Kligler Iron Agar, Modified

Intended Use

Recommended for identification of Yersinia enterocolitica. Also used for differential identification of gram-negative enteric bacilli on the basis of the fermentation of dextrose, lactose and hydrogen sulphide production.

Composition

Final pH (at 25°C): 7.4±0.2

**Formula adjusted, standardized to suit performance parameters # Equivalent to Beef extract

Directions

Suspend 57.41 grams (the equivalent weight of dehydrated medium per litre) in 1000 ml purified/distilled water. Heat to boiling to dissolve the medium completely. Dispense in tubes and sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Allow the tubes to cool in a slanted position to form slope of about one inch butts. Best reactions are obtained on freshly prepared media.

Note: Avoid overheating otherwise may produce precipitate in the medium.

Principle And Interpretation

Kligler Iron Agar is a combination of the lead acetate medium described by Kligler (1) and Russels Double Sugar Agar (2) and is used as a differentiation medium for typhoid, dysentery and allied bacilli (3). Bailey and Lacey substituted phenol red for andrade indicator previously used as pH indicator (4). Kligler Iron Agar differentiates lactose fermenters from the non-fermenters. It differentiates Salmonella Typhi from other Salmonella and also Salmonella Paratyphi A from Salmonella Scottmuelleri and Salmonella Enteritidis (5). Kligler Iron Agar, Modified is used for the identification of Yersinia enterocolitica, as recommended by ISO Committee (6). Fermentation of dextrose results in production of acid, which turns the indicator from red to yellow. Since there is little sugar i.e. dextrose, acid production is very limited and therefore a reoxidation of the indicator is produced on the surface of the medium, and the indicator remains red. However, when lactose is fermented, the large amount of acid produced, avoids reoxidation and therefore the entire medium turns yellow.

Kligler Iron Agar, in addition to tryptone, HM peptone B and yeast extract, contains lactose and glucose (dextrose), which enables the differentiation of species of enteric bacilli. Phenol red is the pH indicator, which exhibits a colour change in response to acid produced during the fermentation of sugars. The combination of or 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 non-fermenters (e.g. Salmonella and Shigella) initially produce a yellow slant due to acid produced by the fermentation of the small amount of glucose (dextrose). When 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). 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. 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.

Pure cultures of suspected organisms from plating media such as MacConkey Agar (M081), Bismuth Sulphite Agar (M027) or Deoxycholate Citrate Agar (M065), SS Agar (M108) etc. are inoculated on Kligler Iron Agar for identification.

Type of specimen

Isolated Microorganism from clinical, food, dairy and water samples.

Specimen Collection and Handling

For clinical samples follow appropriate techniques for handling specimens as per established guidelines (7,8)

For food and dairy samples, follow appropriate techniques for sample collection and processing as per guidelines (9,10,11)

For water samples, follow appropriate techniques for sample collection, processing as per guidelines and local standards.(12)

After use, contaminated materials must be sterilized by autoclaving before discarding.

Warning and Precautions

In Vitro diagnostic use. For professional use only. Read the label before opening the container. Wear protective gloves/ protective clothing/eye protection/face protection. Follow good microbiological lab practices while handling specimens and culture. Standard precautions as per established guidelines should be followed while handling clinical specimens. Safety guidelines may be referred in individual safety data sheets.

Limitations

• Results should be noted after 18-24 hours. Else it might result in erroneous results.
• Straight wire loop should be used for inoculation.
• Pure isolates should be used to avoid erroneous results.

Performance and Evaluation

Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at recommended temperature.

Quality Control

Appearance: Light yellow to pink homogeneous free flowing powder

Gelling: Firm, comparable with 1.5% Agar gel

Colour and Clarity of prepared medium: Red coloured, clear to slightly opalescent gel forms in tubes as slants

Reaction: Reaction of 5.74% w/v aqueous solution at 25°C. pH: 7.4±0.2

pH: 7.20-7.60

Cultural Response

Cultural characteristics observed after an incubation at 35-37°C for 18-48 hours.

Key: (*) Corresponding WDCM numbers. (#) Formerly known as Enterobacter aerogenes

Storage and Shelf Life

Store between 10-30°C in a tightly closed container and the prepared medium at 20-30°C. Use before expiry date on the label. On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in dry ventilated area protected from extremes of temperature and sources of ignition Seal the container tightly after use. Use before expiry date on the label. Product performance is best if used within stated expiry period.

Disposal

User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow established laboratory procedures in disposing of infectious materials and material that comes into contact with clinical sample must be decontaminated and disposed of in accordance with current laboratory techniques (7,8).

Reference

1. Kligler I. J., 1917, Am. J. Publ. Health, 7:1041.
2. Russell F. F., 1911, J. Med. Res., 25:217.
3. Kligler I. J., 1918, J. Exp. Med., 28:319.
4. Bailey S. F. and Lacey G. R., 1927, J. Bacteriol., 13:183.
5. Ewing, 1986, Edwards and Ewings Identification of the Enterobacteriaceae, 4th Ed., Elsevier Science Publishing Co., Inc., N.Y.
6. International Organization for Standardization (ISO), 1994, Draft ISO/DIS 10273.
7. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.
8. 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.
9. American Public Health Association, Standard Methods for the Examination of Dairy Products, 1978, 14th Ed., Washington D.C.
10. Salfinger Y., and Tortorello M.L., 2015, Compendium of Methods for the Microbiological Examination of Foods, 5th Ed., American Public Health Association, Washington, D.C.
11. Wehr H. M. and Frank J. H., 2004, Standard Methods for the Microbiological Examination of Dairy Products, 17th Ed., APHA Inc., Washington, D.C.
12. Lipps WC, Braun-Howland EB, Baxter TE,eds. Standard methods for the Examination of Water and Wastewater, 24th ed. Washington DC:APHA Press; 2023.