Kligler Iron Agar
Intended Use:
Recommended for differential identification of gram-negative enteric bacilli from clinical and non-clinical samples on
the basis of the fermentation of glucose (dextrose), lactose and hydrogen sulphide production.
Composition**
Ingredients Gms / Litre
Peptone 15.000
HM Peptone B # 3.000
Yeast extract 3.000
Proteose peptone 5.000
Lactose 10.000
Dextrose 1.000
Ferrous sulphate 0.200
Sodium chloride 5.000
Sodium thiosulphate 0.300
Phenol red 0.024
Agar 15.000
Final pH ( at 25°C) 7.4±0.2
Directions
Suspend 57.52 grams in 1000 ml purified/distilled water. Heat to boiling to dissolve the medium completely. Mix well and distribute into tubes. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Allow the tubes to cool in slanted position to form slopes with about 1 inch butts. Best reactions are obtained on freshly prepared medium. Do not use screw capped tubes or bottles.
Note: Avoid overheating otherwise it may produce precipitate in the medium.
Principle And Interpretation
Kligler Iron Agar is a combination of the lead acetate medium described by Kligler (1,2) and Russels Double Sugar Agar (3) and is used as a differentiation medium for typhoid, dysentery and allied bacilli (4). 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 Salmonellae and also Salmonella Paratyphi A from Salmonella Scottmuelleri and Salmonella Enteritidis (5). 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 Peptone, 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 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 water samples, follow appropriate techniques for sample collection, processing as per guidelines and local standards (6).
For food and dairy samples, follow appropriate techniques for sample collection and processing as per guidelines (7,8,9). For
clinical samples follow appropriate techniques for handling specimens as per established guidelines (10,11). 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
1. Results should be noted after 18-24 hours to avoid erroneous results.
2. Straight wire loop should be used for inoculation.
3. Pure isolates should be used to avoid erroneous results.
4. Other biochemical and serological tests must be performed for complete identification
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 5.75% 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.
Organism Inoculum Growth(CFU)
Gas H2S Slant Butt
Escherichia coli
ATCC 25922 (00013*) 50-100 luxuriant positive
reaction: negative
reaction, no blackening of medium acidic reaction, yellowing of the medium acidic reaction, yellowing ofthe medium.
