Modified Lysine Iron Agar (MLIA)

Cat. no. G86 Modified Lysine Iron Agar (MLIA), 15x100ml Plate, 18ml 10 plates/bag

INTENDED USE

Hardy Diagnostics Modified Lysine Iron Agar (MLIA) is recommended for the selective and differential isolation of Salmonella spp. from food.

This product is not intended to be used for the diagnosis of human disease.

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. (2) However, some Salmonella spp. could be overlooked on LIA due to their atypical appearance and the overgrowth of non-salmonella Enterobacteriaceae . LIA was modified in 1979 by Rappold and Bolderdijk to provide better detection of hydrogen sulfide-positive and -negative Salmonella spp. through the addition of novobicin, bile salts, lactose, and sucrose. (7)

Hardy Diagnostics MLIA is based upon Rappold and Bolderdijk's modification. The indicator in MLIA 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 the source of hydrogen sulfide, and ferric ammonium citrate is used as an indicator, which forms a black precipitate in the presence of free hydrogen sulfide gas generated by colonies of certain species. Dextrose is the primary carbohydrate source for Salmonella spp., but lactose and sucrose are added to allow differentiation. Enteric organisms that are capable of fermenting dextrose, lactose, or sucrose will produce acid, resulting in yellow media. Lysine is used to show the decarboxylation reaction, which causes an alkaline reaction and the medium color to remain purple. The yellow color is seen only if lysine decarboxylation does not occur, as this alkaline reaction overcomes any acidic (yellow) conditions. If lysine is de-animated in the presence of oxygen, a red color change is exhibited in the colony.

FORMULA

Ingredients per liter of deionized water:*

Lysine Iron Agar 34.0gm
Bile Salts No. 3 1.5gm
Lactose 10.0gm
Sucrose 10.0gm
Sodium thiosulfate 6.76gm
Ferric Ammonium Citrate 0.3gm
Novobiocin 0.015gm

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 2-8ºC. 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: Consult listed references for information on sample collection. (1-4,8)

Samples should be submitted directly to the laboratory without delay and protected from excessive heat and cold.

1. Process the specimen as appropriate.

2. Inoculate a Salmonella enrichment broth (Hanja Tetrathionate Broth, Cat. no. K289, Tetrathionate Broth, Cat. no. K65, or Lactose Broth, Cat. no. K137) and incubate for 18-24 hours at 35ºC.

3. Allow the plates to warm to room temperature, and the agar surface to dry before inoculating.

4. Streak for isolation with a sterile loop or swab.

5. Incubate plates aerobically at 35ºC. for 18-24 hours.

6. Examine colonial morphology, characteristics, and color reactions.

INTERPRETATION OF RESULTS

After 18-24 hours incubation, Salmonella colonies appear as purple colonies with a black-center or black periphery. Escherichia coli , Citrobacter , Enterobacter and Proteus colonies that are not inhibited will be yellow with no blackening. Citrobacter freundii colonies can appear as purple colonies with black-centers and be mistaken for hydrogen sulfide-positive strains of Salmonella .

LIMITATIONS

Citrobacter freundii colonies can appear as purple colonies with black-centers and be mistaken for hydrogen sulfide-positive strains of Salmonella .

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, other culture media such as Tetrathionate Broth (Cat. no. K65) and Lactose Broth (Cat. no. K137), 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 enteritidis
ATCC ® 13076
A 18-24 hrs 35°C Aerobic Growth; Clear colonies with black centers, H 2 S positive, medium remains purple
Escherichia coli
ATCC ® 25922
A 18-24 hrs 35°C Aerobic Partial to complete inhibition; If present, yellow colonies without black centers, H 2 S negative
Proteus mirabilis
ATCC ® 12453
A 18-24 hrs 35°C Aerobic Partial to complete inhibition

USER QUALITY CONTROL

Physical Appearance

Modified Lysine Iron Agar should appear slightly opalescent, and purple in color.

REFERENCES

1. APHA Technical Committee on Microbiological Methods for Foods. Compendium of Methods for the Microbiological Examination of Foods, APHA, Washington, D.C.

2. Tille, P., et al. 2007. Bailey and Scott's Diagnostic Microbiology, 12th ed. C.V. Mosby Company, St. Louis, MO.

3. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

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

5. The Official Compendia of Standards. 2008. USP27-NF22 . United States Pharmacopeial Convention, Rockville, MD.

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

7. Rappold, H., and Bolderdijk, R.F. 1979. Modified lysine iron agar for isolation of Salmonella from food. Appl. Environ. Microbiol. ; 38(1):162-3.

8. U.S. Food and Drug Administration. Bacteriological Analytical Manual. AOAC, Arlington, VA. www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/default.htm

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

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