Co., Inc., N.Y
  • American Public Health Association, Standard Methods for the Examination of Dairy Products, 1978, 14th Ed., Washington D.C
  • Downes F. P. and Ito K., (Ed.), 2001, Compendium of Methods for the Microbiological Examination of Foods, 4th Ed., American Public Health Association, Washington, D.C
  • Isenberg, H.D. Clinical Microbiology Procedures Handbook. 2nd Edition
  • Jorgensen,J.H., Pfaller , M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual of Clinical Microbiology, 11th Edition. Vol. 1.
  • E.W., (Eds.), 2015, Standard Methods for the Examination of Water and Wastewater, 23rd ed., APHA, Washington, D.C.
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About the Author: Arthur Westmann

DEFFE ARTHUR (AMOEBAMANN) is the founder and author of MLTGEEKS and MLTEXPO.He’s from Cameroon and is currently a Final year State Medical Laboratory Technician (MLT MA). Beyond lab works, he’s a passionate internet user with a keen interest in web design and blogging. Furthermore He likes traveling, hanging around with friends and social networking to do in his spare time.

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