LYSINE IRON AGAR (LIA)

Cat. no. L25 LIA, 16x125mm Tube, 8ml Slant 20 or 100 tubes/box
Cat. no. R22 LIA, 13x100mm Tube, 4.5ml Slant 20 or 100 tubes/box

INTENDED USE

Hardy Diagnostics Lysine Iron Agar (LIA) is recommended for use in differentiating certain members of the Enterobacteriaceae, especially Salmonella species, by demonstrating hydrogen sulfide production and the decarboxylation or deamination of lysine.

SUMMARY

Edwards and Fife designed LIA in 1961 to presumptively identify Salmonella species, including lactose fermenting Salmonella arizonae, which has been implicated in food-borne outbreaks of gastroenteritis. (3) This is accomplished by the elimination of lactose from the medium, as some strains of S. arizonae actively ferment lactose, which may suppress the production of hydrogen sulfide. As a result these colonies may be overlooked on mediums containing lactose, since the characteristic black or darkened colonies are not seen. By eliminating lactose, these organisms will produce an abundance of hydrogen sulfide in the medium and may be detected. The incorporation of lysine is another important addition, as most species of Salmonella produce the lysine decarboxylase enzyme. Proteus and Providencia species also may be presumptively identified on this medium, as they produce a red slant and an alkaline butt.

The indicator in LIA is bromcresol purple. An alkaline reaction is seen by the presence of a purple color, and an acidic reaction is indicated by the appearance of a yellow color. Sodium thiosulfate is incorporated into this medium as the source of hydrogen sulfide, and ferric ammonium citrate as the indicator, which turns the butt black in the presence of free hydrogen sulfide gas. Dextrose is the carbohydrate source, and is added in a concentration of 0.1 %. Enteric organisms that are capable of fermenting dextrose will produce acid (a yellow butt and a purple slant), and sometimes gas, seen as cracks and bubbles in the medium. Lysine is added to show the decarboxylation reaction, which causes an alkaline situation to occur, seen as a purple butt. The yellow color is seen only if lysine decarboxylation does not occur, as this reaction overcomes any acidic (yellow) conditions. If lysine is deaminated in the presence of oxygen (the reaction seen in the presence of Proteus and Providencia species), a red color change is seen on the slant.

LIA is contained in a tube, and slanted to form a deep butt and short slant. Inoculation is performed with a straight needle by stabbing to the base of the butt, and streaking the slant when the needle is removed. The cap is replaced loosely to facilitate an aerobic atmosphere.

FORMULA

Ingredients per liter of deionized water:*

L-Lysine Hydrochloride 10.0gm
Peptone 5.0gm
Yeast Extract 3.0gm
Dextrose 1.0gm
Ferric Ammonium Citrate 0.5gm
Sodium Thiosulfate 0.04gm
Bromcresol Purple 0.02gm
Agar 15.0gm

Final pH 6.7 +/- 0.2 at 25ºC.

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

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8 degrees C. 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.

Method of Use:

1. Let the LIA medium warm to room temperature before use.

2. Using an inoculation needle, obtain a pure culture of the organism to be tested. Select well-isolated colonies.

3. Inoculate by stabbing to the bottom of the tube. Streak the surface of the slant while withdrawing the needle.

4. Incubate tubes aerobically with loose caps at 35-37ºC. for 18-48 hours. Examine reaction of medium.

INTERPRETATION OF RESULTS

Lysine decarboxylation is indicated by an alkaline (purple) medium. Deamination of lysine is detected by a red slant. Proteus and Providencia spp. deaminate lysine which results in a distinctive red slant over an acid (yellow) butt. Dextrose fermentation is indicated by a purple slant and a yellow butt. H 2 S production results in a blackening of the medium, especially in the butt. Gas production is demonstrated by the presence of bubbles or cracks in the medium. Consult listed references for the identification of colony morphology and further biochemical tests required for identification. (1,2,4,6)

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.

LIA is not as sensitive in detecting hydrogen sulfide in comparison to other iron containing mediums, such as Sulfide Indole Motility (SIM) Medium (Cat. no. Q30). Thus organisms that have weak hydrogen sulfide production may show only trace hydrogen sulfide activity, or none at all. Some species may require up to seven days incubation time to show this reaction.

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 typhimurium ***
ATCC ® 14028
C 18-48hr 35°C Aerobic Growth; purple slant and butt, blackening with H 2 S positive
Proteus mirabilis ***
ATCC ® 12453
C 18-48hr 35°C Aerobic Growth; red slant, yellow butt, H 2 S negative
Shigella flexneri ***
ATCC ® 12022
C 18-48hr 35°C Aerobic Growth; purple slant, yellow butt, H 2 S negative

*** Recommended QC strains for User Quality Control according to the CLSI document M22 when applicable.

User Quality Control

PHYSICAL APPEARANCE

Lysine Iron Agar (LIA) should appear slightly opalescent, and reddish-purple in color.

S. typhimurium growing on LIA

Salmonella typhimurium (ATCC ® 14028) growing on Lysine Iron Agar (Cat. no. L25). Incubated aerobically for 24 hours at 35ºC.

P. mirabilis growing on LIA

Proteus mirabilis (ATCC ® 12453) growing on Lysine Iron Agar (Cat. no. L25). Incubated aerobically for 24 hours at 35ºC.



S. flexneri growing on LIA

Shigella flexneri (ATCC ® 12022) growing on Lysine Iron Agar (Cat. no. L25). Incubated aerobically for 24 hours at 35ºC.

LIA

Uninoculated tube of Lysine Iron Agar (Cat. no. L25).



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.


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031016gr