KLIGLER IRON AGAR (KIA)

Cat. no. L70 KIA Slant, 16x125mm Tube, 8ml Slant 20 or 100 tubes/box
Cat. no. R70 KIA Slant, 13x100mm Tube, 4.5ml Slant 20 or 100 tubes/box

INTENDED USE

Hardy Diagnostics Kligler Iron Agar (KIA) is recommended for use in differentiating certain members of the Enterobacteriaceae by demonstrating hydrogen sulfide production and the fermentation of dextrose and lactose.

SUMMARY

Kligler Iron Agar (KIA) combines features of Kligler's Lead Acetate medium and Russell's Double Sugar Agar. (8,9) Phenol red is added as the color indicator. The basal medium of KIA is composed of casein and meat peptones with the addition of lactose and dextrose. The production of acid by lactose and/or dextrose fermentation results in color changes of the phenol red pH indicator. Presence of the carbohydrates thus enables the differentiation of species of enteric bacilli.

Non-lactose fermenters initially produce a yellow slant and butt as a result of dextrose fermentation. The concentration of dextrose is only one percent and, therefore, is rapidly exhausted. Once the dextrose is depleted, the reaction reverts to alkaline (red slant) due to the oxidation of acids. Reversion does not occur in the butt of the medium where an acidic environment (yellow butt) is maintained. Lactose fermenting organisms produce yellow slants and butts. There is no reversion to red in the slant because enough acid is produced to maintain an acid pH under aerobic conditions. Non-fermenters produce red slants and butts. H 2 S production results in a blackening of the medium, either throughout the butt or in a ring formation near the top of the butt. Gas production is demonstrated by the presence of bubbles or cracks in the medium.

FORMULA

Ingredients per liter of deionized water:*

Peptone 15.0gm
Lactose 10.0gm
Proteose Peptone 5.0gm
Sodium Chloride 5.0gm
Beef Extract 3.0gm
Yeast Extract 3.0gm
Dextrose 1.0gm
Sodium Thiosulfate 0.3gm
Ferrous Sulfate 0.2gm
Phenol Red 0.024gm
Agar 12.0gm

Final pH 7.4 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-30ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

Specimen Collection: This product is not intended for primary isolation of patient specimens. It should be used only with cultures of isolated organism. This product is used in conjunction with other biochemical tests to identify cultures of isolated organism. Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. If there is to be a delay in processing, the specimen should be inoculated onto an appropriate transport media and refrigerated until inoculation.

1. Allow KIA to warm to room temperature prior to inoculation.

2. Obtain a pure culture of the organism to be tested. Select well-isolated colonies.

3. With an inoculating needle, pick the cener of well-isolated colonies obtained from solid culture media.

4. Stab the center of the medium into the deep of the tube to within 3-5mm from the bottom.

5. Withdraw the inoculating needle and streak the surface of the slant.

6. Loosen closure on the tube before incubating.

7. Incubate aerobically at 35ºC. for 18-48 hours.

8. Read tubes for acid production of the slant/butt, gas, and hydrogen sulfide reactions.

INTERPRETATION OF RESULTS

An alkaline slant-acid butt (red/yellow) indicates fermentation of dextrose only.

An acid slant-butt (yellow/yellow) indicates fermentation of dextrose and lactose.

An alkaline slant-alkaline butt (red/red) indicates that neither dextrose nor lactose was fermented (non-fermenter).

Cracks, splits, or bubbles in the medium indicates gas production.

A black precipitate in the butt indicates hydrogen sulfide production.

LIMITATIONS

It is important to stab the butt of the medium. Failure to stab the butt invalidates this test. The integrity of the agar must be maintained when stabbing. Caps must be loosened during this test or erroneous results will occur.

An organism that produces hydrogen sulfide may mask acid production in the butt of the medium. However, hydrogen sulfide production requires an acid environment, thus the butt portion should be considered acid if hydrogen sulfide is produced.

Certain species or strains may give delayed reactions or completely fail to ferment the carbohydrate in the stated manner. However, in most cases, if the organism fails to ferment dextrose within 48 hours and growth is definitely present, the organism is most likely not in the Enterobacteriaceae family.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, swabs, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Salmonella enterica
ATCC ® 14028
C 18-24hr 35°C Aerobic Growth; red slant, yellow butt, black butt, H 2 S positive, gas positive
Escherichia coli
ATCC ® 25922
C 18-24hr 35°C Aerobic Growth; yellow slant, yellow butt, H 2 S negative, gas positive
Pseudomonas aeruginosa
ATCC ® 27853
C 18-24hr 35°C Aerobic Growth; red slant, red butt, H 2 S negative, gas negative

User Quality Control

PHYSICAL APPEARANCE

Kligler Iron Agar should appear slightly opalescent, and orange-red in color.

S. typhimurium growing in KIA

Salmonella enterica (ATCC ® 14028) growing in Kligler Iron Agar (Cat. no. L70). Incubated aerobically for 24 hours at 35ºC.

E. coli growing in KIA

Escherichia coli (ATCC ® 25922) growing in Kligler Iron Agar (Cat. no. L70). Incubated aerobically for 24 hours at 35ºC.

P. aeruginosa growing in KIA

Pseudomonas aeruginosa (ATCC ® 27853) growing in Kligler Iron Agar (Cat. no. L70). Incubated aerobically for 24 hours at 35ºC.

KIA

Uninoculated tube of Kligler Iron Agar (Cat. no. L70).

REFERENCES

1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

3. Edwards, P.R. and M.A. Fife. 1961. Appl. Microbiol .; 9:478.

4. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

5. MacFaddin, J.F. 1985. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria, Vol. I. Williams & Wilkins, Baltimore, MD.

6. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

7. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

8. J. Med. Res.; 25:217, 1911.

9. Am. J. Public Health; 7:1042, 1917.


ATCC is a registered trademark of the American Type Culture Collection.

030116gr